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Handbook of
INDUSTRIAL
BIOCATALYSIS
© 2005 by Taylor & Francis Group, LLC

Handbook of
INDUSTRIAL
BIOCATALYSIS
Ching T. Hou
Boca Raton London New York Singapore
A CRC title, part of the Taylor & Francis imprint, a member of the
Taylor & Francis Group, the academic division of T&F Informa plc.

Published in 2005 by
CRC Press
Taylor & Francis Group
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CRC Press is an imprint of Taylor & Francis Group
No claim to original U.S. Government works

Printed in the United States of America on acid-free paper
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International Standard Book Number-10: 0-8247-2423-2 (Hardcover)

International Standard Book Number-13: 978-0-8247-2423-8 (Hardcover)
Library of Congress Card Number 2004064922
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Library of Congress Cataloging-in-Publication Data
Hou, Ching T. (Ching-Tsang), 1935-

Handbook of industrial biocatalysis / Ching T. Hou.
p. cm.
Includes bibliographical references and index.
ISBN 0-8247-2423-2 (alk. paper)
1. Enzymes--Biotechnology. I. Title.
TP248.65.E59H68 2005
660.6'34--dc22 2004064922
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Preface
Pasteur initiated the scientific study of fermentation. When it was appreciated that microbes catalyzed
the chemical reactions used in the production of wine, cheese, and other foods, it became reasonable to
expect that they could be put to work in the manufacture of chemicals for industry. Beginning in the
late 1960s, both chemical industry and government agencies moved away from petroleum-based nonre-
newable feedstocks for production of commodity and specialty chemicals to emphasize the use of
renewable resources such as carbohydrates, oils, and fats. In addition, in recent years, the oil and fat
industry has started to emphasize quality rather than quantity of oil and fat for human consumption.
The definition of biocatalysis includes enzyme catalysis, biotransformation, bioconversion, fermentation,
and biotechnology. It deals not only with one-step catalytic reaction, but also includes many sequential
reaction steps to produce a product. Biocatalysis is a bioprocess including molecular manipulation of
enzymes, the reaction itself, and product recovery.
This handbook was assembled with the intent of bringing together all types of industrial biocatalysis.
It consists of 29 chapters whose authors are the world’s most famous and most active researchers in this
field. The basic information and theoretical considerations for a specific topic area or a specific biotech-
nological application are provided, and every effort has been made to include the current information.
This is the most up-to-date handbook on industrial applications of biosciences and biotechnology.
The book is divided into three sections. The first describes the world’s newest biotechnology, including
bioprocesses on producing potential industrial products from hydrophobic substrates such as oils and fats.
The products include healthy food, nutritional supplements, neutraceuticals, chiral synthons, specialty
chemicals, surfactants, biopolymers, and antimicrobial and physiologically active agents. Metabolic path-
ways and function of polyunsaturated fatty acids (PUFAs) in mammals as well as transgenic production of
long-chain PUFA-enriched oils are presented by Vic Huang of Abbott Labs. J. Ogawa and S. Shimizu of
Kyoto University, Japan introduce examples of bioprocess development that started from process design
stemming from the discovery of the unique metabolites hydrantoin, cyclic imide, microbial nucleosides,
and conjugated fatty acids. Tsuneo Yamane of Nagoya University, Japan describes biocatalysis in microaque-
ous organic media including lipase, esterase, protease, and so on. Rolf Schmid of the University of Stuttgart,
Germany contributes a chapter on biocatalysts for the epoxidation and hydroxylation of fatty acid alcohols
including fermentation/bioreactor process using oxygenases. Kumar Mukherjee of Munster, Germany uses
lipase specificities toward fatty acids and their derivatives—alcohols, alkanethiols, and sterols — to enrich
n-3 and n-6 PUFAs, very long-chain monounsaturated fatty acids, other acids, and alcohols. K. Lee and
J. Shaw of the Academia Sinica, Taiwan describe a successful story about recombinant Candida rugosa lipase
with improved catalytic properties and stabilities. Ching Hou of NCAUR, USDA shares his discovery of novel
oxygenated fatty acids and their potential industrial application from vegetable oils. Yuji Shimada of Osaka
Municipal Technical Research Institute (OMTRI) describes many examples of the application of lipase to
industrial-scale purification of oil- and fat-related compounds including production of PUFA-enriched oil,
conversion of waste edible oil to biodiesel fuel, and purification of tocopherols, sterols, and steryl esters.

Casmir Akoh’s group at University of Georgia give a thorough overview on lipase modification of lipids.
Shuji Adachi of Kyoto University describes lipase-catalyzed condensation in an organic solvent including
substrate selectivity of lipase for various carboxylic acids and continuous production of esters such as
acyl ascobates. Naoto Yamada of Kao Corporation, Japan presents enzymatic production of diacyl glycerol
and its beneficial physiological function. Diacyl glycerol functions like oil for all food preparation includ-
ing frying, yet prevents the accumulation of body fat. Satoshi Negishi of Nisshin OilliO, Japan Ltd.,
describes the use of nonimmobilized lipase for industrial esterification of food oils. M. Hosokawa and
K. Takahashi of Hokkaido University, Japan describe their design for industrial production of polyunsat-
urated phospholipids and their biological functions (health application). Dan Solaiman’s group at ERRC,
USDA presents the production of biosurfactants by fermentation of fats, oils, and their coproducts
including microbial glycolipids, sophorolipids, and rhamnolipids. Tsunehiro Aki of Hiroshima University,
Japan describes current metabolic engineering on development and industrialization of transgenic oils.
Tom Foglia’s group at ERRC, USDA presents lipase-catalyzed production of structured lipids as low-
calorie fats. Toro Nakahara of National Institute of Advanced Industrial Science and Technology, Japan
describes microbial polyunsaturated fatty acid production including lipids from bacteria, yeasts, and
fungi. Gudmundur Haraldsson of University of Iceland describes lipase-catalyzed production of EPA or
DHA containing triacylglycerols derived from fish oil. Rich Ashby et al. of ERRC, USDA describe biopoly-
esters derived from the fermentation of renewable resources including polylactic acid, polytrimethylene
terephthalate, and polyhydroxyalkanoates.
The second section of the handbook deals with producing value-added products from carbohydrate
substrates. The scope includes ethanol production, oligosaccharides and glycosides, utilization of hemi-
celluloses, and carbohydrate-active enzymes. Hajime Taniguchi of Chubu University, Nagoya, Japan
presents carbohydrate-active enzymes for the production of oligosaccharides including enzymes for most
of the oligosaccharides, such as isomalto-, nigero-, gentio-, fructo-, galacto-, chitosan-, and xylo-oli-
gosaccharides, trehalose, palatinose, trehalulose, and lactosucrose. Peter Biely and Gregory Cote of
NCAUR, USDA describe a special group of carboxylic acid esters that operate on highly hydrated
substrates such as partially acylated polysaccharides. H. Nakano and S. Kitahata of OMTRI, Osaka, Japan
describe industrial-scale production of various cyclodextrins from starch by cyclodextrin glucotrans-
ferase. Bruce Dien of NCAUR, USDA contributes a review on converting herbaceous energy crops to
bioethanol with emphasis on pretreatment processes. Badal Saha of NCAUR, USDA describes enzymes
as biocatalysts for conversion of lignocellulosic biomass to fermentable sugars. Its substates include
various agricultural residues such as corn fiber, corn stover, wheat straw and rice straw.
The third section deals with other potential industrial bioprocesses. Gregory Zeikus of Michigan State
University describes applications of bioelectrocatalysis for synthesis of chemicals, fuels, and drugs.
Ramesh Patel of Bristol-Myers Squibb, New Jersey uses bioprocesses to synthesize chiral intermediates
for drug development including anticancer drugs (paclitaxel, orally active taxane, deoxyspergualin and
antileukemic agent), antiviral drugs (BMS-186318, HIV protease inhibitor, Atzanavir, crixivan), reverse
transcriptase inhibitor (Abacavir, Lobucavir), antihypertensive drugs (angiotensin converting enzyme
inhibitor, captopril, monopril), neutral endopeptidase inhibitors, squalene synthase inhibitors, throm-
boxane A2 antagonist, calcium channel blockers, potassium channel blockers, β-3-receptor agonists,
melatonin receptor agonists, anti-Alzheimers drugs, anti-infective drugs, respiratory and allergic diseases,
acyloin condensation, enantioselective and enzymatic deprotection. R. Sakaguchi and L. Junejia of Taiyo
Kagaku Company, Japan present a novel nutrition delivery system that also preserves the stability of food
components and flavor. Sima Sariaslani et al. of Dupont Central Research & Development, Experimental
vi

Station, Wilmington, Delaware describe pathway engineering for production of trans-para-hydroxycin-
namic acid from renewable resources. Finally, Chiara Schiraldi and Mario De Rosa of Second University
of Naples, Italy describe industrial applications of extremophiles.
The Handbook of Industrial Biocatalysis is intended for teachers, postdoctorate and graduate students,
and industrial scientists who conduct research in biosciences and biotechnology. It is therefore expected
that it will serve as a valuable reference for researchers in the field and as a complementary text for
graduate-level reading and teaching.
Ching T. Hou, Ph.D.

Peoria, Illinois
vii

Contributors
Shuji Adachi Peter Biely Masashi Hosokawa

Division of Food Science and Institute of Chemistry Graduate School of Fisheries
Biotechnology Slovak Academy of Sciences Sciences
Graduate School of Agriculture Slovakia Hokkaido University
Kyoto University Japan
Japan Gregory L. Côté
National Center for Agricultural
Utilization Research Ching T. Hou
Tsunehiro Aki Agricultural Research Service Microbial Genomics and
Department of Molecular U.S. Department of Agriculture Bioprocessing Research Unit
Biotechnology Peoria, IL National Center for Agricultural
Graduate School of Advanced Utilization Research
Sciences of Matter Bruce S. Dien Agricultural Research Service
Hiroshima University National Center for Agricultural USDA
Japan Utilization Research Peoria, IL
USDA, Agricultural Research
Service
Casimir C. Akoh Peoria, IL Lisa Huang
Department of Food Science Biochemical Sciences &
& Technology Thomas A. Foglia Engineering
University of Georgia Eastern Regional Research DuPont Central Research and
Athens, GA Center Development
ARS Experimental Station
USDA Wyndmoor, PA Wilmington, DE
Richard D. Ashby
Eastern Regional Research Anthony Gatenby
Center Biochemical Sciences & Yung-Sheng Huang
ARS Engineering Ross Products Division
USDA Wyndmoor, PA DuPont Central Research and Abbott Laboratories
Development Columbus, OH
Experimental Station
Arie Ben-Bassat
Wilmington, DE
Biochemical Sciences & Loren B. Iten
Engineering National Center for Agricultural
Gudmundur G.
DuPont Central Research and Utilization Research
Haraldsson
Development Science Institute USDA
Experimental Station University of Iceland Agricultural Research Service
Wilmington, DE Reykjavik, Iceland Peoria, IL
ix

L.R. Juneja Noboru Matsuo Kazuhisa Ono
Nutritional Foods Division Biological Science Laboratories Department of Molecular
Taiyo Kagaku Co. Ltd. Kao Corporation Biotechnology
Mie, Japan Japan Graduate School of Advanced
Sciences of Matter
Steffen C. Maurer Hiroshima University
Seiji Kawamoto
Institute for Technical Japan
Department of Molecular
Biotechnology Biochemistry
University of Stuttgart Ramesh N. Patel
Graduate School of Advanced
Bristol-Myers Squibb
Sciences of Matter Germany
Pharmaceutical Research
Hiroshima University
Institute
Japan Kumar D. Mukherjee New Brunswick, NJ
Institute for Lipid Research
Federal Centre for Cereal Suzette L. Pereira
Sumio Kitahata
Department of Bioscience and Potato and Lipid Research Ross Products Division
Biotechnology Münster, Germany Abbott Laboratories
Shinshu University Columbus, OH
Nagano, Japan Toshihiro Nagao
Osaka Municipal Technical TP Rao
Research Institute Nutritional Foods Division
Guan-Chiun Lee Osaka, Japan Taiyo Kagaku Co. Ltd.
Institute of Botany Mie, Japan
Academia Sinica
Nankang, Taipei, Taiwan Toro Nakahara Badal C. Saha
National Institute of Advanced Fermentation Biotechnology
Industrial Science and Research Unit
Ki-Teak Lee Technology (AIST)
Department of Food Science and National Center for Agricultural
Technology Utilization Research
Chungnam National University Hirofumi Nakano Agricultural Research Service
Yusung-Gu, Taejon Osaka Municipal Technical USDA
Republic of Korea Research Institute Peoria, IL
Osaka, Japan
N. Sakaguchi
Jeung-Hee Lee Nutritional Foods Division
Department of Food Science and Satoshi Negishi Taiyo Kagaku Co. Ltd.
Technology Research Laboratory of The
Mie, Japan
Chungnam National University Nisshin OilliO Group, Ltd.
Yusung-Gu, Taejon Yokosuka, Kanagawa, Japan
Sima Sariaslani
Republic of Korea
Biochemical Sciences &
Jun Ogawa Engineering
Amanda E. Leonard Division of Applied Life Sciences DuPont Central Research and
Ross Products Division Graduate School of Agriculture Development
Abbott Laboratories Kyoto University Experimental Station
Columbus, OH Japan Wilmington, DE

Rolf D. Schmid Christopher D. Skory Yomi Watanabe
Institute for Technical National Center for Agricultural Osaka Municipal Technical
Biochemistry Utilization Research Research Institute
University of Stuttgart USDA, Agricultural Research Osaka, Japan
[email protected] Service
stuttgart.de Peoria, IL
Naoto Yamada
Daniel K.Y. Solaiman Production Quality
Subramani Sellappan Eastern Regional Research Management Division
Department of Food Science Center Kao Corporation
& Technology ARS, USDA Japan
University of Georgia Wyndmoor, PA
Athens, GA
Koretaro Takahashi Tsuneo Yamane
Graduate School of Fisheries
Jei-Fu Shaw Laboratory of Molecular
Institute of Botany Sciences Biotechnology
Academia Sinica Hokkaido University Graduate School of Bio- &
Nankang Japan Agro-Sciences
Taipei, Taiwan Nagoya University
Hajime Taniguchi Japan
Department of Biological
Chwen-Jen Shieh Chemistry
Department of Bioindustrial Chubu University
Teruyoshi Yanagita
Technology Japan
Department of Applied
Dayeh University
Biological Science
Taiwan Tina Van Dyk Saga University
Biochemical Sciences &
Japan
Engineering
Yuji Shimada DuPont Central Research and
Osaka Municipal Technical Development
Research Institute Experimental Station
J. Gregory Zeikus
Japan Department of Biochemistry &
Wilmington, DE
Molecular Biology
Michigan State University
Sakayu Shimizu Takaaki Watanabe East Lansing, MI
Division of Applied Life Sciences Processing Development
Graduate School of Agriculture Research Laboratories
Kyoto University Kao Corporation
Japan Japan
xi

Contents
Chapter 1 Enzymes for the Transgenic Production of Long-Chain

Polyunsaturated Fatty Acid-Enriched Oils
Yung-Sheng Huang, Suzette L. Pereira, and Amanda E. Leonard ..................................... 1-1
Chapter 2 Screening for Unique Microbial Reactions Useful

for Industrial Applications
Jun Ogawa and Sakayu Shimizu ........................................................................................ 2-1
Chapter 3 Biocatalyses in Microaqueous Organic Media

Tsuneo Yamane .................................................................................................................... 3-1
Chapter 4 Biocatalysts for the Epoxidation and Hydroxylation of Fatty Acids

and Fatty Alcohols
Steffen C. Maurer and Rolf D. Schmid ............................................................................... 4-1
Chapter 5 Lipase-Catalyzed Kinetic Resolution for the Fractionation of Fatty

Acids and Other Lipids
Kumar D. Mukherjee ........................................................................................................... 5-1
Chapter 6 Protein Engineering of Recombinant Candida rugosa Lipases

Guan-Chiun Lee, Chwen-Jen Shieh, and Jei-Fu Shaw....................................................... 6-1
Chapter 7 Production of Value-Added Industrial Products from Vegetable Oils:

Oxygenated Fatty Acids
Ching T. Hou and Masashi Hosokawa ............................................................................... 7-1
Chapter 8 Application of Lipases to Industrial-Scale Purification of Oil-

and Fat-Related Compounds
Yuji Shimada, Toshihiro Nagao, and Yomi Watanabe........................................................ 8-1
Chapter 9 Applications of Lipases in Modifications of Food Lipids

Subramani Sellappan and Casimir C. Akoh....................................................................... 9-1
Chapter 10 Lipase-Catalyzed Condensation in an Organic Solvent

Shuji Adachi .................................................................................................................... 10-1
xiii

Chapter 11 Enzymatic Production of Diacylglycerol and Its Beneficial
Physiological Functions
Naoto Yamada, Noboru Matsuo, Takaaki Watanabe, and Teruyoshi Yanagita ............ 11-1
Chapter 12 The Use of Nonimmobilized Lipase for Industrial

Esterification of Food Oils
Satoshi Negishi ................................................................................................................ 12-1
Chapter 13 Preparation of Polyunsaturated Phospholipids

and Their Functional Properties
Masashi Hosokawa and Koretaro Takahashi ................................................................. 13-1
Chapter 14 Production of Biosurfactants by Fermentation of Fats, Oils, and Their

Coproducts
Daniel K.Y. Solaiman, Richard D. Ashby, and Thomas A. Foglia................................. 14-1
Chapter 15 Fatty Acid-Modifying Enzymes: Implications for Industrial Applications

Tsunehiro Aki, Seiji Kawamoto, Seiko Shigeta, and Kazuhisa Ono .............................. 15-1
Chapter 16 Low-Calorie Fat Substitutes: Synthesis and Analysis

Ki-Teak Lee, Thomas A. Foglia, and Jeung-Hee Lee...................................................... 16-1
Chapter 17 Microbial Polyunsaturated Fatty Acid Production

Toro Nakahara ................................................................................................................ 17-1
Chapter 18 Structured Triacylglycerols Comprising Omega-3 Polyunsaturated

Fatty Acids
Gudmundur G. Haraldsson ............................................................................................ 18-1
Chapter 19 Biopolyesters Derived from the Fermentation of Renewable Resources

Richard D. Ashby, Daniel K.Y. Solaiman, and Thomas A. Foglia................................. 19-1
Chapter 20 Carbohydrate Active-Enzymes for the Production of Oligosaccharides

Hajime Taniguchi............................................................................................................ 20-1
Chapter 21 Microbial Hemicellulolytic Carbohydrate Esterases

Peter Biely and Gregory L. Côté ..................................................................................... 21-1
Chapter 22 Application of Cyclodextrin Glucanotransferase to the

Synthesis of Useful Oligosaccharides and Glycosides
Hirofumi Nakano and Sumio Kitahata ......................................................................... 22-1
Chapter 23 Converting Herbaceous Energy Crops to Bioethanol: A Review

with Emphasis on Pretreatment Processes
Bruce S. Dien, Loren B. Iten, and Christopher D. Skory ............................................... 23-1
xiv

Chapter 24 Enzymes as Biocatalysts for Conversion of Lignocellulosic Biomass to
Fermentable Sugars
Badal C. Saha ................................................................................................................. 24-1
Chapter 25 Bioelectrocatalysis: Electroactive Microbial and Enzyme Technologies

for Detection and Synthesis of Chemicals, Fuels, and Drugs
J. Gregory Zeikus............................................................................................................. 25-1
Chapter 26 Biocatalysis: Synthesis of Chiral Intermediates for Pharmaceuticals

Ramesh N. Patel.............................................................................................................. 26-1
Chapter 27 Nutrition Delivery System: A Novel Concept of Nutrient Fortification

T.P. Rao, N. Sakaguchi, and L.R. Juneja ........................................................................ 27-1
Chapter 28 Renewable Resources for Production of Aromatic Chemicals

Sima Sariaslani, Tina Van Dyk, Lisa Huang,
Anthony Gatenby, and Arie Ben-Bassat.............................................................................. 28-1
Chapter 29 Extremophiles from the Origin of Life to Biotechnological

Applications
Chiara Schiraldi and Mario De Rosa............................................................................. 29-1
xv

1
Enzymes for the
Transgenic Production
of Long-Chain
Polyunsaturated Fatty
Acid-Enriched Oils
1.1 Introduction ....................................................................... 1-1

Metabolism of Linoleic Acid and a-Linolenic Acid • Function
of PUFAs • PUFA Production and Chronic Diseases
1.2 Commercial Sources of PUFAs ......................................... 1-3
Fish Oil • Plant Oils • Microbial Oils
1.3 Transgenic Production of PUFAs...................................... 1-4
Yung-Sheng Huang Enzymes Required for Transgenic GLA Production • Enzymes
Required for Transgenic ARA and EPA Production • Enzymes
Required for Transgenic DHA Production • Alternate
Suzette L. Pereira
Enzymes for Transgenic EPA/DHA Production:
The PKS System
Amanda E. Leonard 1.4 Conclusion and Future Perspectives................................. 1-8
1.1 Introduction
Polyunsaturated fatty acids (PUFAs) are fatty acids of 18 carbons or more in length with two or more
methylene-interrupted double bonds in the cis position. Depending on the position of the first double
bond proximate to the methyl end of fatty acids, PUFAs can be designated by the omega (ω-) or (n-)
number, and classified into two major groups: ω6 (or n-6) and ω3 (or n-3) families. For example, linoleic
acid (LA) in the n-6 family is designated as C18:2n-6 to indicate that this fatty acid contains 18 carbons
and two double bonds, with the first double bond at the sixth carbon from the methyl end. Similarly,
α-linolenic acid (C18:3n-3) in the n-3 family has 18 carbons and three double bonds, with the first
double bond located at the third carbon from the methyl end (Figure 1.1).
1.1.1 Metabolism of Linoleic Acid and -Linolenic Acid

Animals are incapable of synthesizing both linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA,
C18:3n-3) due to lack of the ∆12 and ∆15-desaturases. However, animals can metabolize these two fatty
acids obtained from the diet to form longer and more unsaturated PUFAs to meet the metabolic needs.
Since these two fatty acids must be obtained from the diet, they are considered to be essential fatty acids.
1-1

1-2 Handbook of Industrial Catalysis
6 9
COOH
Linoleic
CH3
CH3 3 6 9 COOH
α-Linolenic
FIGURE 1.1 Nomenclature of polyunsaturated fatty acids
In most eukaryotes, the biosynthesis of PUFAs involves a complex series of desaturation and elongation
steps (Figure 1.2).1 For example, eicosapentaenoic acid (EPA, C20:5n-3) is synthesized from ALA by the
addition of a double bond by a ∆6-desaturase to form stearidonic acid (SDA, C18:4n-3); the elongation of
SDA to form ω3-eicosatetraenoic acid (ω3-ETA, C20:4n-3); and the addition of another double bond by a
∆5-desaturase to form EPA.2 The formation of DHA from EPA occurs via different mechanisms in eukary-
otes. In higher eukaryotes like mammals, EPA is elongated to ω3-docosapentaenoic acid (ω3-DPA, C22:5n-
3), which is further elongated to ω3-tetracosapentaenoic acid (ω3-TPA, C24:5n-3). ω3-TPA is then desat-
urated by a ∆6-desaturase to generate ω3-tetracosahexaenoic acid (THA, C24:6n-3) in the microsomes.
The THA is then transported to the peroxisomes, where it is β-oxidized to form DHA.3,4 However, in lower
eukaryotes like the thraustochytrid sp., EPA is elongated to ω3-DPA followed by the addition of a double
bond directly to ω3-DPA, by ∆4-desaturase, to generate DHA.5 The synthesis of long-chain n-6 PUFAs
from LA occurs via similar alternating desaturation and elongation steps (Figure 1.2).
1.1.2 Function of PUFAs

In mammals, PUFAs are important structural components that modulate membrane fluidity and perme-
ability.6 For example, docosahexaenoic acid (DHA, C22:6n-3), a long-chain n-3 PUFA, and arachidonic
ω–3
18:3n-3
18:2n-6 α-linolenic
linoleic
∆6 ∆6
ω–3
18:3n-6 18:4n-3
γ-linolenic stearidonic
elo elo
ω–3
20:3n-6 20:4n-3
dihomo-γ-linolenic eicosatetraenoic
∆5 ∆5
ω–3
20:4n-6 20:5n-3
arachidonic eicosapentaenoic
elo elo
∆4 ∆4
22:5n-6 22:4n-6 22:5n-3 22:6n-3
ω6-docosapentaenoic adrenic ω3-docosapentaenoic docosahexaenoic
elo elo
∆6 ∆6
24:5n-6 24:4n-6 24:5n-3 24:6n-3
ω6-tetracosapentaenoic ω6-tetracosatetraenoic ω3-tetracosapentaenoic ω3-tetracosahexaenoic
FIGURE 1.2 Metabolic pathway of linoleic and α–linolenic acids

Enzymes for the Transgenic Production 1-3
acid (AA, C20:4n-6), a long-chain n-6 PUFA, are found in high proportions in neuronal tissues such as
brain and retina, and testis.7,8 PUFAs also serve as precursors for a number of biologically active molecules,
such as eicosanoids, growth regulators, and hormones.9 In mammals, eicosanoids such as prostaglandins,
leukotrienes, and thromboxanes act locally on various signaling mechanisms that have effects on numer-
ous cellular functions including chemotaxis, vascular permeability, inflammation, vasoconstriction, etc.9
Thus, PUFAs have profound effects on various physiological processes, such as cognitive function, and
immunosuppressive and anti-inflammatory actions.10
1.1.3 PUFA Production and Chronic Diseases

The availability of long-chain PUFAs depends on the diet providing the precursors, such as LA and ALA,
and the activity of enzymes involved in the biosynthesis.11 Generally, only a small proportion of dietary
linoleate and α-linolenate (3.0% and 1.5%, respectively) can be converted to longer PUFAs, with the rest
getting β-oxidized to provide energy.12 The slow formation of PUFAs can be further compromised by
various nutritional and hormonal factors.13 For example, the activity of the ∆6-desaturase in vivo is
regulated by certain dietary components, age, and hormones.14 In addition, in chronic diseases like cancer
and diabetes, altered expression levels of this enzyme in different tissues have been observed.15–21 The
activity of the ∆5-desaturase is also regulated by diet,22 and altered expression levels of this enzyme have
been associated with various disease conditions, including eye disorders, Alzheimer’s disease, and diabe-
tes.23–25 Thus, low levels of long-chain PUFAs have been associated with disorders of the neurovisual
development and other complications of premature birth26–29 as well as implicated with incidence of
chronic diseases, such as diabetes, hypercholesterolemia, rheumatoid arthritis, autoimmune disorders,
Crohn’s disease, and cancer.13,30,31
Clinical evidence has shown that dietary supplementation of PUFAs, such as γ-linolenic acid (GLA,
C18:3n-6) and EPA/DHA, can exert the anti-inflammatory, antithrombotic, and antiarrhythmic activities,
and provide beneficial effects on glucose and lipid metabolism.32–38 These findings have received much
attention from food manufacturers and pharmaceutical companies, as well as the general public. As a
result, sales of these long-chain PUFAs as supplements and fortified foods, such as “DHA plus” eggs, and
DHA- and AA-fortified infant formulas have drastically increased in the past few years.
1.2 Commercial Sources of PUFAs

1.2.1 Fish Oil
Currently, the richest sources of EPA and DHA are derived from fish oils obtained from mackerel, herring,
salmon, and sardines. Fish obtain these long-chain PUFAs (LC-PUFAs) from the LC-PUFA-rich
microalage and phytoplankton they consume. Commercially, fish oils are available in the form of gelatin
capsules or oily preparations. Fish oils obtained from fish liver (e.g., cod liver oil) are rich in vitamin A
and D, and contain lower amounts (13% to 22%) of EPA/DHA. In contrast, fish oils obtained from fish
bodies (e.g., salmon oil) contain 20% to 30% EPA and DHA and are low in cholesterol and vitamin A
and D(reviewed in reference 39). These fish oils are used to enrich food products, animal feeds, and
aquaculture feeds, in addition to their use for direct human consumption. These oils are not very
economical due to the high costs involved in processing, refining, and stabilizing the oils. In addition,
the effects of overfishing and the vulnerability of global fisheries to environmental and climatic changes
have resulted in decreased yields, which has further driven up the cost of fish oils.40
1.2.2 Plant Oils

Plants do not produce EPA or DHA. However, certain plants can produce oils rich in GLA or ALA, and
serve as the current commercial sources of these PUFAs. Plant oils derived from borage, evening primrose,
and black currant are found to be rich in GLA (reviewed in reference 41). Borage oil is derived from the
seed of Borago officinalis and contains ~23% GLA. Evening primrose oil obtained from the seed of

Oenothera biennis contains ~9% GLA. Black currant oil derived from the seeds of Ribes nigrum is attractive
in that it contains 12% ALA in addition to 16% GLA. However, these oils are expensive due to high costs
of cultivation, seed harvesting, and oil extraction. Linseed oil (flax) is the richest source of ALA (57% of
total fatty acids). Dietary ALA can also be obtained from oils of canola, soybean, wheat germ, and walnut.
1.2.3 Microbial Oils

LC-PUFAs can also be extracted from single cell organisms like microalgae and fungi that can be
commercially cultivated in fermentors (heterotrophic producers), or in photoautotrophic cultivation
systems.42,43 Oleagenous fungi such as Mortierella alpina accumulate up to 40% (by wt.) oil, of which up
to 40% represents AA.44 Thus, this organism is commercially used in production of AA.45 Diatoms such
as Nitzschia, a good producer of EPA, and dinoflagellates such as Crypthecodinium cohnii that produce
large amounts of DHA, are currently commercially utilized for the production of EPA- and DHA-enriched
oils.42 Currently, marine protists such as the Thraustochytrids that make large amounts of DHA are being
explored for their potential to make DHA-rich oils for human consumption.42,46,47 The costs of production
of these oils are still considerably high, and further work involving strain-improvement and cultivation-
optimization need to be carried out to make these oils more economical.
1.3 Transgenic Production of PUFAs

Although LC-PUFAs-enriched oils are commercially available as discussed in Section 1.2, the cost of
production of these oils is generally very high, and the sources of supplies are often unreliable or
nonrenewable. The increase in demand for these PUFAs has raised interest in obtaining these from
alternate sources that are more economical and sustainable. One attractive option is the production of
LC-PUFA-enriched vegetable oils in oilseed crops like soybean, canola, and others. Since these plants can
only synthesize 18-carbon (C18) PUFAs such as LA and ALA, it is necessary to genetically manipulate
their lipid biosynthetic pathways in order to produce long-chain PUFAs such as GLA, EPA, and DHA.
For this, genes encoding the enzymes involved in LC-PUFA biosynthesis need to be isolated from LC-
PUFA-rich organisms, and transgenically expressed in oilseed crops. These include the various desaturases
and elongases outlined in Figure 1.3.
Desaturases are enzymes that catalyze the addition of a double bond (unsaturation) in a fatty acyl
chain (reviewed in reference 48). These enzymes are specific to the location, number, and stereochemistry
of double bonds already present in fatty acids.49 In addition, they have specificity for their substrate
carriers, which can be CoA-linked substrates, acyl carrier protein (ACP)-linked substrates, or glycerolipid-
linked substrates.
Elongases are enzymes that are responsible for the addition of two-carbon units to the carboxyl end
of a fatty acid chain. In both plants and animals, the elongase system is composed of four enzymes: a
condensing enzyme, β-ketoacyl CoA synthase (also referred to as elongase), β-ketoacyl CoA reductase,
β-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase. Fatty acid elongation is initiated by the
condensation of malonyl-CoA with a long chain acyl-CoA, yielding a β-ketoacyl-CoA in which the acyl
moiety has been elongated by two carbon atoms. This reaction is catalyzed by the condensing enzyme
β-ketoacyl CoA synthase (also referred to as “elongase”). β-ketoacyl-CoA is then reduced, dehydrated,
and further reduced by the remaining enzymes in the system to yield the elongated acyl-CoA.50 The
condensing enzyme (elongase) is known to be the rate-limiting enzyme,50–52 which regulates the systems
specificity for the fatty acid substrate in term of chain length and degree of unsaturation. The elongases
involved in the elongation of LC-PUFAs are distinct from the plant and yeast elongases that are involved
in the elongation of saturated or monounsaturated fatty acids (reviewed in reference 53).
The following sections will focus on the characterization and transgenic expression of nonmammalian
sources of LC-PUFA biosynthetic genes, with applications for the production of PUFA-enriched trans-
genic oils. Enzymes known to exist in all plants, such as the ∆9- and the ∆12-desaturase that are involved
in LA production (Figure 1.3), will not be described here since these enzymes are highly active in native

n-6 n-3
In Plants
ω-3
∆9 ∆12 18:2 18:3
Desaturase
18:0 18:1
LA ALA
ω-3
∆6-Desaturase ∆6-Desaturase
Desaturase
18:3 18:4
GLA STA
Elongase ω-3 Elongase

Desaturase
20:3 20:4
DGLA ETA
∆5-Desaturase ∆5-Desaturase
ω-3
PKS 20:4 Desaturase
ARA 20:5
EPA
Elongase
22:5
DPA
∆4-Desaturase
22:6
DHA
FIGURE 1.3 Pathway and transgenic production of LC–PUFAs
oil-seed crops. Although the discussion will focus on production of transgenic plant oils, it should be
noted that these enzymes also have applications for the production of transgenic oils in other oleaginous
organisms such Yarrowia and Rhodotorula.
1.3.1 Enzymes Required for Transgenic GLA Production

Commercially available sources of GLA-enriched oils from borage, evening primrose, and black currant54
are not economical for large-scale production. Hence efforts are ongoing to transgenically produce GLA
in an oil-seed crop. The key step in GLA production involves the insertion of a double bond between
carbon #6 and #7 of LA to generate GLA (Figure 1.3). This reaction is mediated by the ∆6-desaturase, a
membrane-bound enzyme located in the endoplasmic reticulum. This enzyme is classified as a “front-
end” desaturase because it is capable of introducing a double bond between a preexisting double bond
and the “front” (carboxyl end) of the fatty acid. It also contains a fused cytochrome b5 domain at the N-
terminus, which plays a role as an electron donor during desaturation, and this domain is essential for
activity.55
∆6-desaturases has been isolated from several fungal, plant, microbial, and mammalian sources that
produce GLA.48 Some of these include M. alpina,56 Mucor rouxii,57 Phytium irregulare,58 Physcomitrella
patens,59 borage,60 Echium plant sp.,61 Primula sp.,62 and Synechcocystis.60,63 Most of these ∆6-desaturases
are thought to act exclusively on the phospholipid-linked LA substrate.44,64 In contrast, the ∆6-desaturases
from mammalian sources are thought to recognize CoA-linked LA substrates.65
Most of the ∆6-desaturases have been functionally expressed in yeast, and many have also been tested in
plants.56,58,60,61,66 In addition, some ∆6-desaturases have also been expressed in oil-seed crops, resulting in
production of GLA in seeds. In the early study conducted in our laboratory, expression of the M. alpina
∆6-desaturase in a low-linolenic acid variety of Brassica napus resulted in the generation of low amounts
(~13%) of GLA in addition to the production of an uncommon fatty acid, ∆6,9-18:2.67,68 This uncommon

fatty acid was derived by the desaturation of oleic acid (OA, 18:1) by the ∆6-desaturase. This problem
was resolved by coexpressing the M. alpina ∆6-desaturase with its ∆12-desaturase, an enzyme that
converts oleic acid to LA (Figure 1.3). This resulted in the accumulation of >40% GLA in the transgenic
canola oil, with no detectable ∆6,9-18:2.67,68 Subsequent studies have been carried out using the Phytium
irregulare ∆6-desaturase in Brassica juncea, and have also resulted in successfully generation of 25% to
40% GLA in the transgenic seed.58 This oil, however, was also found to contain 2% to 10% stearidonic
acid (SDA, 18:4n-3) in addition to the uncommon fatty acid ∆6,9-18:2.58
1.3.2 Enzymes Required for Transgenic ARA and EPA Production

The production of the C20-PUFA arachidonic acid (ARA, 20:4n-6) from LA involves the desaturation of
LA to GLA (see Section 1.3.1.), followed by the elongation of GLA to dihomo-γ-linolenic acid (DGLA,
20:3n-6), and a subsequent desaturation of DGLA to ARA (Figure 1.3). Thus three major enzymes are
involved in this process: a ∆6-desaturase, a C18-PUFA-specific elongase, and a ∆5-desaturase. These same
enzymes also function on the n-3 pathway intermediates and are thus also involved in the biosynthesis
of EPA (20:5n-3) (Figure 1.3). Since ∆6-desaturase has just been discussed in the previous section (Section
1.3.1), this section will focus only on the C18-PUFA-specific elongase and the ∆5-desaturase.
The first C18-PUFA-specific elongase to be isolated was identified in our laboratory from the ARA-
rich fungus, Mortierella alpina.67 This enzyme when tested in baker’s yeast specifically recognized and
elongated the n-6 and n-3 C18-PUFA substrates, GLA and stearidonic acid (SDA, 18:4n-3), respectively,
whereas it demonstrated no activity on monounsaturated or saturated fatty acid substrates.69–72 Enzymes
with similar elongating activity have been isolated from Caenorhabditis elegans,73 Physcomitrella patens,74
and from the marine protist, Thraustochytrium sp.75 In addition, several PUFA-specific elongases have
been isolated from mammalian sources.53 All these PUFA-specific elongases contain five hydrophobic
regions predicted to be membrane-spanning regions. In addition, they contain a highly conserved
histidine-box motif composed of three histidine residues (HXXHH) embedded in the fourth membrane
spanning region.76–78 These features distinguish them from the plant elongases that are involved in
elongation of very long chain saturated and monounsaturated fatty acids, but not PUFAs.79,80 In addition,
the C18-PUFA-specific elongases are thought to recognize CoA-linked substrates64 as opposed to the plant
elongases that recognize acyl carrier protein (ACP)-linked substrates.
The ∆5-desaturase catalyzes the final step in the production of the C20-PUFAs, ARA and EPA. This
enzyme is so called because it introduces a double bond at the ∆5-position of the fatty acid. This desaturase
is also considered a “front-end” desaturase and shares all the conserved structural characteristics displayed
by other front-end desaturases such as the ∆6-desaturase. ∆5-desaturase genes have been identified from
fungi and algae such as Mortierella alpina.81,82 Thraustochytrium sp.,5 and Phaeodactylum tricornutum,83
and these have been functionally characterized in yeast. Additional ∆5-desaturases have been identified
from C. elegans,84 human,85 and rat.86 All the ∆5-desaturases identified so far are capable of desaturating
both the n-6 and n-3 PUFA substrates, DGLA and eicosatetraenoic acid (ETA, 20:4n-3), respectively.
Coexpression of the M. alpina ∆5-desaturase along with its C18-PUFA-specific elongase in yeast, in the
presence of exogenously supplied free fatty acid substrate, resulted in the production of significant amount
of ARA or EPA.69 When introduced into a low linolenic variety of Brassica napus, the M. alpina ∆5-
desaturase was capable of desaturating oleic acid (OA, 18:1n-9) to taxoleic acid (∆5,9-18:2), and LA to
pinolenic acid (∆5,9,12-18:3).81 This demonstrates its functionality in desaturating fatty acids at the ∆5-
position in higher plants, even in the absence of its preferred PUFA substrates.
Since plant oils often contain LA and ALA, the transgenic expression of the ∆6-desaturase, C18 PUFA-
specific elongase, and ∆5-desaturase would result in ARA and EPA. For production of an EPA-enriched
transgenic oil that does not contain ARA, it is necessary to shunt the n-6 PUFA metabolites to their
n-3 counterparts. This reaction is catalyzed by a group of enzymes designated the ω3-desaturases. These
enzymes are absent from mammals, but can be found in some plants, lower eukaryotes, and cyanobac-
teria.87 These enzymes are so called because they introduce a double bond at carbon atom #3 when
counted from the methyl- (ω-) end of the fatty acyl chain. ω3-desaturases share all the conserved features

present in other membrane-bound desaturases. These include the presence of two long stretches of
hydrophobic residues that traverse the lipid bilayer, and three histidine-rich motifs proposed to be
involved in the ligation of iron atoms within the active site domain of these enzymes.88 This protein also
contains the C-terminal motif, KAKSD, proposed to be a retention signal for many transmembrane
proteins in the ER.89 Unlike the front-end desaturases, the ω3-desaturases do not contain a fused cyto-
chrome b5 domain at their N-terminus, and are thus assumed to interact with a separate cytochrome b5
for their activity.
All plant and cyanobacterial ω3-desaturases act exclusively on the C18-PUFA substrate, LA, converting
it to ALA (Figure 1.3). Although many oilseed crops do contain endogenous ω3-desaturases, these
enzymes do not efficiently convert LA to ALA as evidenced by a high LA-to-ALA ratio in their total
lipids.54 Thus for transgenic EPA production, it might be necessary to transgenically express ω3-desatu-
rases with high enzymatic activity, in order to increase the shunt through the n-3 PUFA pathway. A novel
ω3-desaturase was identified from C. elegans that was capable of recognizing multiple n-6 PUFA sub-
strates, which included the C18-PUFAs, LA and GLA, as well as the C20- PUFA, DGLA.90,91 This enzyme
was found to be functional in plants90 and thus has potential applications for transgenic EPA production.
In addition, a novel fungal ω3-desaturase was recently identified in our laboratory that could specifically
convert ARA to EPA, and this enzyme was found to be functional in an oilseed crop.92 This enzyme has
applications for the removal of ARA from EPA- and DHA-enriched transgenic oils, by converting ARA
to EPA. This is especially necessary if the transgenic oils are targeted for adult nutrition, since ARA is a
precursor for synthesis of proinflammatory eicosanoids that are implicated in inflammatory and cardio-
vascular disease development.
Thus the transgenic production of EPA will be contingent on the success in coexpressing at least four
different enzymes in a single system. Coexpression of three of the PUFA biosynthetic enzymes, the ∆6-
desaturase, the PUFA-specific elongase, and the ∆5-desaturase, has been successfully demonstrated in
reconstituted baker’s yeast, resulting in ARA and EPA production when their respective substrate, LA or
ALA, was supplied exogenously.73,83 However, the yields of ARA or EPA obtained in these studies were
poor. Similar results were reported by Domergue et al.64 in their attempt to coexpress the ∆6- and ∆5-
desaturase from P. tricornutum along with the C18-PUFA elongase from P. patens in transgenic linseed.
From these experiments, it appears that there is an accumulation of the ∆6-desaturated fatty acids in the
membrane fractions and almost none in the acyl-CoA pool. It is thought that the ∆6-desaturase from
most fungi and algae function on phospholipid-linked (mainly phosphatidylcholine-linked) LA or ALA
substrates. However, the consecutive step is catalyzed by a PUFA-specific elongase that requires its
substrates to be present in the acyl-CoA pool. Thus it appears that a bottleneck in the pathway is created
due to the inefficient transfer of the ∆6-desaturated products from the phospholipids to the acyl-CoA
pool.64 To overcome this bottleneck, it might be necessary to identify and coexpress additional enzymes
that are involved in the transfer of phospholipid-linked PUFAs to the acyl CoA pools.
1.3.3 Enzymes Required for Transgenic DHA Production

The pathway for the biosynthesis of DHA varies among different groups of organisms. In lower eukaryotes
such as DHA-rich algae and fungi, it is thought that DHA is synthesized from EPA in a two-step process
(Figure 1.3): a) An initial elongation step that is catalyzed by a C20-PUFA recognizing elongase, wherein
EPA is elongated to ω3-DPA; b) The desaturation of ω3-DPA, catalyzed by a ∆4-desaturase, resulting in
the generation of DHA. Thus the production of transgenic DHA would involve coexpressing these two
new genes in addition to the four previously described genes (Section 1.3.2.) needed for EPA production.
The only enzymes identified to date that can recognize and elongate C20-PUFAs are from mammals.53
Expression of these genes in baker’s yeast revealed that some of them could elongate C18-PUFAs in addition
to C20- and C22-PUFAs, whereas others had a specificity for C20- and C22-PUFA substrates. None of these
enzymes acted on monounsaturated fatty acids or saturated fatty acid substrates.53 Attempts are currently
under way to identify similar C20-PUFA recognizing elongases from lower eukaryotes, which can then
be used for the production of transgenic oils.

The first ∆4-desaturase to be described was identified from a marine protist, Thraustochytrium, which
produces copious amounts of DHA.5 Like the ∆5- and ∆6-desaturase, the ∆4-desaturase is also a front-end
desaturating enzyme capable of introducing a double bond at carbon # 4 of ω3-DPA. In addition, this
enzyme can also desaturate the n-6 substrate adrenic acid (ADA, C22:4n-6) to generate ω6-DPA (C22:5n-6)
(Figure 1.3). Expression of the ∆4-desaturase gene in a oilseed crop, Brassica juncea, in the presence of
exogenously supplied ω3-DPA substrate resulted in the production of 3–6% DHA in the leaves, stems,
and roots of the transgenic Brassica.5 A new ∆4-desaturase was recently described from Euglena gracilis,
and this enzyme was found to desaturate C16-fatty acids in addition to C22-PUFAs.93
1.3.4 Alternate Enzymes for Transgenic EPA/DHA

Production: The PKS System
LC-PUFA biosynthesis in bacteria such as Shewanella and Vibrio occurs via a novel polyketide synthase
(PKS) pathway.94–96 This pathway is thought to be analogous to the fatty acid synthase (FAS) pathway
involved in the synthesis of short-chain fatty acids.97 This system was also identified to be involved in
DHA production in the marine eukaryote, Schizochytrium.98 Here, PUFA production is thought to be
initiated by the condensation between a short-chain starter unit like acetyl CoA, and an extender unit
like malonyl CoA. The four-carbon acyl chain formed is covalently attached to an acyl carrier protein
(ACP) domain of the PKS complex, and goes through successive rounds of reduction, dehydration,
reduction, and condensation, with the acyl chain growing by two carbon units with each round. A novel
dehydratase/isomerase has been proposed to exist in this PKS complex that can catalyze trans- to cis-
conversion of the double bonds, thus generating double bonds in the correct position of EPA and DHA.98
Genes involved in this PKS system exist sequentially on long (20–30 Kb) open reading frames (ORFs),
and the identity of every region within these ORFs are still unknown.95,96,98 Expression of the Shewanella
PKS system in an E coli or Synechococcus expression system resulted in EPA production, although the
levels of EPA produced were low.94,98 Although none of these PUFA-PKS genes have been expressed in
plants as yet, this system offers an attractive alternative to the desaturase/elongase system for the pro-
duction of EPA/DHA-enriched transgenic oils.
1.4 Conclusion and Future Perspectives

Fatty acids are critical for the normal development and function of all organisms, and in particular, very
long chain PUFAs are necessary for the health and maintenance of higher organism such as mammals.
Although the biosynthetic pathway of long-chain PUFAs has been studied for a while, detailed biochem-
ical analysis of the enzymatic machinery has been especially hard. This is because the extreme hydro-
phobicity of the desaturases and elongases creates difficulties during purification of large amounts of
these enzymes that are required for biochemical characterization. However, much progress has been made
over the last few years in the cloning and identification of genes encoding the PUFA biosynthetic enzymes
from different organisms. These findings have important biotechnological applications. For example,
these genes can be used in the production of PUFA-rich transgenic oils to meet the increasing demands
of the chemical, pharmaceutical, and nutraceutical industry for therapeutic and prophylactic use.
Advances in understanding gene regulation in PUFA biosynthesis will also impact the single-cell oil
industry. This in turn will affect the marine fish-farming industry, which depends on PUFAs generated
by microalgae and fungi for enhancing the levels of PUFAs in fish.
However, some challenges still need to be overcome with respect to transgenic production of PUFAs.
Although the overall scheme of PUFA biosynthesis appears to be common for most organisms, enzymes
from different organisms may not necessarily be compatible. This may result in unanticipated bottlenecks
in the pathway leading to lower yields of LC-PUFAs. This had already been observed during preliminary
studies on transgenic EPA production (Section 1.3.2). In addition, many unknowns still need to be
addressed with respect to coexpressing several genes from multiple sources simultaneously. It is still
early to predict if the transgenic oils thus generated will have a fatty acid profile reflective of natural fish

oil, without the accumulation of unwanted fatty acid byproducts. In addition, it is not known if the
transgenically produced LC-PUFAs will indeed accumulate in the triacylglycerol (TAG) fraction. Once
these challenges have been overcome however, these transgenic PUFA-enriched oils will afford the public
an economical source of desirable PUFAs that will greatly impact general health and nutrition in the
future.
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2
Screening for Unique
Microbial Reactions
Useful for Industrial
Applications
2.1 Introduction ....................................................................... 2-1

2.2 Analysis and Application of Microbial
Cyclic Amide Metabolism.................................................. 2-2
Overview of Microbial Cyclic Amide Metabolism Analysis
and Application of Microbial Hydantoin Metabolism • Analysis
and Application of Microbial Cyclic Imide Metabolism
2.3 Analysis and Application of Microbial
Nucleoside Metabolism.................................................... 2-10
Overview of Microbial Nucleoside Metabolism • Biochemical
Retrosynthesis of 2'-Deoxyribonucleoside Through Microbial
Nucleoside Metabolism
2.4 Analysis and Application of Microbial Fatty
Acid Metabolism .............................................................. 2-12
Fatty Acid Desaturation Systems for Polyunsaturated Fatty Acid
Production • Fatty Acid Metabolism Useful for Conjugated
Jun Ogawa
Fatty Acid Production
2.5 Conclusions: For Expansion of Biocatalysts for
Sakayu Shimizu Practical Purposes ............................................................ 2-17
2.1 Introduction
In the coming postpetrochemical period, production processes will be required to save energy and to
reduce environmental damage. In this sense, biological reactions are now widely recognized as practical
alternatives to conventional chemical reactions.1,2 On the other hand, novel catalytic procedures are
necessary to produce the emerging classes of organic compounds that are becoming the targets of molecular
and biomedical research. Therefore, screening for novel biocatalysts that are capable of catalyzing new
reactions is constantly needed.
A bioprocess is sometimes designed from an organic chemistry standpoint, regardless of whether or
not a suitable biocatalyst has already been found. This forces screening from a certain level to ascertain
the existence of a desirable biocatalyst. Thus, it is also important to increase the catalog of biocatalysts
to maintain the motivation for such a steady search. One of the most efficient and successful means of
finding new biocatalysts is to screen large numbers of microorganisms, because of their characteristic
diversity and versatility.3–6 The keys for increasing the probability of discovery in the screening are to
examine as many samples as possible and to maintain a thoughtful insight.
2-1

Usually, screening is simply focused on a one-step target reaction. In such screening, the cells of
microorganisms are incubated with target substrates under various reaction conditions and their trans-
formation is monitored. Successful examples of such screening are the finding of novel carbonyl reduc-
tases and lactonohydrolases. You can refer to the recent reviews presenting the details of these enzymes.7,8
Also, information obtained on detailed analysis of a microbial metabolic process leads to unexpected
new reactions and substrates. Examples of the latter, i.e., unique reactions found in the microbial
metabolism of cyclic amides, nucleosides, and fatty acids, are described together with their applications
in this chapter.
2.2 Analysis and Application of Microbial Cyclic

Amide Metabolism
2.2.1 Overview of Microbial Cyclic Amide Metabolism
The chemical structure of cyclic amides can be found in many natural and unnatural compounds. The trans-
formation of naturally occurring cyclic amides of pyrimidines and purines plays an important role in nucleobase
metabolism. These metabolic activities comprise those of various cyclic amide hydrolases (EC 3.5.2.-), such as
dihydropyrimidinase in reductive pyrimidine metabolism,9 barbiturase in oxidative pyrimidine metabolism,10
dihydroorotase in pyrimidine biosynthesis,11 and allantoinase in purine metabolism.12 In the 1970s,
studies on rat liver dihydropyrimidinase showed that the enzyme hydrolyzed 5-monosubstituted hydan-
toin to N-carbamoyl amino acid and that the reaction proceeded D-stereospecifically.13,14 Later on, Yamada
and coworkers showed that microbial cells are good catalysts for this reaction.15 Based on these observations,
hydantoin metabolism in microorganisms was intensively studied and applied to optically active amino
acid production.16,17 Blastobacter sp. A17p-4 was screened from soil as a hydantoin-assimilating bacterium
for the purpose of D-amino acid production from DL-5-monosubstituted hydantoins.18 During the
course of studies on hydantoin metabolism in this bacterium, it was found that it showed not only
hydantoin- but also cyclic imide-metabolizing activity.19 A recent study revealed that the strain has not
only hydantoin-metabolizing enzymes but also enzymes specific to cyclic imide derivatives. Since then,
cyclic imide metabolism and specific enzymes have been widely found and studied in bacteria, yeasts,
and molds.
2.2.2 Analysis and Application of Microbial Hydantoin Metabolism

2.2.2.1 Diversity of Hydantoin-Metabolizing Enzymes
Hydantoin is metabolized to an amino acid through two-step hydrolysis via an N-carbamoyl amino acid.
The enzyme catalyzing the first step, hydrolysis of hydantoin to N-carbamoyl amino acid, is called hydan-
toinase. Three typical hydantoinases with stereospecificity to D-, L-, and DL-5-monosubstituted hydan-
toin are named D-hydantoinase, L-hydantoinase, and DL-hydantoinase, respectively (Figure 2.1).17,20–22
D-Hydantoinases have been found in various genera of bacteria such as Pseudomonas, Bacillus, Blasto-
bacter, and Arthrobacter, and most of them show dihydropyrimidinase activity. The existence of L- and
DL-hydantoinases might be rarer in nature than that of D-hydantoinase. These enzymes can be divided
into two groups: one needing ATP for activity and the other not. The ATP-requiring enzyme from
Pseudomonas putida 77, which functions in creatinine metabolism, showed L-hydantoinase activity.23
The second step of hydantoin metabolism, N-carbamoyl amino acid hydrolysis to amino acid, ammonia,
and carbon dioxide, is catalyzed by carbamoylase. A variety of carbamoylases have been reported
(Figure 2.2). Two typical carbamoylases with stereospecificity to N-carbamoyl D- and L-amino acids are
named D-carbamoylase and L-carbamoylase, respectively.17 D-Carbamoylase generally shows wide substrate
specificity to both aromatic and aliphatic N-carbamoyl-D-amino acids.24 L-Carbamoylase shows rather
limited specificity to aromatic or aliphatic N-carbamoyl-L-amino acids. An L-carbamoylase with rela-
tively broad substrate specificity has been found in Alcaligenes xylosoxidans.25 β-Ureidopropionase from

Screening for Unique Microbial Reactions Useful for Industrial Applications 2-3
D-Hydantoinase L-Hydantoinase
O O H2O
R H2O R COOH H H COOH
NH NH2 NH NH2
H H R R
HN HN HN HN
O O O O
specific for hydantoin without ATP hydrolysis
ATP
O +
O 2 H2O COOH
H2O COOH H H
NH NH2
NH NH2 R R
HN ADP HN
HN HN O + O
O O Pi
with dihydropyrimidinase activity with ATP hydrolysis
O H2O
COOH
R NH R NH2
HN HN
O O
without ATP hydrolysis
ATP
+
O 2H O 2 COOH
R NH R NH2
HN ADP HN
O + O
Pi
with ATP hydrolysis
DL-Hydantoinase
FIGURE 2.1 Reactions catalyzed by typical hydantoinases.
P. putida IFO 12996, which functions in the pyrimidine degradation during N-carbamoyl-β-alanine
hydrolysis, showed broad substrate specificity not only toward N-carbamoyl-β-amino acids, but also toward
N-carbamoyl-γ-amino acids and several N-carbamoyl-α-amino acids.26 The hydrolysis of N-carbamoyl-
α-amino acids is strictly L-stereospecific (Figure 2.2).
2.2.2.2 Optically Active Amino Acid Production by Hydantoin-Metabolizing Enzymes
Different combinations of hydantoin-metabolizing enzymes, i.e., hydantoinases and carbamoylases, pro-
vide a variety of processes for the production of optically pure α-amino acids (Figure 2.3).17 The broad
substrate range of the processes is valuable, especially for the production of D-amino acids and unnatural
L-amino acids.27,28 Some enzymes recognize multichiral centers other than the α-carbons of amino acids,
so they enable simultaneous resolution of multichiral amino acids such as β-methylphenylalanine.29
Recent research on hydantoin-metabolizing enzymes has been concentrated on newly isolated or
improved enzymes, and has included directed evolution techniques, structure elucidation, studies on
fusion proteins, and the use of specially designed whole cell biocatalysts.30
A practical representative is the production of D-p-hydroxyphenylglycine, a building block for
semisynthetic penicillins and cephalosporins. The process involves one chemical step and two enzymatic
steps (Figure 2.4). The substrate, DL-5-(p-hydroxyphenyl)hydantoin, is synthesized by an efficient chem-
ical method involving the amidoalkylation reaction of phenol with glyoxylic acid and urea under acidic
conditions. Then, the D-5-(p-hydroxyphenyl)hydantoin is hydrolyzed enzymatically to N-carbamoyl-D-
p-hydroxyphenylglycine with a thermostable immobilized D-hydantoinase under alkaline conditions.

D-Carbamoylase
R COOH R COOH
NH2 + H2O + NH3 + CO2
H HN H NH2
O
L-Carbamoylase
H COOH H COOH
NH2 + H2O + NH3 + CO2
R HN R NH2
O
β-Ureidopropionase
COOH COOH
NH2 + H2O + NH3 + CO2
HN NH2
O
COOH COOH
NH2 + H2O + NH3 + CO2
HN NH2
O
H COOH H COOH
NH2 + H2O + NH3 + CO2
R HN R NH2
O
FIGURE 2.2 Reactions catalyzed by typical carbamoylases.
Under these conditions, the L-isomer of the remaining 5-(p-hydroxyphenyl)hydantoin is racemized

through base catalysis. Therefore, racemic hydantoins can be converted quantitatively into N-carbamoyl-
D-p-hydroxyphenylglycine through this step. Decarbamoylation to D-p-hydroxyphenylglycine is per-
formed with an immobilized mutant D-carbamoylase. D-Carbamoylase from Agrobacterium sp. KNK712
has been improved into a mutant enzyme showing 20°C-higher thermal stability.31,32 This process
involving immobilized hydantoinase and carbamoylase has been used for the commercial production
of D-p-hydroxyphenylglycine since 1995.
2.2.3 Analysis and Application of Microbial Cyclic Imide Metabolism

2.2.3.1 Analysis of Microbial Cyclic Imide Metabolism
Based on the finding of cyclic imide-hydrolyzing activity in Blastobacter sp.,19 the metabolism of various
cyclic imides by microorganisms was systematically investigated. The fact that Blastobacter sp. grows well
in a synthetic minimum medium containing succinimide as the sole carbon source indicates that the
bacterium has a metabolic system for the assimilation of cyclic imides as energy sources and nutrients.33
The bacterium can metabolize various cyclic imides with structures similar to that of succinimide such as
maleimide, 2-methylsuccinimide and glutarimide, and sulfur-containing cyclic imides such as 2,4-thiaz-
olidinedione and rhodanine. Further investigation of the metabolic fate of these cyclic imides showed that
they were metabolized through a novel metabolic pathway (Figure 2.5). This pathway comprises in turn
the hydrolytic ring-opening of cyclic imides into half-amides, hydrolytic deamidation of the half-amides
to dicarboxylates, and dicarboxylate transformation similar to that in the tricarboxylic acid (TCA) cycle.
Two novel enzymes, imidase and half-amidase, and D-hydantoinase were found to function in this pathway.
The nature of imidase, which hydrolyzes a cyclic imide to a half-amide, was further investigated in
detail. Three types of enzymes with different substrate specificities were found (Figure 2.6). An imidase

Screening for Unique Microbial Reactions Useful for Industrial Applications
D-Hydantoinase
identical with
dihydropyrimidinase D-Carbamoylase
5-Monosubstituted Pseudomonas N-Carbamoyl-D- Comamonas
D-Amino acid
D-hydantoin Blastobacter etc. amino acid Blastobacter
specific for D-hydan- Agrobacterium etc.
toin Agrobacterium N-Carbamoylsarcosine
DL-Hydantoinase amidohydrolase
ATP-independent D-specific for
R
Racemization
Base-catalysis R
Bacillus N-carbamoyl-
Hydantoin HN H Arthrobacter HN H α-amino acids H NH2
racemase Pseudomonas R C COOH
O ATP-dependent COOH
Pseudomonas N O O NH2 L-Carbamoylase
Pseudomonas
Arthrobacter H Alcaligenes
L-Hydantoinase
Flavobacterium
ATP-independent Arthrobacter
Arthrobacter Pseudomonas
ATP-dependent
Bacillus
Bacillus
Bacillus β-Ureidopropinase
N-Methylhydantoin L-specific for
5-Monosubstituted N-Carbamoyl-L-
amidohydrolase L-Amino acid
L-hydantoin amino acid N-carbamoyl-
ATP-dependent and α-amino acids
specific for L-hydan- Pseudomonas
toin Pseudomonas Chemical
decabamoylation
Hydantoin
hydrolysis
N-Carbamoyl amino acid
hydrolysis
FIGURE 2.3 Processes for optically active α-amino acid production with combinations of various hydantoin–metabolizing enzymes.
2-5
NH2 OH
CHO
+ CO +
COOH
NH2
Amidoalkylation
CO HO
H Spontaneous CO
NH racemization
HO
HN CO NH
H
HN CO
D-Hydantoinase
HO
COOH
H NHCONH2
D-Carbamoylase
HO
COOH
H NH2
FIGURE 2.4 Industrial process for D-p-hydroxyphenylglycine production with hydantoin-metabolizing enzymes.
with specificity toward simple cyclic imides was purified from Blastobacter sp.34 This enzyme is also active
toward sulfur-containing cyclic imides such as 2,4-thiazolidinedione and rhodanine. However, bulky
cyclic imides or monosubstituted cyclic ureides are not hydrolyzed. Bulky cyclic imides are hydrolyzed
by the D-hydantoinase of Blastobacter sp. and mammalian dihydropyrimidinases.35 Another imidase,
phthalimidase, with specificity toward phthalimide derivatives was found in Alcaligenes ureafaciens.36
Half-amides, the products of imidase, were further metabolized to dicarboxylates by half-imidase. The
enzyme was purified from Blastobacter sp. and found to be specific toward half-amides.37 These enzyme
activities were widely distributed among bacteria, yeast, and molds.38 Cyclic imide metabolism and
the enzymes involved have practical potential for the production of high-value organic acids such as pyruvate
from cyclic imides or their metabolites, and also the stereo- and regiospecific production of half-amides
and dicarboxylates.
2.2.3.2 Application of Microbial Cyclic Imide Metabolism to Pyruvate Production

Cyclic imide metabolism has been applied to the production of a high-value organic acid, pyruvate.39
The commercial demand for pyruvate has been increasing because of its use as an effective precursor in
the synthesis of various drugs and agrochemicals in addition to as a component of mammalian-cell

COOH COOH
H3C H3C
O O
COOH COOH
HOOC OH HOOC OH
TCA cycle-like
reaction
COOH COOH H3C COOH
HOOC HOOC HOOC
H3C
HOOC COOH HOOC COOH HOOC COOH HOOC COOH HOOC COOH
Half-amidase
H 3C CH3
COOH COOH or
O O COOH O COOH O COOH
NH2 NH2 O NH2 NH
NH2 HOOC CONH2
Imidase
CH3
O O O O O O
N N N O O
H N
H H H O N O
H
succinimide maleimide 2-methylsuccinimide glutarimide phthalimide
FIGURE 2.5 Pathways of microbial cyclic imide-metabolism.
2-7
2-8
Mammalian dihydropyrimidinase
Blastobacter sp. imidase Blastobacter sp. D-hydantoinase Alcaligenes ureafaciens imidase

O
O NH O
H3C H2N
NH O O N-OH
O O H3C O
O NH O O
NH NH NH O
O N-NH2
S O HN O NH O
O O
NH O N-S
N O
HN O O O
O O R O O
O NH
NH O N-Br O
NH H NH NH
S NH HN O N O O
S O O
HN O O N
O CH3
Sulfur-containing Simple Simple Substituted Bulky N-CH3
cyclic imdes cyclic imdes cyclic ureides cyclic ureides cyclic imides O
Phthalimide
Handbook of Industrial Catalysis

derivatives
FIGURE 2.6 Substrate spectra of typical imidases.

COOH
Pyruvate H3C
O
Acetyl-CoA
COOH
HOOC OH
COOH
Fumarate TCA cycle
HOOC
HOOC COOH
O COOH
NH2
Succinimide O O
N
H
FIGURE 2.7 Pyruvate production from fumarate through microbial cyclic imide metabolism.
culture media. Pseudomonas putida s52 isolated with succinimide as the sole carbon source exhibits highly
active cyclic imide metabolism. This activity has been used for pyruvate production from fumarate, a
cheap cyclic imide metabolism intermediate (Figure 2.7). Using cells cultivated in medium containing
2% (w/v) fumarate as the catalyst, 286 mM pyruvate was produced from 500 mM fumarate in 27 h.
Bromopyruvate, a malic enzyme inhibitor, inhibited the pyruvate production and also the growth of
Pseudomonas putida s52 in the medium with fumarate as the sole carbon source. Bromopyruvate-resistant
mutants were derived from Pseudomonas putida s52, and their pyruvate production was examined. One
of the mutants showed much higher pyruvate production than the parent strain. Using the mutant cells
cultivated in medium containing 2% (w/v) fumarate as the catalyst, 770 mM pyruvate was produced
from 1000 mM fumarate in 96 h.40
2.2.3.3 Application of the Imidase-Catalyzing Reaction to Fine Organic Synthesis

In case of a half-amide, a useful building block for organic synthesis, there is synthetic difficulty in selective
amidation at one of two equivalent carboxyl groups. 3-Carbamoyl-α-picolinic acid (α-3CP) is one of the
regioisomeric half-amides of 2,3-pyridinedicarboxylic acid (PDC). α-3CP is a promising intermediate
for modern insecticide synthesis. Chemical synthesis of α-3CP from PDC via the dimethylester involves
troublesome regiospecific diester hydrolysis to the half-ester. Enzymatic regiospecific hydrolysis of
2,3-pyridinedicarboximide (PDI) (Figure 2.8) is one of the attractive methods for overcoming this
disadvantage. Some imidases and D-hydantoinases (dihydropyrimidinases) have been found to hydro-
lyze aryl-substituted cyclic imides such as PDI, 3,4-pyridinedicarboximide and phthalimide.35,41 Based
on these findings, potential imidases that are applicable to the regiospecific hydrolysis of PDI to α-3CP
were screened for. Phthalimide-assimilating microorganisms have been isolated as possible catalysts for
the regiospecific hydrolysis of PDI to α-3CP. Arthrobacter ureafacience O-86 was selected as the best strain
and applied to the cyclohexanone-water two-phase reaction system, pH 5.5, where the spontaneous
random hydrolysis of PDI was avoided and the enzyme maintained its activity. Under the optimized
conditions, with the periodical addition of PDI (in total, 40 mM), 36.6 mM α-3CP accumulated in the
water phase with a molar conversion yield of 91.5% and a regioisomeric purity of 94.5% in 2 h.36

NH2
N COOH
O
3-carbamoyl-α-picolinic acid
NH (α-3CP)
N O
2, 3-pyridinedicarboximide COOH
(PDI) NH2
N O
2-carbamoyl-β-picolinic acid
(β-2CP)
FIGURE 2.8 Imidase-catalyzing regioselective hydrolysis of 2,3-pyridinedicarboximide (PDI) to 3-carbamoyl-

α-picolinic acid (α-3CP).
2.3 Analysis and Application of Microbial Nucleoside

Metabolism
2.3.1 Overview of Microbial Nucleoside Metabolism
Recently, nucleosides and a variety of chemically synthesized nucleoside analogs have attracted a great
deal of interest as they have antibiotic, antiviral, and antitumor effects.42 In light of this trend, the
microbial metabolism of nucleosides was reevaluated in detail, although it had been well studied as an
assimilation or salvage pathway.43 The first reaction in nucleoside metabolism is N-riboside cleavage. Two
kinds of enzymes, nucleosidase (nucleoside hydrolase; EC 3.2.2.-) and nucleoside phosphorylase (EC
2.4.2.-), are known to catalyze this reaction (Figure 2.9). Nucleosidase catalyzes the irreversible hydrolysis
of nucleosides and participates mainly in the assimilation pathway. A nucleosidase from Ochrobactrum
anthropi, which specifically catalyzes the N-riboside cleavage of purine nucleosides, has been purified
and characterized.44 The enzyme was revealed to be useful for the decomposition of purine nucleosides
in foodstuffs, with these nucleosides causing hyperuricemia, an increasingly common disease in adults.45
On the other hand, nucleoside phosphorylase catalyzes the phosphorolytic cleavage of nucleosides and
shows ribosyl transferase activity.46 This enzyme functions mainly in the salvage pathway. Nucleoside
phosphorylase has been well studied and used as a catalyst for the synthesis of nucleoside analogs through
base exchange reactions.46,47
In the case of 2'-deoxyribonucleoside degradation, the product of the nucleoside phosphorylase reac-
tion is 2-deoxyribose 1-phosphate. This is further transformed into D-glyceraldehyde 3-phosphate and
acetaldehyde via 2-deoxyribose 5-phosphate. These reactions are reversible and successively catalyzed
Nucleosidase OH
O
HO
H2O
HO OH
O B B
HO
B
HO OH
O O P
P
HO
Nucleoside
phosphorylase HO OH
FIGURE 2.9 Reactions catalyzed by nucleosidase and nucleoside phosphorylase.

CH2OH
O
OH Glucose 1111 mM
HO OH
OH
O Glycolysis
PO HO OP 356 mM
AMP turnover: 47.5
OH
HO FDP yield as to glucose: 32.0%
CHO
CH2OH 178 mM
G3P CHOH C= O DHAP
CH2O P CH2O P
CH3CHO
DERA
Acetaldehyde
OH 246 mM
O
yield as to FDP: 69.1%
P O
DR5P yield as to glucose: 22.1%
HO 12.3 mM
PPMase
O O P
HO DR1P
HO Adenine
NPase
P
B
O 9. mM
HO
yield as to DR5P: 80.2%
2'-Deoxyinosine yield as to glucose: 17.8%
HO
FIGURE 2.10 Biochemical retrosynthesis of 2'-deoxyribonucleoside.
by phosphopentomutase and deoxyriboaldolase, and the products of these reactions, D-glyceraldehyde

3-phosphate and acetaldehyde, finally flow into a central metabolic process such as the glycolytic pathway.
Recently, a unique application of these reversible reactions to nucleoside synthesis was investigated, as
described below.
2.3.2 Biochemical Retrosynthesis of 2'-Deoxyribonucleoside Through

Microbial Nucleoside Metabolism
With the spread of PCR techniques and new antiviral nucleosides, and the advent of antisense drugs for
cancer therapy, there will be an urgent need for a DNA building block, 2'-deoxyribonucleoside, on a
large scale in the near future. Classical 2'-deoxyribonucleoside sources are hydrolyzed herring and salmon
sperm DNA. These sources, however, will not allow us to meet future demands for a stable and economical
supply of 2'-deoxyribonucleoside. A possible microbial method for 2'-deoxyribonucleoside production
from easily available materials, glucose, acetaldehyde, and a nucleobase, has been examined, that is, the use of
reversible reactions involved in nucleoside degradation. In this process, microorganisms possessing glycolytic
enzymes, deoxyriboaldolase, phosphopentomutase, and nucleoside phosphorylase, were used as catalysts. The
glycolytic enzymes produce D-glyceraldehyde 3-phosphate from glucose. Subsequently, deoxyriboaldolase,
phosphopentomutase, and nucleoside phosphorylase cooperatively produce 2'-deoxyribonucleosides from
D-glyceraldehyde 3-phosphate, acetaldehyde, and a nucleobase via 2-deoxyribose 5-phosphate (Figure 2.10).
A deoxyriboaldolase suitable for 2-deoxyribose 5-phosphate synthesis with tolerance to acetaldehyde has
been found on screening.48 A potential enzyme was found in Klebsiella pnuemoniae and transformed into

E coli.49 Using the E coli transformant as a source of glycolytic enzymes and deoxyriboaldolase, 2-deoxyribose
5-phosphate was produced from glucose and acetaldehyde in the presence of ATP, which is required for
D-glyceraldehyde 3-phosphate generation from glucose by glycolytic enzymes. The energy derived from
ATP was replaced by the energy derived from alcohol fermentation by baker’s yeast in the form of fructose
1,6-diphosphate, which was further utilized as a D-glyceraldehyde 3-phosphate precursor by the E coli
transformant.50 The 2-deoxyribose 5-phosphate produced was further transformed to 2'-deoxyribonu-
cleoside by E coli transformants expressing phosphopentomutase and nucleoside phosphorylase.51 Typical
results of such synthesis are also presented in Figure 2.10. It is noteworthy that the glycolytic pathway
supplies the important substrates, fructose 1,6-diphosphate and D-glyceraldehyde 3-phosphate, for
2'-deoxyribonucleoside synthesis via 2-deoxyribose 5-phosphate.
2.4 Analysis and Application of Microbial Fatty Acid

Metabolism
2.4.1 Fatty Acid Desaturation Systems for Polyunsaturated Fatty Acid

Production
C20 polyunsaturated fatty acids (PUFAs), such as 5,8,11-cis-eicosatrienoic acid (mead acid, MA),
dihomo-γ-linolenic acid, arachidonic acid, and 5,8,11,14,17-cis-eicosapentaenoic acid (EPA), exhibit
unique biological activities. Because food sources rich in these PUFAs are limited to a few seed oils and
fish oils, the screening for alternative sources of these PUFAs in microorganisms has been conducted,
which resulted in the isolation of an arachidonic acid-producing fungus, Mortierella alpina 1S-4.52 This
fungus produces 30–60 g/l of mycelia (dry weight) containing about 60% lipids. The lipids mainly consist
of triacylglycerol, which contains arachidonic acid. The amount of arachidonic acid is 40–70% of the
lipids—approximately 13 kg/kl on large-scale fermentation. The fungus operates unique fatty acid
desaturation systems involving at least five desaturases with different specificities. EPA has been produced
through the ω3 route from α-linolenic acid via successive ∆6 desaturation, elongation and ∆5 desaturation
using the same fungus.53 Further investigation led to the isolation of desaturase-defective mutants and
opened routes for the production of other PUFAs (Figure 2.11).54,55 For example, dihomo-γ-linolenic
acid and MA can be produced through the ω6 and ω9 routes using ∆5 and ∆12-desaturase-defective
mutants, respectively. A recent review presented the details of this research.56
2.4.2 Fatty Acid Metabolism Useful for Conjugated Fatty Acid Production
Conjugated fatty acids have attracted much attention as a novel type of biologically beneficial functional
lipids. For example, dietary conjugated linoleic acid (CLA) reduces carcinogenesis, atherosclerosis, and
body fat.57,58 Today, CLA is produced through chemical isomerization of linoleic acid, which results in
the by-production of unexpected isomers. Considering the use of CLA for medicinal and nutraceutical
purposes, an isomer-selective and safe process is required. A bioprocess is a potential alternative for this
purpose.
Dairy products are among the major natural sources of CLA, of which cis-9,trans-11-octadecadienoic
acid is the main isomer.59 CLA has been shown to be produced from polyunsaturated fatty acids by
certain rumen microorganisms such as Butyrivibrio species. cis-9,trans-11-Octadecadienoic acid has been
suggested as an intermediate in the biohydrogenation of linoleic acid to octadecaenoic acid by the
anaerobic rumen bacterium Butyrivibrio fibrisolvens.60 It has also been reported that Propionibacterium
freudenreichii, which is commonly used as a dairy starter culture, can produce CLA from free linoleic
acid.61 Recently, the ability to produce CLA from linoleic acid was extensively screened for in lactic acid
bacteria.62 Many strains were found to produce CLA from linoleic acid, and the mechanism of CLA
production was investigated with Lactobacillus acidophilus AKU 1137 as a representative strain.63 The

COOH COOH COOH
COOH COOH
18:0 18:1n-9 18:2n-9 20:2n-9 20:3n-9,MA ω9
∆9
∆12
COOH COOH COOH

COOH COOH
20:3n-6( ∆5) 20:2n-6 18:2n-6 18:3n-6 20:3n-6,DGLA COOH 20:4n-6,AA ω6
ω3 ω3
COOH COOH COOH

COOH COOH COOH
20:4n-3( ∆5) 20:3n-3 18:3n-3 18:4n-3 20:4n-3 20:5n-3,EPA ω3
∆5 EL ∆6 EL ∆5
FIGURE 2.11 Polyunsaturated fatty acid–synthesizing systems in M. alpina 1S–4 and mutants of it. EL, elongase; AA, arachidonic acid; MA, Mead acid;
ALA, α-linolenic acid; EPA, 5,8,11,14,17-cis-eicosapentaenoic acid; DGLA, dihomo-γ-linolenic acid.
2-13
CLAs produced by L. acidophilus were identified as cis-9,trans-11-octadecadienoic acid (CLA1) and trans-
9,trans-11-octadecadienoic acid (CLA2).64 Preceding the production of CLA, hydroxy fatty acids identified
as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid were accumulated.
The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed to CLA on incubation with washed
cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA pro-
duction from linoleic acid (Figure 2.12). Based on these results, the transformation of hydroxy fatty acids
by lactic acid bacteria was investigated. Lactic acid bacteria transformed ricinoleic acid (12-hydroxy-cis-
9-octadecaenoic acid) into CLA (a mixture of CLA1 and CLA2).65 There are two possible pathways for
CLA synthesis from ricinoleic acid by lactic acid bacteria: 1) direct transformation of ricinoleic acid into
CLA through dehydration at the ∆11 position, and 2) dehydration of ricinoleic acid at the ∆12 position
to linoleic acid, which is a potential substrate for CLA production by lactic acid bacteria (Figure 2.12).
In a similar manner on linoleic acid transformation to CLA, lactic acid bacteria transformed α- and
γ-linolenic acid into the corresponding conjugated trienoic acids.66,67 Those produced from α-linolenic
acid were identified as cis-9,trans-11,cis-15-octadecatrienoic acid (18:3) and trans-9,trans-11,cis-15-18:3,
and those from γ-linolenic acid as cis-6,cis-9,trans-11-18:3 and cis-6,trans-9,trans-11-18:3 (Figure 2.13).
Washed cells of lactic acid bacteria exhibiting high productivity of conjugated fatty acids were obtained
by cultivation in medium supplemented with polyunsaturated fatty acids such as linoleic acid and
α-linolenic acid, indicating that these enzyme systems are induced by polyunsaturated fatty acids, maybe
for their detoxication.60
2.4.2.1 Preparative CLA Production from Linoleic Acid by Lactic Acid Bacteria
After screening 14 genera of lactic acid bacteria, L. plantarum AKU 1009a was selected as a potential
strain for CLA production from linoleic acid.62 Washed cells of L. plantarum exhibiting a high level of
CLA production were obtained by cultivation in a nutrient medium containing linoleic acid. Under the
optimum reaction conditions with the free form of linoleic acid as the substrate, washed cells of L. plantarum
produced 40 mg/ml CLA (33% molar yield) from 12% (w/v) linoleic acid in 108 h. The resulting CLA
comprised a mixture of CLA1 (38% of total CLA) and CLA2 (62% of total CLA), and accounted for 50%
of the total fatty acids obtained. A higher yield (80% molar yield as to linoleic acid) was attained with
2.6% (w/v) linoleic acid as the substrate in 96 h, resulting in CLA production of 20 mg/ml [consisting of
CLA1 (2%) and CLA2 (98%)] and accounting for 80% of the total fatty acids obtained. Most of the CLA
produced was associated with the washed cells, and mainly as a free form.62
2.4.2.2 Preparative CLA Production from Ricinoleic Acid and Castor Oil by
Lactic Acid Bacteria
The ability to produce CLA from ricinoleic acid is widely distributed in lactic acid bacteria. Washed cells
of L. plantarum JCM 1551 were selected as a potential catalyst for CLA production from ricinoleic acid.68
Cells cultivated in a medium supplemented with a mixture of α-linolenic acid and linoleic acid showed
enhanced CLA-productivity. Under the optimum reaction conditions, with the free acid form of ricinoleic
acid as the substrate and washed cells of L. plantarum as the catalyst, 2.4 mg/ml CLA was produced from
3.4 mg/ml ricinoleic acid in 90 h, the molar yield as to ricinoleic acid being 71%. The CLA produced,
which was obtained in the free fatty acid form, consisted of CLA1 (21% of total CLA) and CLA2 (79%
of total CLA), and accounted for 72% of the total fatty acids obtained.68
Ricinoleic acid is abundant in a plant oil, castor oil. Castor oil is an economical source of ricinoleic
acid. About 88% of the total fatty acids in castor oil is ricinoleic acid. Unfortunately, CLA cannot be directly
produced from castor oil by lactic acid bacteria. Lactic acid bacteria only use the free form of ricinoleic
acid for CLA production, i.e., not its triacylglycerol form, which is mainly found in castor oil. However,
in the presence of lipase, castor oil became an effective substrate for CLA production by lactic acid
bacteria.65 The addition of a polyhydroxy-type detergent enhanced the CLA production from castor oil.
Under the optimum conditions with castor oil as the substrate and washed cells of L. plantarum JCM 1551
as the catalyst, 2.7 mg/ml CLA was produced from 5.0 mg/ml castor oil in 99 h. The CLA produced
accounted for 46% of the total fatty acids obtained, and consisted of CLA1 (26%) and CLA2 (74%).69

Linoleic acid (cis-9, cis-12-octadecadienoic acid) Ricinoleic acid (12-hydroxy-cis-9-octadecaenoic acid)
O ∆9 ∆12 ∆12 dehydration O ∆9 OH
HO–C HO–C
∆9 hydration
HY2 (10-hydroxy-cis-12-octadecaenoic acid) HY1 (10-hydroxy-trans-12-octadecaenoic acid)

O ∆12 O ∆12 ∆11 dehydration
HO–C HO–C
OH OH
Dehydration,
isomerization
CLA 1 (cis-9, trans-11-octadecadienoic acid) CLA 2 (trans-9, trans-11-octadecadienoic acid)

O ∆9 ∆11 O
∆9 ∆11
HO–C HO–C
FIGURE 2.12 Conjugated linoleic acid (CLA)–producing systems in lactic acid bacteria.
2-15
2-16
O ∆6 ∆9 ∆11
γ-Linolenic acid HO–C
(cis-6,cis-9, cis-12-octadecatrienoic acid)
cis-6,cis-9, trans-11-octadecatrienoic acid
O
∆6 ∆9 ∆12
HO–C
O ∆6 ∆9 ∆11
HO–C
cis-6,trans-9, trans-11-octadecatrienoic acid
O
∆9 ∆11 ∆15
α-Linolenic acid HO–C
(cis-9, cis-12,cis-15-octadecatrienoic acid)
O
cis-9 ,trans-11,cis-15-octadecatrienoic acid
∆9 ∆12 ∆15
HO–C
O ∆9 ∆11 ∆15
HO–C
Handbook of Industrial Catalysis

trans-9 ,trans-11,cis-15-octadecatrienoic acid
FIGURE 2.13 α-and γ-Linolenic acid transformation into conjugated fatty acids by lactic acid bacteria.

2.5 Conclusions: For Expansion of Biocatalysts for Practical

Purposes
In this chapter, the authors introduced several examples of unique microbial reactions useful for practical
purposes. The reactions resulted from detailed observation of microbial cyclic amide, nucleoside, and
fatty acid metabolisms, and have paved the way to new bioprocesses for the production of optically active
amino acids, organic acids, half-amides, 2'-deoxyribonucleosides, polyunsaturated fatty acids, conjugated
fatty acids, etc.
Modern society requests the development of processes exhibiting environmental harmonization,
economic efficiency, and specificity. This trend is causing the application of biological reactions to a
greater variety of industries. Future bioprocesses will generally not be limited by the available technology
or the nature of the substrates and products. Instead, the feasibility of new bioprocesses will often be
determined by the availability of biocatalysts, the search for which needs patience for steady research but
has a deep impression when a new biocatalyst is encountered.
Recently, some rational methods creating new biocatalysts have been rapidly developed. Modern gene
technology, crystal structure analysis, and bioinformatics enable the modulation of enzyme function
through site-directed mutation, DNA shuffling, etc. One example is modification of the substrate specificity
of a monooxygenase, P450 BM-3, from Bacillus megaterium. P450 BM-3 is a fatty acid monooxygenase.
Its substrate specificity was expanded to aromatic hydrocarbons, and phenolic and arylalkyl compounds
by means of crystal structure-based directed mutation, which resulted in the creation of novel catalysts
for regiospecific and stereospecific alcohol synthesis.70–72 However, “rationality” is not the only answer
for developing new biocatalysts. Classical screening of microbial diversity and versatility is still important.
Such screening is something like a midnight walk without moonlight; however, detailed observation and
deep insight with a well-considered strategy will lead to a new biocatalyst. This philosophy has now been
succeeded by in vitro random evolution technology.73
The industrial success of biocatalysts unfortunately depends on the economics of the specific processes.
However, once successful, it provides enormous opportunities. With the introduction of each new process
accumulating experience and confidence, it becomes easier to develop and justify the next new bioprocess.
Thus, it is important to increase the catalog of biocatalysts waiting to be examined for practical purposes.
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enzymes from the origin of life? Appl Microbiol Biotechnol 51:293–309, 1999.
23. J Ogawa, JM Kim, W Nirdnoy, Y Amano, H Yamada, S Shimizu. Purification and characterization
of an ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, from Pseudomonas
putida 77. Eur J Biochem 229: 284–290, 1995.
24. J Ogawa, S Shimizu, H Yamada. N-Carbamoyl-D-amino acid amidohydrolase from Comamonas
sp. E222c: Purification and characterization. Eur J Biochem 212:685–691, 1993.
25. J Ogawa, H Miyake, S Shimizu. Purification and characterization of N-carbamoyl-L-amino acid
amidohydrolase with broad substrate specificity from Alcaligens xylosoxidans. Appl Microbiol Biotechnol
43:1039–1043, 1995.
26. J Ogawa, S Shimizu. β-Ureidopropionase with N-carbamoyl-α-L-amino acid amidohydrolase aciti-
vity from an aerobic bacterium, Pseudomonas putida IFO 12996. Eur J Biochem 223:625–630, 1994.
27. C Syldatk, R Müller, M Pietzsch, F Wagner. Microbial and enzymatic production of D-amino acids
from D,L-monosubstituted hydantoins. In: D Rozzell, F Wagner, eds. Biocatalytic Production of
Amino Acids and Derivatives. München: Hanser Publishers, 1992, pp. 75–128.
28. C Syldatk, R Müller, M Siemann, K Krohm, F Wagner. Microbial and enzymatic production of
L-amino acids from D,L-monosubstituted hydantoins. In: D Rozzell, F Wagner, eds. Biocatalytic
Production of Amino Acids and Derivatives.München: Hanser Publishers, 1992, pp. 129–176.
29. J Ogawa, A Ryono, SX Xie, RM Vohra, R Indrati, H Miyakawa, T Ueno, S Shimizu. Separative
preparation of the four stereoisomers of β-methylphenylalanine with N-carbamoyl amino acid
amidohydrolases. J Molec Catal B: Enzymatic 12:71–75, 2001.
30. J Altenbuchner, M Siemann-Herzberg, C. Syldatk. Hydantoinases and related enzymes as biocatalysts
for the synthesis of unnatural chiral amino acids. Curr Opin Biotechnol 12:559–563, 2001.
31. H Nanba, Y Ikenaka, Y Yamada, K Yajima, M Takano, K Ohkubo, Y Hiraishi, K Yamada, S Takahashi.
Immobilization of N-carbamyl-D-amino acid amidohydrolase. Biosci Biotechnol Biochem 62:1839–
1844, 1998.

32. Y Ikenaka, H Nanba, K Yajima, Y Yamada, M Takano, S Takahashi. Thermostability reinforcement

through a combination of thermostability-related mutations of N-carbamyl-D-amino acid
amidohydrolase. Biosci Biotechnol Biochem 63:91–95, 1999.
33. J Ogawa, CL Soong, M Honda, S Shimizu. Novel metabolic transformation pathway for cyclic
imides in Blastobacter sp. A17p-4. Appl Environ Microbiol 62:3814–3817, 1996.
34. J Ogawa, CL Soong, M Honda, S Shimizu. Imidase, a dihydropyrimidinase-like enzyme involved
in the metabolism of cyclic imides. Eur J Biochem 243:322–327, 1997.
35. CL Soong, J Ogawa, M Honda, S Shimizu. Cyclic-imide-hydrolyzing activity of D-hydantoinase
from Blastobacter sp. strain A17p-4. Appl Environ Microbiol 65:1459–1462, 1999.
36. J Ogawa, CL Soong, M Ito, T Segawa, T Prana, MS Prana, S Shimizu. 3-Carbamoyl-a-picolinic
acid production by imidase-catalyzed regioselsective hydrolysis of 2,3-pyridinedicarboximide in a
water-organic solvent, two-phase system. Appl Microbiol Biotechnol 54:331–334, 2000.
37. CL Soong, J Ogawa, S Shimizu. A novel amidase (half-amidase) for half-amide hydrolysis involved
in the bacterial metabolism of cyclic imides. Appl Environ Microbiol 66:1947–1953, 2000.
38. CL Soong, J Ogawa, H Sukiman, T Prana, MS Prana, S Shimizu. Distribution of cyclic imide-transforming
activity in microorganisms. FEMS Microbiol Lett 158:51–55, 1998.
39. J Ogawa, CL Soong, M Ito, S Shimizu. Enzymatic production of pyruvate from fumarate–an
application of microbial cyclic-imide-transforming pathway. J Molec Catal B: Enzymatic 11:355–
359, 2001.
40. J Ogawa, W Tu, CL Soong, M Ito, T Segawa, S Shimizu. Pyruvate production through microbial
cyclic imide transformation. Proceedings of 4th International Symposium on Green Chemistry in
China, Jinan, 2001, p. 291.
41. YS Yang, S Ramaswamy, WB Jakoby. Rat liver imidase. J Biol Chem 268:10870–10875, 1993.
42. H Mitsuya, S Border. Inhibition of the in vivo infectivity and cytopathic effect of human T-lymphotrophic
virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides. Proc Natl
Acad Sci USA 83:1911–1915, 1986.
43. S Koga, J Ogawa, LY Chen, YM Choi, H Yamada, S Shimizu. Nucleoside oxidase, a hydrogen
peroxide–forming oxidase, from Flavobacterium meningosepticum. Appl Environ Microbiol 63:4282–
4286, 1997.
44. J Ogawa, S Takeda, SX Xie, H Hatanaka, T Ashikari, T Amachi, S Shimizu. Purification, charac-
terization, and gene cloning of purine nucleosidase from Ochrobactrum anthropi. Appl Environ
Microbiol 67:1783–1787, 2001.
45. NL Edwards, IH Fox. Disorders associated with purine and pyrimidine metabolism. Spec Top
Endocrinol Metab 6:95–140, 1984.
46. TA Krenitsky, GW Koszalka, JV Tuttle. Purine nucleoside synthesis, an efficient method employing
nucleoside phosphorylases. Biochemistry 20:3615–3621, 1981.
47. K Yokozeki, H Shirae, K Kubota. Enzymatic production of antivial nucleosides by the application
of nucleoside phosphorylase. Ann N Y Acad Sci 613:757–759, 1990.
48. J Ogawa, K Saito, T Sakai, N Horinouchi, T Kawano, S Matsumoto, M Sasaki, Y Mikami, S Shimizu.
Microbial production of 2-deoxyribose 5-phosphate from acetaldehyde and triosephosphate for
the synthesis of 2'-deoxyribonucleosides. Biosci Biotechnol Biochem 67:933–936, 2003.
49. N Horinouchi, J Ogawa, T Sakai, T Kawano, S Matsumoto, M Sasaki, Y Mikami, S Shimizu. Cons-
truction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose
5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production. Appl
Environ Microbiol 69:3791–3797, 2003.
50. N Horinouchi, S Sakai, T Kawano, J Ogawa, S Shimizu. Efficient production of 2-deoxyribose
5-phosphate from glucose using glycolysis of baker’s yeast. Proceedings of Annu Meet 2002 Soc
Biotechnol Japan, Osaka, 2002, p. 125.
51. J Ogawa, T Kawano, N Horinouchi, S Sakai, S Shimizu. Enzymatic synthesis of 2'-deoxyribonucleoside
from microbially prepared 2-deoxyriobse 5-phosphate. Proceedings of Annu Meet Soc Biosci
Biotechnol Agrochem Japan, Tokyo, 2003, p. 182.

52. Y Shinmen, S Shimizu, K Akimoto, H Kawashima, H Yamada. Production of arachidonic acid by

Mortierella fungi: selection of a potent producer and optimization of culture conditions for large-
scale production. Appl Microbiol Biotechnol 31:11–16, 1989.
53. S Shimizu, H Kawashima, K Akimoto, Y Shinmen, H Yamada. Conversion of linseed oil to an
eicosapentaenoic-acid containing oil by Mortierella alpina 1S-4 at low temperature. Appl Microbiol
Biotechnol 21:1–4, 1989.
54. M Certik, E Sakuradani, S Shimizu. Desaturase defective fungal mutants: useful tools for the
regulation and overproduction of polyunsaturated fatty acids. Trends Biotechnol 16:500–505, 1998.
55. M Certik, S Shimizu. Biosynthesis and regulation of microbial polyunsaturated fatty acid produc-
tion. J Biosci Bioeng 87:1–14, 1999.
56. J Ogawa, E Sakuradani, S Shimizu. Production of C20 polyunsaturated fatty acids by an arachidonic
acid-producing fungus Mortierella alpina 1S-4 and related strains. In: TM Kuo, HW Gardner, eds.
Lipid Biotechnology. New York: Mercel Dekker, 2002, pp. 563–574.
57. MW Pariza, Y Park, ME Cook. The biologically active isomers of conjugated linoleic acid. Prog
Lipid Res 40:283–298, 2001.
58. JM Ntambi, Y Choi, Y Park Y, JM Peters, MW Pariza. Effects of conjugated linoleic acid (CLA) on
immune responses, body composition and stearoyl-CoA desaturase. Can J Appl Physiol 27:617–628,
2002.
59. SF Chin, W Liu, JM Storkson, YL Ha, MW Pariza. Dietary sources of conjugated dienoic isomers
of linoleic acid, a newly recognized class of anticarcinogens. J Food Compos Anal 5:185–197,
1992.
60. CR Kepler, KP Hirons, JJ Mcneill, SB Tove. Intermediates and products of the biohydrogenation
of linoleic acid by Butyrivibrio fibrisolvens. J Biol Chem 241:1350–1354, 1966.
61. J Jiang, L Bjorck, R Fonden. Production of conjugated linoleic acid by dairy starter cultures. J Appl
Microbiol 85:95–102, 1998.
62. S Kishino, J Ogawa, Y Omura, K Matsumura, S Shimizu. Conjugated linoleic acid production from
linoleic acid by lactic acid bacteria. J Am Oil Chem Soc 79:159–163, 2002.
63. J Ogawa, K Matsumura, S Kishino, Y Omura, S Shimizu. Conjugated linoleic acid accumulation
via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by
Lactobacillus acidophilus. Appl Environ Microbiol 67:1246–1252, 2001.
64. S Kishino, J Ogawa, A Ando, T Iwashita, T Fujita, H Kawashima, S Shimizu. Structural analysis
of conjugated linoleic acid produced by Lactobacillus plantarum, and factors affecting isomer
producton. Biosci Biotechnol Biochem 67:179–182, 2003.
65. S Kishino, J Ogawa, A Ando, Y Omura, S Shimizu. Ricinoleic acid and castor oil as substrates for
conjugated linoleic acid production by washed cells of Lactobacillus plantarum. Biosci Biotechnol
Biochem 66:2283–2286, 2002.
66. S Kishino, J Ogawa, S Shimizu. α- and γ-Linolenic acid transformation to conjugated fatty acids
by Lactbacillus plantarum AKU1009a. Proceedings of Annu Meet Soc Biosci Biotechnol Agrochem
Japan, Sendai, 2002, p. 296.
67. S Kishino, J Ogawa, A Ando, S Shimizu. Conjugated α-linolenic acid production from α -linolenic
acid by Lactbacillus plantarum AKU1009a. Eur J Lipid Sci Technol 105:572–577, 2003.
68. A Ando, J Ogawa, S Kishino, S Shimizu. Conjugated linoleic acid production from ricinoleic acid
by lactic acid bacteria. J Am Oil Chem Soc 80:889–894, 2003.
69. A Ando, S Kishino, S Sugimoto, J Ogawa, S Shimizu. Conjugated linoleic acid production from
castor oil by Lactobacillus plantarum JCM 1551. Enzyme Microb Technol 35:40–45.
70. QS Li, J Ogawa, S Shimizu. Critical role of the residue size at position 87 in H2O2-dependent
substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3. Biochem Biophys
Res Commun 280:1258–1261, 2001.
71. QS Li, J Ogawa, RD Schmid, S Shimizu. Engineering cytochrome P450 BM-3 for oxidation of
polycyclic aromatic hydrocarbons. Appl Environ Microbiol 67:5735–5739, 2001.

72. QS Li, J Ogawa, RD Schmid, S Shimizu. Residue size at position 87 of cytchrome P450 BM-3
determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation. FEBS Lett
508:249–252, 2001.
73. FH Arnold. Combinatorial and computational challenges for biocatalyst design. Nature 409:253–
257, 2001.

3
Biocatalyses
in Microaqueous
Organic Media
3.1 Introduction ....................................................................... 3-1

3.2 Enzymes .............................................................................. 3-2
Techniques Enabling Biocatalyst Highly Active in Organic
Media • Reactivation • Purity • Properties of Enzymes
3.3 Water ................................................................................... 3-5
The “Microaqueous” Concept • Existing Places of Water and
Equilibrium • Water Activity, aw
3.4 Organic Solvents................................................................. 3-9
Hydrophobicity, LogP • Dielectric Constant
(or Dipole Moment), ε (or D)
3.5 Bioreactor System of Microaqueous Organic Media....... 3-10
Tsuneo Yamane Esterification • Transesterification
3.1 Introduction
Enzymatic or microbial reactions are generally performed by enzymes or microorganisms that exist in
the large excess of water. However, there are cases where yield or rate or productivity increases significantly
by reducing water content in the reaction system. Most biocatalytic reactions using organic solvents are
involved in this category.
Performing enzymatic or microbial reactions in organic media has several advantages as opposed to
using aqueous medium, including the following:
• Shifting of thermodynamic equilibrium to favor synthesis over hydrolysis.
• Reduction in water-dependent side reaction (such as hydrolysis reaction in transfer reactions).
• Immobilization of the enzyme is often unnecessary (even if it is desired, merely physical deposition
onto solid surfaces is enough).
• Elimination of microbial contamination (quite critical in view of industrial scale process).
• Increasing solubility of hydrophobic substrates.
• Recovery of product from low boiling-point solvents is easy and also the insoluble biocatalyst is
easily separated.
Here organic media as the reaction system are classified into two categories:1
1. Solvent systems
2. Solvent-free systems
3-1

In the former system one or more substrates are dissolved in an inert organic solvent that does not
participate in the reaction in any respect, but to provide an environment for the biocatalyst to exert its
action on the dissolved substrate(s).
There are a number of cases where the former is the system of choice: for example, when the substrate
is solid at the temperature of the reaction, when high concentration of the substrate is inhibitory for the
reaction, when the solvent used yields a better environment (accelerating effect) for the enzyme, and so forth.
In the latter system, no other compounds but substrate(s) and enzyme are present in a reactor. In
principle, one substrate can be used in a large excess over another, and if so, it may also act as a solvent
for the other reactant. This may be alternatively named “neat” biotransformation.
One of the big attractions of enzymatic solvent-free synthesis is potentially very high volumetric
productivity. This, however, does not apply to all reactions, and in many instances it may actually take
a longer time to achieve the desired degree of conversion in the absence of added solvent. In this case,
volumetric productivity in the reactor [(mass of product formed) (reactor volume)−1 (time)−1] should
be calculated for both solvent-based and solvent-free systems using the same volume of the reaction
mixture and the same amount of the enzyme in order to make an economically justified choice between
the two. Similarly, no risk of solvent-induced inactivation of the biocatalyst in solvent-free system is
another advantage, but the overall loss of the enzyme activity can still be significant if the reaction time
is too long. It should be added that avoidance of organic solvents is particularly advantageous to the food
industry where stringent legal regulations related to the use of organic solvents are in force. Also, no
fireproof and explosion-proof equipment/procedures are necessary for the solvent-free processing and
the environment in the factory is less hazardous to the health of workers.
Biocatalytic reactions in organic media are currently being studied very actively as interdisciplinary fields
between organic chemistry and enzyme engineering, and between oleochemistry and enzyme engineering
to synthesize or convert lipids, saccharides, peptide, chiral compounds, polymers, etc. A comprehensive
monograph was published in 1996 reviewing the progress,2 and methods and protocols related to this topic
were summarized in monographs.3–5
Types of reactions involve: 1) bond formations of ester, amide, and glycoside by synthetic reaction as
reverse ones of hydrolyses or by transfer reactions (transesterification, transpeptidylation, transglycosy-
lation, etc), 2) oxidation and reduction, 3) C-O and C-N bond formation by addition/substitution
reactions, 4) C-C bond formation, 5) polymerizations, etc. The use of biocatalysts for these reactions
results in the best advantages in their selectivities involving enantio-selectivity, regio-selectivity, functional
group selectivity, etc. The process may be made simpler because of these selectivities without having to
introduce a protecting group and its deprotection afterward.
3.2 Enzymes
Many enzymes have been used for studying their actions in organic media, including lipases whose origins
are bacteria, molds, yeasts, and mammals and plants to lesser extents, esterases, proteases such as
thermolysin, -chymotrypsin, and subtilisin, peroxidase, phenol oxidase, alcohol dehydrogenase (yeast),
and so on. As a microorganism in organic media, baker’s yeast has been most often used.
3.2.1 Techniques Enabling Biocatalyst Highly Active in Organic Media

A number of techniques have been developed that enable biocatalysts active in organic media. These include:
1. Enzyme molecules are dissolved freely (some enzymes are soluble in glycerol or dimethylsulfoxide).
2. Enzyme molecules are derivatized with polyethylene glycol (PEG), or are formed as a complex
with surfactant (they are called lipid-coated enzyme or surfactant-modified enzymes, or surfactant-
enzyme complexes). All these modified enzymes are soluble in some organic solvents.
3. Enzymes are suspended or dispersed in organic media. If their dispersion is not uniform, it is
deposited on the surface of fine solid particles beforehand.
4. The enzymes are confined in a reverse micelle.
\

Biocatalyses in Microaqueous Organic Media 3-3
5. The enzymes exist in aqueous phase inside micropores of porous solid particles. Enzymes are
either free or immobilized.
6. The enzyme or microbial cells are entrapped in hydrophobic gel.
7. Microbial cells (wet or dry) having a particular enzyme are suspended in organic media, or they
are first immobilized in macroporous supports followed by suspension or packed in a bioreactor.
Since a number of techniques have been developed as mentioned above, it can be said confidently that
any enzyme can make its activity high in organic media. Techniques #2 and #4 exert high activity of the
enzyme preparation, but its recovery or continuous usage will be difficult. Technique #3 will give us
biocatalysts suitable for industrial usage because of its ease in recovery or of ability of continuous operation,
although one must invent a method of pretreatment or addition of activity enhancer to get higher activity.
3.2.2 Reactivation
Powders of some commercial enzyme preparations exhibit low activities when they are suspended in
organic media, but they are drastically reactivated when they have been re-lyophilized from their aqueous
solutions containing proper amount of sugar alcohols,6–8 surfactants,9,10 fatty acids,11 hydrocarbons,12 etc.
This phenomenon is related to the second technique mentioned above.
3.2.3 Purity
A factor that many researchers have paid little attention to, but that is very important, is the purity of the
enzyme preparations they use. As will be mentioned later, a profile of the effect of a trace amount of water
depends on the purity of the enzyme preparation, and the enzyme molecules are surrounded by large
amounts of impurities remaining around them and influence directly the enzyme catalysis when the crude
enzyme preparations are suspended in organic media (Figure 3.1a).13 Therefore their catalytic activities
are affected strongly by the nature and amount of the impurities. Crude enzyme powder exhibit enough
activity when it is uniformly dispersed in organic media, but pure enzyme powder is not uniformly
dispersed, and shows no activity unless three factors are optimized: water content, proper support
material, and the addition of a reactivator (or activity enhancer) (Figure 3.1b).6 Figure 3.2 indicates that
A B
Water-immiscible Water-immiscible
Organic Solvent
Organic Solvent
Impurities
Enhancer
Water Water
Enzyme Enzyme
Disperser
FIGURE 3.1 Schematic pictures of crude enzyme powder particles suspended in microaqueous medium (a), and
the system of (pure enzyme + activity enhancer) deposited on a disperser particle which is suspended in microaqueous
medium (b).6

FIGURE 3.2 Effect of additive on the lactonization activity of the pure enzyme at various concentrations of free
water. The vertical arrow indicates the solubility of water in benzene at 40°C (0.118%). —∆—, no addition, i.e.,
enzyme plus celite; —❍—, enzyme plus arabitol plus celite; ——, enzyme plus sorbitol plus celite; ——; enzyme
plus erythritol plus celite; —●—, enzyme plus phosphotidylcholine plus celite; —▲—, enzyme plus lactose plus
celite; —×—, enzyme plus BSA plus celite; —■—, enzyme plus casein plus celite; —∇—, enzyme plus PVA plus
celite; —❑—, enzyme plus dextran plus celite.
the rate of lactonization by Pseudomonas fluorescens lipase is increased significantly when a sugar alcohol
such as erythritol, arabitol, or sorbitol is added before freeze-drying together with celite powder.6 In this
case, the pure lipase is not dispersed at all without celite powder, and even if the enzyme is deposited on
the celite powder, the catalytic activity is very low.
3.2.4 Properties of Enzymes

Properties of an enzyme change more or less in organic media, including thermal stability and various
specificities (selectivities):
1. Thermal stability (half life), t1/2—Inactivation of an enzyme is regarded as change of its three-
dimensional structure from an active form to an inactive form, and often involves water molecules
during this structural change. Such structural changes do not occur in a complete anhydrous state
so that its thermostability is enhanced. Therefore, the value of its half life, t1/2, becomes much
longer in very anhydrous conditions than in an aqueous solution, and approaches to t1/2 value of
its aqueous solution when the water content increases.14 Bear in mind, however, that its catalytic
activity in nearly complete anhydrous state drops down to a low level, as will be mentioned later.
In any cases, t1/2 should be evaluated under a strict microaqueous condition, also taking into
consideration the nature of the organic solvent.
2. Substrate specificity, kcat /Km—Substrate specificity of a given enzyme is quantitatively evaluated
by the value of kcat /Km. In nonaqueous enzymology its value changes in various organic solvents.15
This implies that the specificity of an enzyme can be varied by selecting the kind of organic solvent
without applying protein engineering and/or screening another new enzyme from nature.
3. Enantioselectivity, E—Enantioselectivity of a given enzyme is quantitatively evaluated by the value
of the so-called enatiomeric ratio, or E value, which is defined as the relative ratio of kca /Km values
\

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For her heart in his grave is lying!
11. She sings the wild song of her dear native plains,
Every note which he loved awaking;
Ah! little they think who delight in her strains,
How the heart of the Minstrel is breaking!
12. He had lived for his love, for his country he died,
They were all that to life had entwined him;
Nor soon shall the tears of his country be dried,
Nor long will his love stay behind him.
13. O! make her a grave where the sunbeams rest,

When they promise a glorious morrow;
They’ll shine o’er her sleep, like a smile from the West,
From her own loved island of sorrow!
[89] Tragˊ
-i-cal, mournful, calamitous.
[90] Exˊ
-e-cuted (here means) put to death.
[91] Re-pelledˊ
, resisted.
[92] Ferˊ
vor, warmth, ardor.
[93] Portˊ
al, an entrance, a gateway.
[94] Sen-si-bil-i-tyˊ
, delicate feeling, tenderness.
[95] Disˊ
-si-pate, to disperse, to scatter.
[96] Solˊ
-i-tude, loneliness, seclusion.
[97] Blandˊ
-ish-ments, artful caresses, soothing words.
[98] Mas ˊ
-quer-ade ˊ
, an assemblage of persons for amusement in
which masks are worn.
[99] Ab-stracˊ
-tion, absence of mind.
[100] Orˊ
-ches-tra, a place or gallery for musicians.
[101] Garˊ
-ish, gaudy, showy.
[102] Ca-priˊ
-cious-ness, freak, whimsicalness.
XIII.—LITTLE VICTORIES.
Remark.—In conversational pieces like the following, the manner
of each speaker should be imitated, as in a dialogue.
1. “Oh, mother, now that I have lost my limb, I can never be a

soldier or a sailor; I can never go round the world!” And Hugh burst
into tears, now more really afflicted than he had ever been yet. His
mother sat on the bed beside him, and wiped away his tears as they
flowed, while he told her, as well as his sobs would let him, how long
and how much he had reckoned on going round the world, and how
little he cared for anything else in future; and now this was the very
thing he should never be able to do!
2. He had practiced climbing ever since he could remember, and
now this was of no use; he had practiced marching, and now he
should never march again. When he had finished his complaint,
there was a pause, and his mother said, “Hugh, you have heard of
Huber.”
“The man who found out so much about bees?” said Hugh.
“Bees and ants. When Huber had discovered[103] more than had
ever been known about these, and when he was sure that he could
learn still more, and was more and more anxious to peep into their
tiny[104] homes, and curious ways, he became blind.”
3. Hugh sighed, and his mother went on.
“Did you ever hear of Beethoven? He was one of the greatest
musical composers[105] that ever lived. His great, his sole delight,
was in music. It was the passion of his life. When all his time and all
his mind were given to music, he suddenly became deaf, perfectly
deaf; so that he never more heard one single note from the loudest
orchestra[106]. While crowds were moved and delighted with his
compositions[107] it was all silence to him.” Hugh said nothing.
4. “Now do you think,” asked his mother—and Hugh saw that a
mild and gentle smile beamed from her countenance—“do you think
that these people were without a heavenly Parent?”
“Oh no! but were they patient?” asked Hugh.
“Yes, in their different ways and degrees. Would you suppose that
they were hardly treated? Or would you not rather suppose, that
their Father gave them something better to do, than they had
planned for themselves?”
5. “He must know best, of course; but it does seem very hard,
that that very thing should happen to them. Huber would not have
so much minded being deaf, perhaps; or that musical man being
blind.”
“No doubt their hearts often swelled within them, at their
disappointments; but I fully believe that they very soon found God’s
will to be wiser than their wishes. They found, if they bore their trial
well, that there was work for their hearts to do, far nobler than any
the head could do through the eye, or the ear. And they soon felt a
new and delicious pleasure, which none but the bitterly disappointed
can feel.”
“What is that?”
6. “The pleasure of rousing the soul to bear pain, and of agreeing
with God silently, when nobody knows what is in the breast. There is
no pleasure like that of exercising one’s soul in bearing pain, and of
finding one’s heart glow with the hope that one is pleasing God.”
“Shall I feel that pleasure?”
“Often and often, I have no doubt; every time you can willingly
give up your wish to be a soldier, or a sailor, or any thing else you
have set your mind upon, you will feel that pleasure. But I do not
expect it of you yet. I dare say, it was long a bitter thing to
Beethoven to see hundreds of people in raptures[108] with his music
when he could not hear a note of it.”
7. “But did he ever smile again?” asked Hugh.
“If he did, he was happier than all the fine music in the world
could have made him,” replied his mother.
“I wonder, oh, I wonder if I shall ever feel so!”
“We will pray to God that you may. Shall we ask him now?”
Hugh clasped his hand. His mother kneeled beside the bed, and,
in a very few words, prayed that Hugh might be able to bear his
misfortune[109] well, and that his friends might give him such help
and comfort as God should approve.
[103] Dis-covˊ
-ered, found out.
[104] Tinˊ
-y, very small.
[105] Com-posˊ
er, an author.
[106] Orˊ
-ches-tra, a body of musicians.
[107] Com-po-siˊ
-tions, musical pieces.
[108] Raptˊ
-ures, extreme delight.
[109] Mis-forˊ
-tune, calamity.
XIV.—LITTLE VICTORIES.—Continued.
8. Hugh found himself subject to very painful feelings sometimes,

such as no one quite understood, and such as he feared no one was
able to pity as they deserved. On one occasion, when he had been
quite merry for a while, and his mother and sister Agnes were
chatting[110] they thought they heard a sob from the sofa. They
spoke to Hugh, and found that he was indeed crying bitterly.
“What is it, my dear,” said his mother. “Agnes, have we said
anything that could hurt his feelings?”
“No, no,” sobbed Hugh. “I will tell you presently.”
9. And presently he told them, that he was so busy listening to
what they said, that he forgot every thing else, when he felt as if
something got between two of his toes; unconsciously he put down
his hand, as if his foot was there! Nothing could be plainer than the
feeling in his toes; and, then, when he put out his hand, and found
nothing, it was so terrible! it startled him so! It was a comfort to find
that his mother knew about this. She came, and kneeled by his sofa,
and told him that many persons who had lost a limb, considered this
the most painful thing they had to bear, for some time; but that,
though the feeling would return occasionally through life, it would
cease to be painful.
10. Hugh was very much dejected[111], and when he thought of
the months and years, to the end of his life, and that he should
never run and play, and never be like other people, he almost
wished that he was dead.
Agnes thought that he must be miserable indeed, if he could
venture to say this to his mother. She glanced at her mother’s face,
but there was no displeasure there. On the contrary, she said this
feeling was very natural. She had felt it herself, under smaller
misfortunes than Hugh’s; but she had found, though the prospect
appears all strewn[112] with troubles, that they come singly, and are
not so hard to bear, after all.
11. She told Hugh, that when she was a little girl, she was very
lazy, fond of her bed, and not at all fond of dressing or washing.
“Why, mother! you?” exclaimed Hugh.
“Yes; that was the sort of little girl I was. Well, I was in despair,
one day, at the thought that I should have to wash and clean my
teeth, and brush my hair, and put on every article of dress, every
morning as long as I lived.”
“Did you tell any body?” asked Hugh.
12. “No; I was ashamed to do that; but I remember I cried. You
see how it turns out. When we have become accustomed to any
thing, we do it without ever thinking of the trouble, and, as the old
fable tells us, the clock, that has to tick so many millions of times,
has exactly the same number of seconds to do it in. So will you find,
that you can move about on each separate occasion, as you wish,
and practise will enable you to do it without any trouble or thought.”
“But this is not all, nor half what I mean,” said Hugh.
13. “No, my dear, nor half what you will have to bear. You resolved
to bear it all patiently, I remember. But what is it you dread the
most?”
“Oh! all manner of things. I can never do like other people.”
“Some things,” replied his mother. “You can never play cricket, as
every Crofton boy would like to do. You can never dance at your
sister’s Christmas parties.”
14. “Oh! mamma!” cried Agnes, with tears in her eyes, and with
the thought in her mind, that it was cruel to talk so.
“Go on! go on!” cried Hugh, brightening. “You know what I feel,
mother; and you don’t keep telling me, as others do, and even sister
Agnes, sometimes, that it will not signify much, and that I shall not
care, and all that; making out that it is no misfortune, hardly, when I
know what it is, and they don’t. Now then, go on, mother! What
else?”
15. “There will be little checks and mortifications continually, when
you see little boys leaping over this, and climbing that, and playing
at the other, while you must stand out, and can only look on. And
some people will pity you, in a way you will not like; and some may
even laugh at you.”
“Oh mamma!” exclaimed Agnes.
“Well, and what else?” said Hugh.
16. “Sooner or later, you will have to follow some way of life
determined by this accident, instead of one that you would have
liked better.”
“Well, what else?”
“I must ask you, now. I can think of nothing more; and I hope
there is not much else; for, indeed, I think here is quite enough for a
boy, or any one else, to bear.”
“I will bear it, though; you will see.”
17. “You will find great helps. These misfortunes, of themselves
strengthen one’s mind. They have some advantages, too. You will be
a better scholar for your lameness, I have no doubt. You will read
more books, and have a mind richer in thoughts. You will be more
beloved by us all, and you yourself will love God more for having
given you something to bear for His sake. God Himself will help you
to bear your trials. You will conquer your trials one by one, and by a
succession of little victories, will, at last, completely triumph over
all.”
[110] Chatˊ
-ting, talking familiarly.
[111] De-jectˊ
-ed, discouraged, low-spirited.
[112] Strewn, scattered.
XV.—OUR TITLES.
1. Are we not Nobles? we who trace

Our Pedigree[113] so high,
That God for us and for our race
Created Earth and Sky,
And Light, and Air, and Time, and Space,
To serve us, and then die.
2. Are we not Princes? we who stand

As heirs beside the Throne;
We who can call the promised land
Our Heritage,[114] our own;
And answer to no less command
Than God’s, and His alone.
3. Are we not Kings? both night and day,

From early until late,
About our bed, about our way,
A guard of Angels wait;
And so we watch, and work, and pray
In more than royal state.
4. Are we not holy? do not start;

It is God’s sacred will
To call us Temples set apart
His Holy Ghost may fill;
Our very food.... Oh hush, my heart,
Adore It and be still!
5. Are we not more? Our life shall be

Immortal[115] and divine;
The nature Mary gave to Thee,
Dear Jesus, still is Thine;
Adoring in Thy Heart I see
Such blood as beats in mine.
6. O God! that we can dare to fail,

And dare to say we must!
O God, that we can ever trail
Such banners in the dust,
Can let such starry honors pale,
And such a Blazon[116] rust!
7. Shall we upon such Titles bring

The taint of sin and shame?
Shall we, the children of the King
Who hold so grand a claim,
Tarnish, by any meaner thing,
The glory of our name?
[113] Pedˊ
-i-gree, lineage, line of descent from a progenitor.
[114] Her ˊ-it-age, an estate that passes from an ancestor to an
heir.
[115] Im-morˊ
-tal, exempt from death.
[116] Blaˊ
-zon, a coat of arms.
XVI.—THE WIDOW OF THE PINE
COTTAGE.
1. It was Saturday night, and the widow of the Pine Cottage sat by
her blazing fagots,[117] with her five tattered children at her side,
endeavoring, by listening to the artlessness, of their prattle,[118] to
dissipate[119] the heavy gloom that pressed upon her mind. For a
year, her own feeble hand had provided for her helpless family, for
she had no supporter: she thought of no friend in all the wide,
unfriendly world around.
2. But that mysterious Providence, the wisdom of whose ways is
above human comprehension, had visited her with wasting sickness,
and her little means had become exhausted. It was now, too, mid-
winter, and the snow lay heavy and deep through all the surrounding
forests, while storms still seemed gathering in the heavens, and the
driving wind roared among the neighboring pines, and rocked her
puny[120] mansion.
3. The last herring smoked upon the coals before her; it was the
only article of food she possessed, and no wonder her forlorn,
desolate state brought up in her lone bosom all the anxieties of a
mother, when she looked upon her children; and no wonder, forlorn
as she was, if she suffered the heart-swellings of despair to rise,
even though she knew that He, whose promise is to the widow and
to the orphan, can not forget his word.
4. Providence had, many years before, taken from her her eldest
son, who went from his forest home to try his fortune on the high
seas, since which she had heard no tidings of him; and, later still,
the hand of death deprived her of the companion and staff of her
earthly pilgrimage,[121] in the person of her husband. Yet to this
hour she had upborne; she had not only been able to provide for her
little flock, but had never lost an opportunity of ministering to the
wants of the miserable and destitute.
5. The indolent may well bear with poverty, while the ability to
gain sustenance remains. The individual who has but his own wants
to supply, may suffer with fortitude the winter of want; his affections
are not wounded, his heart not wrung. The most desolate in
populous cities may hope, for charity has not quite closed her hand
and heart, and shut her eyes on misery.
6. But the industrious mother of helpless and depending children,
far from the reach of human charity, has none of these to console
her. And such a one was the widow of the Pine Cottage; but as she
bent over the fire, and took up the last scanty remnant of food, to
spread before her children, her spirits seemed to brighten up, as by
some sudden and mysterious impulse, and Cowper’s beautiful lines
came uncalled across her mind:
Judge not the Lord by feeble sense,

But trust Him for His grace;
Behind a frowning Providence
He hides a smiling face.
7. The smoked herring was scarcely laid upon the table, when a
gentle rap at the door, and loud barking of a dog, attracted the
attention of the family. The children flew to open it, and a weary
traveler, in tattered garments, and apparently indifferent health,
entered and begged a lodging, and a mouthful of food. Said he, “It
is now twenty-four hours since I tasted bread.” The widow’s heart
bled anew as under a fresh complication[122] of distresses; for her
sympathies[123] lingered not around her fireside. She hesitated not
even now; rest and a share of all she had she proffered to the
stranger. “We shall not be forsaken,” said she, “or suffer deeper for
an act of charity.”
8. The traveler drew near the board, but when he saw the scanty
fare, he raised his eyes toward heaven with astonishment: “And is
this all your store?” said he, “and a share of this do you offer to one
you know not? then never saw I charity before! but, madam,” said
he, continuing, “do you not wrong your children by giving a part of
your last mouthful to a stranger?”
9. “Ah,” said the poor widow, and the tear-drops gushed[124] into
her eyes as she said it, “I have a boy, a darling son, somewhere on
the face of the wide world, unless heaven has taken him away, and I
only act toward you, as I would that others should act toward him.
God, who sent manna[125] from heaven, can provide for us as He
did for Israel; and how should I this night offend Him, if my son
should be a wanderer, destitute as you, and He should have
provided for him a home, even poor as this, were I to turn you
unrelieved away.”
10. The widow ended, and the stranger, springing from his seat,
clasped her in his arms: “God, indeed, has provided your son a
home, and has given him wealth to reward the goodness of his
benefactress: my mother! oh, my mother!” It was her long-lost son,
returned to her bosom from the Indies. He had chosen that disguise
that he might the more completely surprise his family; and never
was surprise more perfect, or followed by a sweeter cup of joy.
11. That humble residence in the forest was exchanged for one
comfortable, and indeed beautiful, in the valley; and the widow lived
long with her dutiful son, in the enjoyment of worldly plenty, and in
the delightful employments of virtue; and, at this day, the passer-by
is pointed to the willow that spreads its branches above her grave.
[117] Fagˊ
-ots; bundles of sticks used for fuel.
[118] Pratˊ
-tle; trifling talk.
[119] Disˊ
-si-pate; to scatter, to disperse.
[120] Puˊ
-ny; small and weak.
[121] Pilˊ
-grim-age; the journey of human life.
[122] Com-pli-ca ˊ-tion; the act of mingling together several
things.
[123] Symˊ
-pa-thies; compassion.
[124] Gushed; flowed copiously.
[125] Man ˊ-na; food miraculously provided by God for the
Israelites.
XVII.—A PSALM OF LIFE.
longfellow.
Henry Wadsworth Longfellow was born at Portland, Maine, in
1807. He is one of the most popular of living poets.
1. Tell me not, in mournful numbers,

Life is but an empty dream!
For the soul is dead that slumbers,
And things are not what they seem.
2. Life is real! Life is earnest!

And the grave is not its goal;
Dust thou art, to dust returnest,
Was not spoken of the soul.
3. Not enjoyment, and not sorrow,

Is our destined[126] end or way,
But to act, that each to-morrow
Find us farther than to-day.
4. Art is long, and time is fleeting;

And our hearts, though stout and brave,
Still, like muffled[127] drums, are beating
Funeral marches to the grave.
5. In the world’s broad field of battle,

In the bivouac[128] of life,
Be not like dumb, driven cattle!
Be a hero in the strife!
6. Trust no Future, howe’er pleasant,

Let the dead past bury its dead!
Act—act in the living Present!
Heart within, and God o’erhead.
7. Lives of great men all remind us,

We can make our lives sublime;[129]
And, departing, leave behind us
Foot-prints[130] on the sands of time;—
8. Foot-prints, that, perhaps, another,

Sailing over life’s solemn[131] main,[132]
A forlorn[133] and shipwrecked brother,
Seeing, shall take heart again.
9. Let us, then, be up and doing,

With a heart for any fate;
Still achieving,[134] still pursuing,[135]
Learn to labor, and to wait.
[126] Des’-tin-ed, fated; appointed.

[127] Muf’-fled, covered; wrapped up.
[128] Biv’-ouac (biv’-wak), encampment without tents; a
watching.
[129] Sub-lime’, lofty; grand.
[130] Foot-prints, impression of the foot.
[131] Sol’-emn, grave; serious.
[132] Main, open sea; ocean.
[133] For’-lorn, forsaken; helpless.
[134] A-chiev’-ing, performing; doing.
[135] Pur-su’-ing, following up.
XVIII.—LET VIRTUE BE YOUR AIM.
1. Whatever be thy lot on earth,

Thy mission[136] here below,
Though Fame may wreathe[137] her laurels[138] fair,
Around your youthful brow,—
If you would rise from earthly things,
And win a deathless name,
Let all your ways be just and right—
Let virtue be your aim.[139]
2. Though cherished[140] friends may traitors[141] prove.

Their kindness all depart,
And leave a mournful spell around
Thy sad and bleeding heart;
Though you may oft be scorned[142] by men,
Or those who bear the name,
Let all your ways be just and right—
Let virtue be your aim.
3. Oh! ye who dwell in stately[143] halls,

Where wealth and fame[144] are known,
Remember you may yet be poor,
Neglected and alone!
But, oh! remember this broad truth,
Ere others’ faults you scan,[145]
Your wealth may make a thousand fools—
But virtue makes the man.
[136] Mis’-sion, errand; business.

[137] Wreathe, entwine.
[138] Lau’-rels, flowers for garlands.
[139] Aim, purpose; intention.
[140] Cher’-ish-ed, dear; loved.
[141] Trai’-tors, betrayers.
[142] Scorn’-ed despised; disdained
[143] State’-ly, magnificent; grand
[144] Fame, renown; glory.
[145] Scan, examine critically.
XIX. -THE BOBOLINK.
w. irving.
1. The happiest bird of our spring, and one that rivals[146] the
European lark, in my estimation,[147] is the Bobolink. He arrives at
that choice portion of the year, which, in this latitude, answers to the
description[148] of the month of May, so often given by the poets.
With us, it begins about the middle of May, and lasts until nearly the
middle of June.
2. Earlier than this, winter is apt to return on its traces, and to
blight[149] the opening beauties of the year; and later than this,
begin the parching, and panting, and dissolving[150] heats of
summer. But, in this genial[151] interval,[152] Nature is in all her
freshness and fragrance; “the rain is over and gone, the flowers
appear on the earth, the time of the singing of birds is come, and
the voice of the turtle[153] is heard in our land.”
3. The trees are now in their fullest foliage[154] and the brightest
verdure; the woods are gay with the clustered[155] flowers of the
laurel; the air is perfumed by the sweet-brier and the wild rose; the
meadows are enameled[156] with clover-blossoms; while the young
apple, the peach, and the plum, begin to swell, and the cherry to
glow among the green leaves.
4. This is the chosen season of revelry[157] of the Bobolink. He
comes amidst the pomp and fragrance of the season; his life seems
all sensibility[158] and enjoyment, all song and sunshine. He is to be
found in the soft bosoms of the freshet and sweetest meadows; and
is most in song when the clover is in blossom.
5. He perches[159] on the topmost twig of a tree, or on some
flaunting weed, and, as he rises and sinks with the breeze, pours
forth a succession[160] of rich, tinkling notes, crowding one upon
another, like the outpouring melody of the sky-lark, and possessing
the same rapturous[161] character.
6. Sometimes he pitches from the summit of a tree, begins his
song as soon as he gets upon the wing, and flutters tremulously[162]
down to the earth, as if overcome with ecstasy[163] at his own
music. Sometimes he is in pursuit of his paramour;[164] always in full
song, as if he would win her by his melody; and always with the
same appearance of intoxication[165] and delight.
7. Of all the birds of our groves and meadows, the Bobolink was
the envy of my boyhood. He crossed my path in the sweetest
weather, and the sweetest season of the year, when all Nature called
to the fields, and the rural[166] feeling throbbed in every bosom; but,
when I, luckless urchin, was doomed to be mewed[167] up, during
the livelong day, in a school-room, it seemed as if the little
varlet[168] mocked at me, as he flew by in full song, and sought to
taunt me with his happier lot. O, how I envied him! No lessons, no
tasks, no schools; nothing but holiday, frolic, green fields, and fine
weather.
[146] Ri’-vals, strives to excel.

[147] Es-ti-ma’-tion, opinion; esteem.
[148] De-scrip’-tion, account.
[149] Blight, blast; destroy.
[150] Dis-solv’-ing, melting.
[151] Ge’-ni-al, fruitful; productive.
[152] In’-ter-val, space between.
[153] Tur’-tle, species of dove.
[154] Fo’-li-age, leaves collectively.
[155] Clus’-ter-ed. growing in bunches.
[156] En-am’-el-ed, inlaid; variegated.
[157] Rev’-el-ry, festive mirth; jollity.
[158] Sens-i-bil’-ty, delicate feeling.
[159] Perch’-es, alights.
[160] Suc-ces’-sion, series.
[161] Rap’-tur-ous, joyful; thrilling.
[162] Trem’-u-lous-ly, tremblingly.
[163] Ec’-sta-sy, excessive joy.
[164] Par’-a-mour, lover.
[165] In-tox-i-ca’-tion, high excitement.
[166] Ru’-ral, pertaining to the country.
[167] Mew’-ed, shut up; confined.
[168] Var’-let, scamp; rascal.
XX.—THE BOBOLINK.—Continued.
1. Farther observation and experience have given me a different

idea of this little feathered voluptuary,[169] which I will venture to
impart, for the benefit of my school-boy readers, who may regard
him with the same unqualified envy and admiration which I once
indulged.
2. I have shown him only as I saw him at first, in what I may call
the poetical part of his career, when he, in a manner, devoted
himself to elegant pursuits and enjoyments, and was a bird of music,
and song, and taste, and sensibility, and refinement. While this
lasted, he was sacred from injury; the very school-boy would not
fling a stone at him, and the merest rustic[170] would pause to listen
to his strain.
3. But mark the difference. As the year advances, as the clover-
blossoms disappear, and the spring fades into summer, his notes
cease to vibrate[171] on the ear. He gradually gives up his elegant
tastes and habits, doffs his poetical and professional suit of black,
assumes a russet,[172] or rather a dusky garb, and enters into the
gross enjoyments of common, vulgar birds.
4. He becomes a bon vivant, a mere gormand;[173] thinking of
nothing but good cheer, and gormandizing on the seeds of the long
grasses, on which he lately swung and chanted so musically. He
begins to think there is nothing like “the joys of the table,” if I may
be allowed to apply that convivial[174] phrase to his indulgences. He
now grows discontented with plain, every-day fare, and sets out on
a gastronomical[175] tour, in search of foreign luxuries.[176]
5. He is to be found in myriads[177] among the reeds of the
Delaware, banquetting[178] on their seeds; grows corpulent[179] with
good feeding, and soon acquires the unlucky renown of the Ortolan.
[180] Wherever he goes, pop! pop! pop! the rusty firelocks of the
country are cracking on every side; he sees his companions falling
by thousands around him; he is the reed-bird, the much-sought for
tit-bit of the Pennsylvanian epicure.[181]
6. Does he take warning and reform? Not he! He wings his flight
still farther south in search of other luxuries. We hear of him
gorging[182] himself in the rice swamps; filling himself with rice
almost to bursting; he can hardly fly for corpulency. Last stage of his
career, we hear of him spitted by dozens, and served up on the table
of the gormand, the most vaunted[183] of southern dainties, the
rice-bird of the Carolinas.
7. Such is the story of the once musical and admired, but finally
sensual[184] and persecuted[185] Bobolink. It contains a moral,
worthy the attention of all little birds and little boys, warning them to
keep to those refined and intellectual[186] pursuits, which raised him
to such a pitch of popularity, during the early part of his career; but
to eschew[187] all tendency to that gross and dissipated[188]
indulgence, which brought this mistaken little bird to an untimely
end.
[169] Vo-lupˊ
-tu-a-ry, one given to pleasure.
[170] Rusˊ
-tic, dweller in the country.
[171] Viˊ
-brate, quiver.
[172] Rusˊ
-set, reddish brown.
[173] Gorˊ
-mand, glutton.
[174] Con-vivˊ
-i-al, festal; social.
[175] Gas-tro-nomˊ
-ic-al, pertaining to good eating.
[176] Luxˊ
-u-ries, dainties.
[177] Myrˊ
-i-ads, tens of thousands.
[178] Banˊ
-quet-ting, feasting.
[179] Corˊ
-pu-lent, fleshy; fat.
[180] Orˊ
-to-lan, delicate, small bird.
[181] Epˊ
-i-cure, one given to luxury.
[182] Gorgˊ
-ing, swallowing greedily.
[183] Vauntˊ
-ed, boasted.
[184] Sensˊ
-u-al, luxurious.
[185] Perˊ
-se-cu-ted, harassed; vexed.
[186] In-tel-lectˊ
-u-al, mental.
[187] Es-chewˊ
, avoid; shun.
[188] Disˊ
-si-pa-ted, loose; abandoned.
XXI.—WHO IS MY NEIGHBOR?
1. Thy neighbor? It is he whom thou

Hast power to aid[189] and bless,
Whose aching heart, or burning brow
Thy soothing[190] hand may press.
2. Thy neighbor? ’Tis the fainting poor,

Whose eye with want is dim,
Whom hunger sends from door to door—
Go thou, and succor[191] him.
3. Thy neighbor? ’Tis that weary[192] man,

Whose years are at their brim,
Bent low with sickness, cares and pain—
Go thou, and comfort him.
4. Thy neighbor? ’Tis the heart bereft[193]

Of every earthly gem:
Widow[194] and orphan helpless left—
Go thou, and shelter them.
5. Whene’er thou meet’st a human form

Less favored[195] than thine own,
Remember ’tis thy neighbor worm,
Thy brother or thy son.
6. Oh! pass not, pass not heedless by;

Perhaps, thou canst redeem
The breaking heart from misery—
Go, share thy lot with him.
[189] Aid, help; assist.

[190] Soothˊ
-ing, solacing.
[191] Sucˊ
-cor, help; relieve.
[192] Weaˊ
-ry, tired; fatigued.
[193] Be-reftˊ
, deprived.
[194] Widˊ
-ow, a woman whose husband is dead.
[195] Faˊ
-vor-ed, benefitted.
XXII.—PORTRAIT OF A VIRTUOUS
AND ACCOMPLISHED WOMAN.
fenelon.
1. Antiope is mild, simple, and wise; her hands despise not labor;
she foresees things at a distance; she provides against
contingencies[196] she knows when it is proper to be silent; she acts
regularly and without hurry; she is continually employed, but never
embarrassed,[197] because she does every thing in its proper
season.
2. The good order of her father’s house is her glory, it adds
greater luster[198] to her than beauty. Though the care of all lies
upon her, and she is charged with the burden of reproving,[199]
refusing, retrenching[200] (things which make almost all women
hated), yet she has acquired the love of all the household; and this,
because they do not find in her either passion, or conceitedness,
[201] or levity, or humors as in other women. By a single glance of
her eye, they know her meaning, and are afraid to displease her.
3. The orders she gives are precise; she commands nothing but
what can be performed; she reproves with kindness, and in
reproving encourages. Her father’s heart reposes upon her as a
traveler, fainting beneath the sun’s sultry rays, reposes himself upon
the tender grass under a shady tree.
4. Antiope is a treasure worth seeking in the most remote corners
of the earth. Neither her person nor her mind is set off with vain
ornaments; and her imagination, though lively, is restrained by her
discretion. She never speaks but through necessity; and when she
opens her mouth, soft persuasion and simple graces flow from her
lips. When she speaks, every one is silent; and she is heard with
such attention, that she blushes, and is almost inclined to
suppress[202] what she intended to say; so that she is rarely ever
heard to speak at any length.
[196] Con-tinˊ
-gen-cies, chances, casual events, possibilities.
[197] Em-barˊ
-rassed, perplexed, confused.
[198] Lusˊ
-ter, brightness, splendor, brilliancy.
[199] Re-proveˊ
, to censure to one’s face, to reprimand.
[200] Re-trenchˊ
, to lessen, to curtail.
[201] Con-ceitˊ
-ed-ness, pride, vanity.
[202] Sup-pressˊ
, to restrain, to conceal.
XXIII.—THE WORK OF TO-DAY.
charles mackay.
1. If Fortune with a smiling face,

Strew roses on our way,
When shall we stoop to pick them up?
To-day, my friend, to-day.
But should she frown with face of care.
And talk of coming sorrow,
When shall we grieve if grieve[203] we must?
To-morrow, friend, to-morrow.
2. If those who’ve wronged us, own their fault,

And kindly pity pray,
When shall we listen, and forgive?
But, if stern[204] Justice urge[205] rebuke,[206]
And warmth from Memory borrow,
When shall we chide,[207] if chide we dare?
3. If those to whom we owe a debt,

Are harmed unless we pay,
When shall we struggle to be just?
But, if our debtor[208] fail our hope,
And plead his ruin thorough,[209]
When shall we weigh his breach[210] of faith?
4. For virtuous[211] acts, and harmless joys,

The minutes will not stay;
We’ve always time to welcome them,
But care, resentment,[212] angry words,
And unavailing sorrow,
Come far too soon, if they appear
[203] Grieve, mourn, sorrow.

[204] Stern, severe, rigid.
[205] Urge, press, impel.
[206] Re-bukeˊ
, reproof, reprehension.
[207] Chide, blame, reproach.
[208] Debtˊ
-or, one that owes.
[209] Thorˊ
-ough, complete, perfect.
[210] Breach, non-fulfillment, violation.
[211] Virˊ
-tuous, morally good.
[212] Re-sentˊ
-ment, retaliation.
XXIV.—THE AVARICIOUS MILLER.
goldsmith.
Oliver Goldsmith was born in 1731, at Pallasmore, County of
Longford, Ireland, and died in 1774. The works of Goldsmith are
more popular to-day than those of any English author of the
Eighteenth Century. He was equally successful as a novelist and a
poet. His “Vicar of Wakefield”, “Traveller,” and “Deserted Village”
are each models of excellence in their way. His essays, also, have
rare merit.
1. Whang, the miller, was naturally avaricious;[213] nobody loved

money better than he, or more respected those that had it. When
people would talk of a rich man in company, Whang would say, “I
know him very well; he and I have been very long acquainted; he
and I are intimate.”[214]
2. But, if a poor man was mentioned, he had not the least
knowledge of the man; he might be very well, for aught he knew;
but he was not fond of making many acquaintances, and loved to
choose his company.
3. Whang, however, with all his eagerness[215] for riches, was
poor. He had nothing but the profits of his mill to support him; but,
though these were small, they were certain: while it stood and went,
he was sure of eating; and his frugality[216] was such, that he, every
day, laid some money by; which he would, at intervals, count and
contemplate with much satisfaction.
4. Yet still his acquisitions[217] were not equal to his desires; he
only found himself above want, whereas he desired to be possessed
of affluence.[218] One day, as he was indulging these wishes, he was
informed that a neighbor of his had found a pan of money under
ground, having dreamed of it three nights in succession.[219]

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© 2005 by Taylor & Francis Group, LLC

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No claim to original U.S. Government works

International Standard Book Number-10: 0-8247-2423-2 (Hardcover)

Library of Congress Cataloging-in-Publication Data

Hou, Ching T. (Ching-Tsang), 1935-

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© 2005 by Taylor & Francis Group, LLC

Ching T. Hou, Ph.D.

© 2005 by Taylor & Francis Group, LLC

Shuji Adachi Peter Biely Masashi Hosokawa

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Chapter 1 Enzymes for the Transgenic Production of Long-Chain

Chapter 2 Screening for Unique Microbial Reactions Useful

Chapter 3 Biocatalyses in Microaqueous Organic Media

Chapter 4 Biocatalysts for the Epoxidation and Hydroxylation of Fatty Acids

Chapter 5 Lipase-Catalyzed Kinetic Resolution for the Fractionation of Fatty

Chapter 6 Protein Engineering of Recombinant Candida rugosa Lipases

Chapter 7 Production of Value-Added Industrial Products from Vegetable Oils:

Chapter 8 Application of Lipases to Industrial-Scale Purification of Oil-

Chapter 9 Applications of Lipases in Modifications of Food Lipids

Chapter 10 Lipase-Catalyzed Condensation in an Organic Solvent

© 2005 by Taylor & Francis Group, LLC

Chapter 12 The Use of Nonimmobilized Lipase for Industrial

Chapter 13 Preparation of Polyunsaturated Phospholipids

Chapter 14 Production of Biosurfactants by Fermentation of Fats, Oils, and Their

Chapter 15 Fatty Acid-Modifying Enzymes: Implications for Industrial Applications

Chapter 16 Low-Calorie Fat Substitutes: Synthesis and Analysis

Chapter 17 Microbial Polyunsaturated Fatty Acid Production

Chapter 18 Structured Triacylglycerols Comprising Omega-3 Polyunsaturated

Chapter 19 Biopolyesters Derived from the Fermentation of Renewable Resources

Chapter 20 Carbohydrate Active-Enzymes for the Production of Oligosaccharides

Chapter 21 Microbial Hemicellulolytic Carbohydrate Esterases

Chapter 22 Application of Cyclodextrin Glucanotransferase to the

Chapter 23 Converting Herbaceous Energy Crops to Bioethanol: A Review

© 2005 by Taylor & Francis Group, LLC

Chapter 25 Bioelectrocatalysis: Electroactive Microbial and Enzyme Technologies

Chapter 26 Biocatalysis: Synthesis of Chiral Intermediates for Pharmaceuticals

Chapter 27 Nutrition Delivery System: A Novel Concept of Nutrient Fortification

Chapter 28 Renewable Resources for Production of Aromatic Chemicals

Chapter 29 Extremophiles from the Origin of Life to Biotechnological

© 2005 by Taylor & Francis Group, LLC