ENZYMES

IN
DETERGENCY
 SURFACTANT SCIENCE SERIES

CONSULTING EDITORS

MARTINJ. SCHICK FREDERICK M. FOWKES

Consultant (1915-1990)
New York. New York

1. Nonionic Surfactants, edited by Martin J. Schick (see also Volumes 19, 23,
and 60)
2. Solvent Properties of Surfactant Solutions, edited by Kozo Shinoda (see
Volume 55)
3. Surfactant Biodegradation, R. D. Swisher(see Volume 18)
4. Cationic Surfactants, edited by Eric Jungermann (see also Volumes 34, 37,
and 53)
5. Detergency: Theory· and Test Methods (in three parts), edited by W G.
Cutler and R. C. Davis {see also Volume 20)
6. Emulsions and Emulsion Technology (in three parts), edited by Kenneth J.
Ussant
7. Anionic Surfactants (in two parts), edited by Warner M. Unfield (see Volume
56)
8. Anionic Surfactants: Chemical Analysis, edited by John Cross (out of print)
9. Stabilization of Colloidal Dispersions by Polymer Adsorption, Tatsuo Sato
and Richard Ruch (out of print)
10. Anionic Surfactants: Biochemistry, Toxicology, Dermatology, edited by
Christian Gloxhuber (see Volume 43)
11. Anionic Surfactants: Physical Chemistry of Surfactant Action, edited by E. H.
Lucassen-Reynders (out of print)
12. Amphoteric Surfactants, edited by B. R. Bluestein and Clifford L. Hilton (see
Volume 59)
13. Demulsification: Industrial Applications, Kenneth J. Ussant {out of print)
14. Surfactants in Textile Processing, Arved Datyner
15. Electrical Phenomena at Interfaces: Fundamentals, Measurements, and
Applications, edited by Ayao Kitahara and Akira Watanabe
16. Surfactants in Cosmetics, edited by Martin M. Rieger (out of print)
17. Interfacial Phenomena: Equilibrium and Dynamic Effects, Clarence A. Ml11er
andP. Neogi
18. Surfactant Biodegradation: Second Edition, Revised and Expanded, R. D.
Swisher
19. Nonionic Surfactants: Chemical Analysis, edited by John Cross
20. Detergency: Theory and Technology, edited by W Gale Cutler and Erik
Kiss a
21. Interfacial Phenomena in Apolar Media, edited by Hans-Friedrich Eicke and
Geoffrey D. Parfitt
22. Surfactant Solutions: New Methods of Investigation, edited by Raoul Zana
23. Nonionic Surfactants: Physical Chemistry, edited by Martin J. Schick
24. Microemulsion Systems, edited by Henri L. Rosano and Marc Clausse
25. Biosurfactants and Biotechnology, edited by Nairn Kosaric, W. L. Cairns, and
NeH C. C. Gray
26. Surfactants in Emerging Technologies, edited by Milton J. Rosen
27. Reagents in Mineral Technology, edited by P. Somasundaran and Brij M.
Moudgil
28. Surfactants in Chemical/Process Engineering, edited by Darsh T. Wasan,
Martin E. Ginn, and Dinesh 0. Shah
29. Thin Liquid Films, edited by I. B. Ivanov
30. Microemulsions and Related Systems: Formulation, Solvency, and Physical
Properties, edited by Maurice Bourret and Robert S. Schechter
31. Crystallization and Polymorphism of Fats and Fatty Acids, edited by Nissim
Garti and Kiyotaka Sato
32. Interfacial Phenomena in Coal Technology, edited by Gregory D. Botsaris
and Yuli M. Glazman
33. Surfactant-Based Separation Processes, edited by John F. Scamehom and
Jeffrey H. Harwell .
34. Cationic Surfactants: Organic Chemistry, edited by James M. Richmond
35. Alkylene Oxides and Their Polymers, F. E. Bailey, Jr., and Joseph V.
Koleske
36. Interfacial Phenomena in Petroleum Recovery, edited by Norman R. Morrow
37. Cationic Surfactants: Physical Chemistry, edited by Donn N. Rubingh and
Paul M. Holland
38. Kinetics and Catalysis in Microheterogeneous Systems, edited by M. Gratzel
and K Ka/yanasundaram
39. Interfacial Phenomena in Biological Systems, edited by Max Bender
40. Analysis of Surfactants, Thomas M. Schmitt
41. Light Scattering by Liquid Surfaces and Complementary Techniques, edited
by Dominique Langevin
42. Polymeric Surfactants, ltja Piirma
43. Anionic Surfactants: Biochemistry, Toxicology, Dermatology. Second Edition,
Revised and Expanded, edited by Christian Gloxhuber and Klaus Kiinstler
44. Organized Solutions: Surfactants in Science and Technology, edited by Stig
E. Friberg and BjtJm Undman
45. Defoaming: Theory and Industrial Applications. edited by P. R. Garrett
46. Mixed Surfactant Systems, edited by Keizo Ogino and Masahiko Abe
47. Coagulation and Flocculation: Theory and Applications, edited by Bohus/av
Dobias
48. Biosurfactants: Production • Properties • Applications, edited by Nairn
Kosaric
49. Wettability, edited by John C. Berg
50. Fluorinated Surfactants: Synthesis • Properties • Applications, Erik Kissa
51. Surface and Colloid Chemistry in Advanced Ceramics Processing, edited by
Robert J. Pugh and Lennart Bergstrom
52. Technological Applications of Dispersions, edited by Robert B. McKay
53. Cationic Surfactants: Analytical and Biological Evaluation, edited by John
Cross and Edward J. Singer
54. Surfactants in Agrochemicals, Tharwat F. Tadros
55. Solubilization in Surfactant Aggregates, edited by Sherril D. Christian and
John F. Scamehom
56. Anionic Surfactants: Organic Chemistry, edited by Helmut W. Stache
57. Foams: Theory, Measurements, and Applications, edited by Robert K. Prud'-
homme and Saad A Khan
58. The Preparation of Dispersions in Liquids, H. N. Stein
59. Amphoteric Surfactants: Second Edition, edited by En"c G. Lomax
60. Nonionic Surfactants: Polyoxyalkylene Block Copolymers, edited by
Vaughn M. Nace
61. Emulsions and Emulsion Stability, edited by Johan Sjoblom
62. Vesicles, edited by Morton Rosoff
63. Applied Surface Thermodynamics, edited by A. W. Neumann and Jan K.
Spe!t
64. Surfactants in Solution, edited by Arun K. Chattopadhyay and K. L.
Mittal
65. Detergents in the Environment, edited by Milan Johann Schwuger
66. Industrial Applications of Microemulsions, edited by Conxita Solans and
Hironobu Kunieda
67. Liquid Detergents, edited by Kuo-Yann Lai
68. Surfactants in Cosmetics: Second Edition, Revised and Expanded, edited
by Martin M. Rieger and Linda D. Rhein
69. Enzymes in Detergency, edited by Jan H. van Ee, Onno Misset, and Erik
J. Baas

ADDITIONAL VOLUMES IN PREPARATION

Structure-Performance Relationships in Surfactants, edited by Kunia

Esumi and Minoru Ueno
Powdered Detergents, edited by MichaelS. Showell
 ENZYMES
IN
DETERGENCY
edited by
Jan H. van Ee
Cargill B. V.
Amsterdam, The Netherlands

Onno Misset
Genencor International B. V.
Delft, The Netherlands

Erik J. Baas
Business Innovation Partners B. V.
Amsterdam, The Netherlands

0 ~"~~,~~:~~.
Boca Raton London New York

CRC Press is an imprint of the

Taylor & Francis Group, an informa business
Reprinted 2010 by CRC Press
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Preface

Household cleaning is a daily worldwide activity, which means that billions of

people use detergents to clean their clothes, dishes, and so forth. In addition to the
major ingredients, which are the surfactants and builders, enzymes are increas-
ingly added to achieve "catalytic cleaning," thereby creating smaller fragments of
the stain constituents, which in tum facilitates the chemical cleaning action of
surfactants. The use of enzymes is growing constantly, not only in volume but
also in types of enzyme; since the initial introduction of proteolytic enzymes
some 40 years ago, present-day detergents have come to contain lipases,
amylases, and cellulases. Household detergents are the most important applica-
tion of industrial enzymes, representing approximately 50% of total sales.
Much progress has been made in the last decades in elucidating the structure-
function relations of enzymes. Also, more has become known about the cleaning
mechanisms of enzymes, and production and formulation techniques have also
been considerably improved. This volume of the Surfactant Science Series con-
tains overviews of all these different aspects as they relate to the use of enzymes
in laundry and dishwasher detergents: their application, development, screening,
protein engineering, manufacturing, safety, markets, and such. The combination
of all these different subjects in one volume renders this book unique because no
other publication of comparable broadness and depth exists.
The editors were very pleased with the overwhelmingly positive response of
the major companies involved-both detergent manufacturers and enzyme sup-
pliers-in participating in the realization of this book. Needless to say, this broad
participation augments the book's value. The authors are all authorities in their
respective fields. As in other volumes of this series, chapters were not required to

iii
lv Preface

adhere to the same fonnat. The authors were free to present and interpret data
from their own viewpoint and experience.
During the preparation of this book, which began in the summer of 1994, major
changes occurred in the world of enzyme manufacturers. The editors and authors
who originally worked for Gist-Brocades moved to Genencor International in
June 1995, when the latter company acquired the industrial enzymes business.
Likewise, the authors originally working for Solvay became Genencor employees
in July of 1996. Consequently, the book now contains many chapters that are
written by Genencor authors; nevertheless, the "history" of those authors ensures
that knowledge of detergent enzymes not only from Genencor International, but
also from both Gist-Brocades and Solvay is represented in this work.
Finally, the editors express their deep gratitude to all the authors involved and
to Joseph Stubenrauch of Marcel Dekker, Inc. for his encouragement and helpful
suggestions. The support of Gist-Brocades in the initial stage of the editing
process and later Genencor International, Cargill B.V., and Business Innovation
Partners B.V. in pennitting the editors to undertake their task is highly
appreciated.

Jan H. vanEe
OnnoMisset
ErikJ. Baas
Contents

Preface iii
Contributors vii

I. Enzymes in Detergency, Past to Present

1. Historical Overview 1
Jan H. vanEe
2. Detergent Enzymes' Market 11
Joel H. Houston
3. Enzymes: Their Applications and Biochemical Characterization 23
ErikJ. Baas, Marc M.P. Bollier. Peter F. Plank, and
Deborah S. Winetzky

II. Enzymes in High Duty Detergents

II.A Proteases
4. Application of Pro teases in Detergents 61
Maarten R. Egmond
5. Development of New Proteases for Detergents 75
Richard Bott

IT.B Lipases
6. Application of Lipases in Detergents 93
Ann M. Wolffand MichaelS. Showell
7. Development of New Lipases 107
OnnoMisset
v
vi Contents

ll.C Cellulases
8. Application of Cellulases That Contribute to Color Revival and
Softening in Detergents 133
Beatrix Kottwitz and Horst Upadek
9. Application of Alkaline Cellulases That Contribute to Soil Removal
in Detergents 149
Eiichi Hoshino and Susumu Ito
10. Development of New Cellulases 175
Karl-Heinz Maurer

ILD Amylases
11. Application of Amylases in Detergents 203
Horst Upadek and Beatrix Kottwitz
12. Development of New Amylases 213
Wolfgang Aehle

DI. Enzymes in Automatic-Dishwashing Detergents

13. Application of Enzymes in Automatic-Dishwashing Detergents 231
H. Magg and G. Wiischenbach

IV. Manufacturing and Safety Aspects of Detergent Enzymes

14. Manufacturing and Downstream Processing of Detergent
Enzymes 251
Hubert A. Hernnann, Ivan Good, and A. Laufer
15. Formulation of Detergent Enzymes 299
Todd Becker, Gene Park, and Alfred L Gaertner
16. Worker Safety: How to Establish Site Enzyme Capability 327
Gordon Peters and D. P. Mackenzie
17. Life Cycle Assessment ofProteases Used in Detergents 341
Michael Bahn and Klaus Intemann

V. Future Developments
18. Outlook and Expectations 359
Jan H. van Ee, Onno Missel, and Erik J. Baas

Index 373
Contributors

Wolfgang Aehle Research and Development, Genencor International B.V.,

Delft, The Netherlands
Erik J. Baas Business Innovation Partners B.V., Amsterdam, The Netherlands
Michael Bahn COGNIS Industrial Consulting GmbH, Dusseldorf, Germany

Todd Becker Genencor International, Inc., Palo Alto, California

Marc M.P. Bollier Genencor International B.V., Delft. The Netherlands

Richard Bott Genencor International, Inc., Palo Alto, California

Maarten R. Egmond Unilever Research Laboratorium, Vlaardingen, The

Netherlands

Alfred L. Gaertner Genencor International, Inc., Palo Alto, California

Ivan Good Research and Development. Solvay Enzymes, Inc., Elkhart,

Indiana

Hubert A. Herrmann Research and Development, Genencor International

GmbH, Nienburg, Germany

Eiichi Hoshino Tochigi Research Laboratories, Kao Corporation, Haga,

Tochigi, Japan

Joel H. Houston Colin A. Houston & Associates, Inc., Mamaroneck, New

York
vii
viii Contributors

Klaus Intemann COGNIS Industrial Consulting GmbH, Dusseldorf,

Germany
Susumu Ito Tochigi Research Laboratories, Kao Corporation, Haga, Tochigi,
Japan
Beatrix Kottwitz Applied Research Detergents, Henkel KGaA, Dusseldorf,
Germany
A. Uiufer Solvay Arzneimittel, Hannover, Germany
D. P. Mackenzie Global Medical, The Procter & Gamble Company, Cin-
cinnati, Ohio
H. Magg Benckiser Productions GmbH, LudwigshafenJRhein, Germany
Karl-Heinz Maurer COGNIS Gesellschaft fiir Biotechnologie mbH, DUssel-
dorf, Germany
Onno Misset* Research and Development, Genencor International B.V.,
Delft, The Netherlands
Gene Park Genencor International, Inc., Palo Alto, California
Gordon Peters Industrial Hygiene, The Procter & Gamble Company, Cin-
cinnati, Ohio
Peter F. Plank Applications Development-Enzyme Cleaning Products,
Genencor International, Inc., Palo Alto, California
Michael S. Showell Fabric & Hard Surface Technology, The Procter &
Gamble Company, Cincinnati, Ohio
Horst Upadek Applied Research Detergents, Henkel KGaA, DUsseldorf,
Germany
Jan H. van Ee Research and Development, Cargill B.V., Amsterdam, The
Netherlands
G. Waschenbach Benckiser Productions GmbH, LudwigshafenJRhein,
Germany
Deborah S. Winetzky Applications Development-Enzyme Cleaning Prod-
ucts, Genencor International, Inc., Palo Alto, California
Ann M. Wolff Fabric & Hard Surface Technology, The Procter & Gamble
Company, Cincinnati, Ohio

*Current affiliation: Gist·Brocades, Delft, The Netherlands.

 ENZYMES
IN
DETERGENCY
1
Historical Overview
JAN H. VAN EE Research and Development. Cargill B.V., Amsterdam,
The Netherlands

I. Introduction 1
II. Prehistoric Times
ITI. From Romans Until Rohrn 4
IV. Modem Times 4
References 9

I. INTRODUCTION
This chapter gives a historical overview of the development of washing habits
and detergent enzyme usage from prehistoric times up to modem times. For
the sake of convenience the most important dates and events are summarized in
Table 1.

II. PREHISTORIC TIMES

In prehistoric times people protected themselves against cold with warm gar-
ments, which basically were made from sheep fleeces. These fleeces can be quite
smelly, especially when wear is in a rather wet climate. Therefore, it was felt
necessary to clean these "garments," one way or the other. This was not an easy
1
2 vanEe

TABLE 1 Historical Overview of Detergent Enzymes Development

Prehistoric times
5000 BC Freshwater scouring in the river
Mud trampling
Feed mechanics
3500BC Soapworth, wood ashes, soda, bat
2100BC Soap from olive oil, wood ash
1000 BC Fat, oil, and natron
From Romans until Rohm
Stale urine presoak, mud trampling
700 Soap from quicklime and fragrances
1700 Soap from fat and soda ash
1900 Soap, silicate, perborate
1928 Alky1sulfates, synthetic detergents
Modern times
1931 Pancreatic enzymes/Burnus; Otto Rohm
1959-1965 Alcalase, Maxatase/Bio 40, Biotex; Schnyder, Kortman
1971 Termamy1, Maxamyl; Novo, Gist-Brocades
Mid-1970s Maxatase L, Alcalase L; Gist-Brocades, Novo
1982-1985 Savinase, Maxacal; Novo, Gist-Brocades
Optic1ean; Solvay
BLAP; Henkel
Purafact; Genencor Int.
1986 Celluzyme; Novo
BPN' protease; P&G-Genencor Int.
1987-1989 KACellulase; Kao
1988 Maxapem; Gist-Brocades
Lipolase; Novo
1989 Durazym:.Novo
1992 Carezym; Novo-P&G
1994-1995 Purafect OxP; Genencor Int.
Lipornax; Gist-Brocades
Lumafast; Genencor Int.
Properase; Gist-Brocades-Genencor Int.

task, because fleeces contain dirt, such as fecal matter, plant remnants, dried
sweat, and in particular (wool) grease, with all the adventitious filth stuck to it.
Originally, primitive humans (read women) took their "clothes" to the river for
freshwater scouring, seeking places with very soft water, because grease removal
in hard water was much more difficult [1].
Historical Overview 3

In about 5000 BC, it was discovered that woolen garments were deaned more
effectively when they had been trampled into the river mud and rinsed several
times afterward to remove the mud, which carried away most grease and the dirt
attached to it. Later on, this water- and clay-based-cleaning process was carried
out by bare-legged women standing in a wide wooden tub, supplying with their
feet the mechanics of this prototype washing machine. Around 3500 Be, it became
common practice to use other "detergent" ingredients, such as soapworth, wood
ashes, and soda, which gave extra-cleaning power, owing to a higher alkalinity.
This resulted in the use of a special bat (Fig. I) to beat the clothes, because the
alkali would have damaged the skin of the feet [ 1].
From the historical Sumerian Lagash, it is known that at about 2100 Be, the
Sumerians, who lived in the watershed of the Euphrates and the Tigris rivers,
made soap from olive oil and wood ash. Before boiling, these two raw materials
were to be mixed in fully described proportions, the recipe of which, in fact,
represents the oldest record of a chemical reaction. The resultant soap was used
mainly for the washing of woolen clothes. In Egypt, the most common detergent,
at about 1000 nc, was a mixture of animal fat or vegetable oil with natron (which
was essentially sodium carbonate found in the Wadi Natrun Desert), optionally
further mixed with a kind of fuller's oil, that seems to have been what nowadays
is called lanolin [ 1].

FIG. 1 Women washing at the river. Woodcut by Hans Franck (1526). (From CIBA
Review 5:2037.) ·
4 vanEe

Ill. FROM ROMANS UNTIL ROHM

The Romans did not have soap. They used to presoak pieces of cloth in stale
urine, which was collected systematically in large vessels placed at the comers of
the streets. Subsequently, the fabrics were trampled on in a vat containing water
and mud, after which several rinses were given with clean water [1]. Not until the
seventh century, did the use of soap, as an important aid for cleaning clothes,
grow significantly. At that time, it was discovered by the Arabs that the use of
quicklime allowed the preparation of much harder soaps. This technology spread
all over Europe, but specifically, in the Mediterranean area the manufacturing of
notably cosmetic soap was blooming, owing to the availability of fragrant plants.
It took until the late 1700s, when new technologies for soda ash manufacturing
had been developed, that soap became available to people of all classes. Soap
powders became very popular in the late 1800s, mainly for reasons of con-
venience; because new mixing methods for soda, silicate, and perborate allowed
one-step washing and bleaching. The next step in the further improvement of
detergents, was to find a better alternative for soap to overcome its biggest
disadvantages, such as the high alkaline reaction and the sensitivity toward hard
water. After a series of attempts, which led to good wetting agents with rather
poor detergency properties, the first synthetic substances with excellent wetting
and detergency characteristics were discovered in 1928 by the sulfation of fatty
alcohols. After this discovery, low-cost quantities of these alkylsulfates rapidly
became available through large-scale chemical reduction of fatty acid esters into
fatty alcohols. By this route commercial volumes of syndets (synthetic deter-
gents) could be produced (about 1930), which had better-cleaning properties than
natural soap.

IV. MODERN TIMES

At the same time, in 1931 to be precise, the original idea of using enzymes was
described by Dr. Otto Rohm. He patented [2] the use of pancreatic enzymes in
presoak detergent compositions, to improve their ability to remove stubborn
proteinaceous stains. Also in that same year, the first enzymatic detergent, named
"Bumus," was launched. Despite the theoretical possibilities, the practical use of
enzymes did not quite become a success because the enzymes could be made
available by extraction of pancreatic glands in only limited amounts. Moreover,
the functional enzymes (e.g., trypsin and chymotrypsin), with their pH optimum
between 7 and 9, are not optimally suited for use in alkaline (presoak) detergents.
Because of these limitations, these products were sold only moderately until the
late 1940s. The first detergent containing a bacterial protease-Bio 40, produced
by Schnyder in Switzerland-appeared on the market in 1959, quickly followed
Historical Overview 5

by the very successful launch of Biotex in 1963, by Korman and Schulte in

the Netherlands (Fig. 2). The enzymes used at that time were alkaline serine
proteases, from the bacterium Bacillus lichenifonnis, market by the Danish com-
pany Novo Nordisk under the trade name Alcalase, and by the Dutch company
Gist-Brocades, under the trade name Maxatase [3]. The better compatibility of
these enzymes with the detergent matrices greatly spurred the development of
other enzymatic detergents, and enzyme sales in the period 1965-1970 grew very
fast. In 1970, this rapid growth of enzyme usage was set back dramatically (see
Fig. 4), especially in the United States, by the strongly negative publicity on the
dustiness of the enzyme formulations, with the concomitant development of
allergies by some workers in detergent manufacturing plants. Because this situa-
tion was taken very seriously by both enzyme producers and detergent manufac-
turers, the problem was solved relatively quickly by the development of reduced-
dust enzyme granulates (Fig. 3), that provided safe handling. Consequently, since
1971, enzyme sales have again increased steadily (Fig. 4), also because of the
application of amylases, such as Maxamyl by Gist-Brocades and Termamyl by
Novo Nordisk, which obtained better removal of carbohydrates, such as food
stains and chocolate.

.
...................... ,.
~-

FIG. 2 Biotex in 1963. (From "100 jaar Kortman Nederland B.V.," 1987.)
6 vanEe

FIG. 3 Maxatase priUs "for a dime."

In the mid-1970s, the share of liquid detergents became more and more impor-
tant, notably in the United States and Europe, leading to the market introduction
of specially developed liquid proteases, such as Maxatase LS and Alcalase L for
application among others in Procter & Gamble's liquid Tide.
In the late 1970s, starting in Japan and Europe, detergents were gradually, but
severely, reformulated to cope with the growth in environmental awareness,
which led to the replacement of phosphates by other builders. Also, to deal with
the energy crisis, the detergent manufactures were forced to develop and incor-
porate low-temperature bleach activators, such as TAGU (Henkel) and TAED
(Lever).
Because of these developments, new proteases were needed that were more
alkaline (pH 9-11), and showed a better performance at 40°-60°C. The answer
came in 1982-1985 by the development and market introduction of the detergent
enzymes Savinase by Novo Nordisk and Maxacal by Gist-Brocades [4] later in
 Historical Overview 7
500

l 450
!.,
! 400

350

300

250

200

150

100

50

0
1964 1969 1974 1919

FIG. 4 Market growth of detergent enzymes.

that decade, followed by the introduction of Opticlean by Solvay, Purafect by

Genencor International, and Blap by HenkeUCognis [5].
In the late 1980s to early 1990s, several major changes in the detergent industry
took place, such as nontower detergent manufacturing (dry mixing of com-
posites), the development of concentrated heavy-duty powder detergents (ini-
tiated in Japan), the development of softening through the wash (STW) deter-
gents, the development of concentrated/structured/nonaqueous liquid detergents,
reformulation of automatic dishwasher detergents, and others. Also, a clear ten-
dency was noted toward cleaning at lower wash temperatures, gradually shifting
from 60° to 40°C, specifically in Europe.
As a consequence, the detergent enzyme industry was faced with several chal-
lenging opportunities to develop new enzymes to fill in the performance gaps that
otherwise would have arisen in these new detergent formulations. Consequently,
a whole series of new detergent enzymes appeared on the market, some manufac-
tured by new entrants in the industry. For instance in 1986 Novo Nordisk intro-
duced a cellulase, called Celluzyme, to improve textile color maintenance and to
8 vanEe

allow softening through the wash [6,7]. About that same year Procter & Gamble
(P&G) introduced a new mild alkaline protease derived from Bacillus amylo-
liquefaciens, specifically suited for boosting the perfonnance of their liquid Tide
[8]. In 1987-1989 Kao, in Japan, introduced their K.AC (Kao alkaline cellulase)
to boost secondary detergency at low temperatures [9,10]. To be compatible with
the high concentration of (low-temperature-activated) bleach in compact deter-
gents, new proteases were designed by new genetic technologies. As a result,
Gist-Brocades in 1988, introduced their Maxapem, which was the first bleach-
resistant detergent protease, obtained through protein engineering, that shows
significantly improved perfonnance, specifically after (household) storage of the
bleach-containing detergents [11-13]. Novo followed about one year later with
their version, called Durazym, to be followed a few years later by Purafect OxP
from Genencor International.
Because of the lower wash temperature, some of the original detergent
ingredients showed reduced cleaning efficacy, especially in removing fatty food
stains and sebum. In 1988, Novo made the first attempt to solve this problem by
the market introduction of a detergent lipase, which they called Lipolase.
Although this enzyme may be used in various detergents, its perfonnance is
heavily dependent on the detergent matrix-surfactant composition, and shows
benefits only after several wash cycles [14]. A few years later (1992), Novo, in
close cooperation with P&G introduced a second-generation cellulase, called
Carezym, with significantly improved color-brightening and softening properties.
In 1994, Gist-Brocades introduced the second-generation lipase, which is
called Lipomax, and which has the capability of efficiently removing fat and
sebum stains in one single-cycle wash procedure [15]. At about that same time the
lipase of Genencor International, called Lumafast, was introduced.
Lately, one of the most exciting new opportunities for the use of detergent
enzymes was the opening up of India, South America, and notably China, as
witnessed by the vast growth in interests that, for example, P&G, Unilever, and
Henkel, have taken in these geographic areas. Because of the specific wash
conditions, such as low surfactant concentration, very low temperature, presoak-
ing, and such, new enzymes were again needed to cope with these rather stringent
circumstances. Therefore, Gist-Brocades in 1994, introduced a new detergent
protease, called Properase, which is very efficient in low-temperature areas and in
low-budget detergents [16,17].
So it is seen that, over the last decade, quite a few new types of detergent
enzymes have entered the marketplace. Today it is not really an exception that
modern types of heavy-duty powder detergents contain one or more different
proteases for protein stain removal, an amylase for starchy food stain removal, a
cellulase for color revival, softening, or secondary detergency, and a lipase for the
efficient removal of fat and sebum stains.
Historical Overview 9

Automatic dishwasher detergents (ADDs) have also changed rather substan-

tially during the last decade. Traditionally, ADDs contained high concentrations
of phosphate (as builder), sodium dichloroisocyanurate (as bleach), and sodium
metasilicate (as bleach stabilizer), having a pH well above 11.5. Again, owing to
growing environmental concern and public awareness, in the early 1980s, ADD
manufacturers were forced to modify their formulations quite fundamentally.
By adapting the good experience with laundry detergents, nowadays ADDs do
contain activated peroxy-bleach, and builders, such as citrate or other polycar-
boxylic salts, and do have a moderate pH. As a consequence, the application of
enzyme technology became possible, leading to modem-type ADDs that contain
proteases and amylases with excellent cleaning properties [18,19].
Likewise, a growth can be noted in the application of enzymes in industrial
and institutional cleaning (1&1), which sectors traditionally use rather strong
chemical cleaning agents, but that are gradually now moving toward more
"enzyme friendly'' cleaning processes.
Therefore, it may be concluded that enzymatic cleaning is broadly applied and
is expected to grow even further, specifically when new functionalities will be
incorporated or even more stressful wash conditions are to be overcome every-
where in the worldwide marketplace. The only way to cope with this challenge
for enzyme producers is to strive to be globally present, as exemplified by the
teaming up, both from a marketing and a research and development point of view,
in the recent acquisition of the detergent enzyme business of Gist-Brocades and
Solvay by Genencor International

REFERENCES
1. R. H. S. Robertson, in Fuller's Earth a History, Voltuma Press, Hythe, Kent, 1986,
pp. 1-82.
2. German Patent DE 283923 to Otto Rohm (1913).
3. C. Dambmann, P.. Holm, V. Jensen, and M. H. Nielsen, How enzymes got into
detergents, Dev. Ind. Microbiol. 12:11 (1971).
4. J. H. van Ee and G. J. Verschoor, J. Com. Esp. Deterg. 17:499 (1986).
5. C. R. Wilson, B. F. Ladin, J. R. Mielenz, S. S. M. Hom, D. Hansen, R. B. Reynolds,
N. Kennedy, J. Schindler, M. Bahn, R. Schmid, M. Markgraf, and C. Paech, PCT
patent appl. World Patent 9102792 (1991).
6. P.. N. Christensen and V. Donalyan, Chim. Oggi 8:41 (1990).
7. P. N. Christensen, K. Thomas, and S. Branner, Development of detergent enzymes,
Proceedings of the Second World Conference on Detergents, American Oil Chemist
Society, 1986, pp. 181-186.
8. M. Showell, Protein engineered detergent proteases, presented at the 82 Annual Meet-
ing of the American Oil Chemist Society, Chicago IL, May 1991.
10 vanEe

9. M. Murata, A new detergency mechanism based on alkaline cellulase, Proceedings of

the Second World Surfactant Conference, CESIO, 1988, pp. 548-553.
10. S. Ito, S. Shikata, K. Ozaki, K. Okamoto, S. Inoue, A. Takei, Y. Ohta, and T. Satoh,
Agric. Bioi. Chern. 53:1275 (1989).
11. J. H. van Ee, L. S. J. M. Mulleners, W. C. van Rijswijk, and R. A. Cuperus, J. Com.
Esp. Deterg. 19:257 (1988).
12. J. H. van Ee, J. Com. Esp. Deterg. 22:83 (1991).
13. J. H. van Ee, Chim. Oggi 9:31 (1991).
14. A. Aaslyng, E. Gormsen, and H. Malmos, SCI, Book of Abstracts on Recent Advances
in the Detergent Industry, Cambridge, 1990, p. 59.
15. M. Crossin and H. B. M. Lenting, Inform 6:473 (1995).
16. J. H. van Ee, J. M. van der Laan, K. P. Broekhuizen, E. J. Baas, M. Bollier, and
L. J. S.M. Mulleners, J. Com. Esp. Deter. 25:123 (1994).
17. J. H. van Ee,J. M. vanderLaan,E.J. Baas, and L. J. S.M. Mulleners,lnform5: p. 542
(1994).
18. J. H. van Ee, W. C. van Rijswijk, and M. Bollier, Chim.-Oggi ]]:21 (1992).
19. J. H. van Ee, W. C. van Rijswijk, and M. Bollier, Proceedings of the Third World
Conference on Detergents, 1993, p. 204.
2
Detergent Enzymes' Market
JOEL H. HOUSTON Colin A. Houston & Associates, Inc., Mamaroneck,
New York

I. Summary 11
II. Introduction 12
A. Evolution: 1960s-1980s 12
B. Second-generation and new enzymes 12
ill. Detergent Market: 1995 14
IV. Detergent Enzyme Markets and Producers 14
A. Products 17
B. Market size 17
V. Conclusion 20

References 21

I. SUMMARY
The market for detergent enzymes has always had periods of rapid growth in
response to the introduction of technical advances to the marketplace. Initially,
rapid growth came through the introduction of protease; recent rapid growth has
been driven by introductions of cellulase and lipase. From 1990 to 1995, the
detergent enzyme market experienced an annual growth rate of nearly 16%; this
translates into a world value of 515 million dollars today. This 515 million dollar
market is expected to continue to grow at a slower rate until the next series of
enzyme innovations are commercialized-then once again the market growth
will escalate rapidly.
11
12 Houston

II. INTRODUCTION
A. Evolution: 1960s-1980s
Since 1961 when Bio 40 was introduced with Novo's Alcalase, detergent
enzymes have proceeded on a tremendous growth track [1,2]. By 1969, at least
50% of European detergents were offering "biological attack"; the usage of which
continued in these detergents at a 50% penetration rate through the 1970s and
reached almost 75% in 1987. In Japan, proteolytic enzymes were introduced by
Lion in 1979, and achieved a great commercial success.
After a rapid start in the United States, enzyme growth suffered a setback in
1971, when factory workers developed health problems, and detergent producers
withdrew the enzymes from their products [1,3,4]. Enzyme manufacturers
remedied the problem by introducing a dust-free, prilled product form, and deter-
gent manufacturers introduced effective dust-handling systems [1,3]. Detergents
with enzymes remained a minor factor in the U.S. detergent market through the
1970s and into the early 1980s, with only 5-6% of detergents containing enzymes
in 1982.
The resurrection of enzymes began in the early 1980s [2] with
1. Procter & Gamble's new Era Plus liquid-containing enzymes, which used a new
nonphosphate builder system, went into test market in 1982.
2. Other new product forms, concentrated powders such as Colgate-Palmolive's
Fresh Start and Procter & Gamble's Ariel, containing enzymes were then
offered in the United States.
3. Procter & Gamble's liquid Tide was introduced in 1985 with enzyme, and
powder Tide with its 23% market share was considered a candidate for conver-
sion to enzymes.
4. By 1986, the U.S. share of detergents using enzymes had leapt to over 40%,
up from the 15-20% share held in 1984.
Although Colgate-Palmolive and Procter & Gamble embraced enzymes in the
early 1980s, Lever Brothers held back its flagship brands for several years-both
in the United States and the United Kingdom. Protease enzymes' next major U.S.
development was full acceptance by Lever, which finally came in the late 1980s.

B. Second-Generation and New Enzymes

In the early 1980s, lipase enzymes were identified for fat splitting, and these were
introduced fll'St in detergents in Japan in 1988 and into Europe in April1990. In
1987 in Japan, Kao had achieved great success with the first alkaline cellulase for
detergents [4]. Europe saw some use of cellulase in detergents in 1990, but they
remained a novelty through 1991. The use of cellulase in the United States did
Detergent Enzymes' Market 13

not appear until 1993. Cellulase was initially proposed as a product for fabric
softeners in the 1960s, but found its first success in providing antipilling and
color revival in laundry detergents. In 1993, relatively high dosages of enzymes
were seen in bleach-alternative liquid products. The bleach alternative with high
levels of enzyme blends marked the beginning of the current period, which is
noted for the popularity of enzyme "cocktails."
Another growth market has been European automatic dishwasher detergents
(ADDs); beginning in 1990, many have been reformulated to contain enzymes
[5]. The shift in ADD formulations was driven by the need to reduce the use of
harsh sodium silicates, and this became an opportunity to address other issues.
European formulators moved to abandon phosphate and chlorine in the new
formulations. Europeans were able to eliminate phosphate because, unlike other
regions, separate water-softening devices based on ion-exchange are included in
their machines. Efforts to introduce similar formulations in the United States have
been largely ineffective because the United States does not have the ion-exchange
capacity in its machines. New attempts to broaden the use of enzymes in U.S.
dishwashing can be expected.
In the next decade, there is likely to be a continuation of the decline in clothes
wash temperatures [6]. Wash temperatures fell in Europe in the 1970s and 1980s
and consumers moved away from 95°C washes. Temperatures of 40° and 60°C
are now common, but further declines are expected. In the United States, efforts
are underway to lead machine manufacturers to lower-temperature wash stan-
dards [7]. This could come through legislation, or through a market pull approach
from utilities or other groups. With lower temperatures, reliance on enzymes,
such as lipase, to remove fatty soils will increase as existing chemical systems
have difficulty with these stains at lower temperatures [8]. The other types of
enzymes are also likely to benefit.
Other consumer developments are expected to drive greater reliance on
enzymes in detergents. With the decline in time available for housekeeping
chores owing to dual-career households, expectations are rising for detergents to
remove problem stains without special treatments. Furthermore, with the rise in
the cost of garments, consumers want to see extended use and, hence, will expect
less aggressive detergents, for instance ones operating at lower pH, or that contain
"color guard" systems.
Recently, the practice of formulating with multienzyme laundry detergent
packages has increased the use of amylase, an enzyme that works on starches and
one that has been available for years. Use of cellulase in fabric softener products
may be possible in the near future. The introduction of peroxidase enzymes,
first to prevent dye transfer, and second possibly to provide bleaching, may be
expected.
14 Houston

Ill. DETERGENT MARKET: 1995

The laundry detergent markets are widely varied from area to area. In all,
17.9 million tons of laundry detergent products will be consumed in 1995.
Europe and North America represent half of the market, but only about 12%
of the population. Powder produEts predominate, whereas liquids (Table 1)
hold about 10% of the market. Other product forms include detergent pastes
and bars.
Regionally, Western Europe is the most important market, having the highest
value, [five times higher dosage, see Chap. 3], the most competition, and the most
developed products. North America and Europe are well-developed markets,
characterized by low growth, equivalent to that of population growth. In the other
regions, Eastern Europe holds the largest portion, representing over half of the
total for the area (Fig. 1).
Other regions represent the important areas where growth is based on increased
per capita use and the substitution of soap. Laundry soap represents nearly a
5 million ton market worldwide and is important still in Asia. Over the next 15
years, the substitution of soaps will provide an important engine for growth to
detergent and enzyme makers.
Enzymes are also found in other detergent products, ranging from automatic
dishwasher detergents to dry bleaches, stain removers, and boosters. Autodish
products are found largely in Europe and North America, where together they are
a 700,000 ton market. Dry bleaches are primarily found in North America and
represent an annual market of over 100,000 tons.

IV. DETERGENT ENZYME MARKETS AND

PRODUCERS
Novo Nordisk developed the frrst alkaline-stable enzyme, Alcalase, in 1958. It
was developed for meat and fish packers, and since 1961-1962, when it was first
used in laundry applications, Novo has held the leading market share [1,9]. Today
Novo holds over 50% of the 515 million dollar detergent market (Fig. 2). Other

TABLE 1 World Laundry Detergents:

1995 (million tons)
Heavy-duty powders 6,500
Heavy-duty liquid 1,700
Others and unspecified 9,700
Total 17.900
Detergent Enzymes' Market 15
West Europe 19%

Latin America 10% ~orth America 17%

Other Regions 20%

FIG. 1 Regional laundry detergent markets (1995) on a volume basis: Total =

17.9 million tons.

Novo 57%

Other 4%

Showa Denko 6%

FIG. 2 Detergent enzyme merchant producer shares: 1995. (From Colin A. Houston &
Associates estimates.)
16 Houston

producers include Genencor International, Solvay, and Showa Denko. There is

captive protease production by Henkel and Kao. Kao also makes and sells an
alkaline cellulase.
Although Genencor International is a modem, technology-driven company,
it has a long history in enzymes, with roots tracing back to Rohm and Haas
Enzymes, which was sold to Coming Glass in 1981. In 1982, Genentech and
Coming created a new company, Genencor. In 1984, A. E. Staley made a substan-
tial investment in Genencor to become a partner with Coming and Genentech.
Eastman bought Staley's share and Genencor became part of Eastman Kodak. In
1990, Cultor, the former Finn Sugar, joined Eastman. to form Genencor Inter-
national. In 1995, Genencor International purchased portions of Gist-Brocades
N.V.'s industrial enzyme business, including the detergent enzyme sector. Gist-
Brocades is a classical company driven by fermentation, similar to Novo Nordisk.
In December 1995, Genencor International announced that it had reached an
agreement to purchase the enzyme business of Solvay [10).
There were earlier consolidations within the industry. Solvay purchased
Miles Enzymes in 1990 and added it to its Kali Chemie enzyme business in
Europe.
Another interesting feature of this market is the captive positions held by
Henkel and Kao. Henkel produces and uses its enzymes primarily in the
European region, but does some research in the United States. Kao has developed
materials captively and is now looking for merchant sales opportunities. Kao was
the first to succeed with an alkaline cellulase and now offers these products to
other detergent producers.
Enzyme research is difficult and expensive. Close cooperation between cus-
tomer and supplier is often required. At times Novo has worked with Unilever to
develop protease, lipase, and cellulase products for detergents. A cellulase was
developed years later in another joint effort, this time between Novo and Procter
& Gamble (P&G). Today Novo's arrangement with P&G on cellulases with
exclusive rights to Novo's Carezyme package is resulting in significant benefits.
Between February 1995 when P&G introduced this technology, and October
1995, the Tide brand gained 3 percentage points to reach a 30% share of U.S.
laundry detergent sales. Increasingly, producers are finding the development of
new enzymes to be risky and costly; therefore, they have increased the success
potential of projects through joint development agreements. The need to differen-
tiate laundry products remains high and leaves detergent producers very willing
to support research for one of the leading formulating technologies.
Today, production facilities are sited around the world, Novo has four locations
and Genencor has four. Solvay produces in Germany, the United States and
Argentina. Novo, Kao, and Showa Denko are in Japan, and Henkel is in Austria.
Table 2 lists the production locations of the producers.
Detergent Enzymes' Market 17

TABLE 2 Enzyme Production Facilities

Producer Locations Processes

Novo Nordisk Kalundborg, Denmark Fermentation, granulation

Granldinton, North Carolina Fermentation, granulation
Hokkaido, Japan Fermentation
Aravcaria, Brazil Fermentation, granulation
Genencor International Cedar Rapids, Iowa Fermentation, granulation
Brugge, Belgium Fermentation, granulation
Hanko, Finland Fermentation, graimlation
Jamsankoshi, Finland Fermentation, granulation
Solvay Elkhardt, Indiana Fermentation
Arroyito, Argentina Fermentation
Nienburg, Germany Fermentation, granulation
Showa Denko Ohito, Japan Fermentation, granulation
Bioz.ym BV (HenkeVSandoz.) Kundl, Austria Fermentation, granulation
Kao Wakayama, Japan Fermentation, granulation

A. Products
Each of the producers tends to have a broad product line and offers a range of
strengths for each type of enzyme. Other variations include products targeting
different operating conditions. For instance, some products operate well at a pH
of 11, whereas others are designed to operate better at pH 9. Additional features
include improved bleach or color stability [11]. Coating systems for liquid
products are another point of differentiation. The strength of the enzymes is
measured by proprietary methods that prevent direct comparison of enzymes
between producers. Table 3 indicates the trade names offered by the producers by
enzyme type.

B. Market Size
In the 1960s, enzymes experienced phenomenal growth and, in fact, were the
performance growth engine to the detergent industry that optical brighteners had
been in the 1950s [2]. The 1970s were a disappointment in the United States, and
European enzymes held a steady 50% of detergents through the decade. The
1980s saw strong growth in the United States and Japan and from the introduction
of new types; cellulase and lipase. European use of enzymes reached 75% of
detergents during the 1980s and is at 95% today [9]. In another development, the
encapsulation of enzymes opened up the liquid detergent market as an important
new outlet. Cellulase and lipase enzymes did not really affect the Western
 .....
CD

TABLE3 Enzyme Product Names by Type and Supplier: 1995

Protease Amylase
--
High Bleach- Cold Bleach-
Producer Alkaline alkaline stable water Conventional stable Lipase Cellulase

Merchant market
Novo Nordisk Alcalase Savinase Durazym - Termarnyl Durarnyl Lipolase Carezym
Esperase - - - - Lipolase ultra Celluzyme
Genencor Maxatase Maxacal Maxapem Properase Maxarnyl Purafect OxAm Lipomax
International -Optimase Purafect Purafect OxP - - - Lumafast
Solvay Opticlean Opticlean+ - ArnylaseMT
ShowaDenko - Kazusase
Captive market
Kao - KAP - - KAA - - KAC
Henkel Biozym Blap Blap+

%
0

i
::J
Detergent Enzymes' Market 19

markets until the 1990s, when they provided an important growth driver
[8,12,13]. Today, enzymes are the foremost performance tool available to for-
mulators and are the basis of strategic initiatives by the detergent producers.
World use of detergent enzymes is reported to have reached over 250 million
dollars in 1990 [12]. Today, the market is 515 million dollars, a growth of
nearly 16% per year. This unusually high growth is due to the addition of the
autodish market and to the new functionalities provided by lipase and cellulase
finding use in Western markets. Such growth is unlikely to be repeated in the
near future.
Geographically, the use of enzymes is shown in Figure 3. There is a high level
of penetration of enzyme detergents in most areas, including Europe, Japan, and
North and South America. Penetration in the United States is 75%, compared
with 95% for Europe and Japan. The high value in Europe is mainly due to high
levels of detergent use and high enzyme concentrations (see Chap. 3).
The mainstay of the market has been the protease types. fu the United States,
these products have been evolving towards "pH-neutral" types. In Europe, the
pH of systems remains relatively high. Protease prices have been declining, but
with increased usage, the total market value has held steady in most areas [9].
Proteases now offer very good performance value that has been seized on in the
introduction of "bleach-altemati ve" formulations.

West Europe $185

FIG. 3 Regional detergent enzyme market values: 1995, in millions of dollars.

20 Houston

The value of protease sales worldwide is in the region of 320 million dollars.
Lowest concentration proteases sell for $1.60-$2.00flb. Typically, pricing
depends on
Concentration
Form
Market location
Exchange rates
Customer sizes
Level of competition
The determination of the lipase and cellulase markets size is awkward because
of proprietary arrangements stemming from enzyme producers and consumers
undertaking joint development efforts. Lipases have grown rapidly since 1990
[8,13] to reach an estimated 55 million dollars today. Cellulase enzymes, in
contrast with lipase, have grown more rapidly since 1993 and are estimated to
have reached 110 million dollars in 1995. Lipases sell for over $4.00flb and the
market is dominated by Novo. Gist-Brocades/Genencor International has intro-
duced a new, more active lipase (Lipomax) product that has generated great
interest.
Table 4 breaks out regional market segments by type of enzyme. It is clear from
the table that enzymes in detergents have become an important market. Given
the speed of growth by the new materials-lipase and cellulase-considerable
growth potential remains. Cellulase and lipase growth should continue to drive
the market for several more years.

V. CONCLUSION
Today, enzymes have gained wide approval and are used in detergents throughout
the world. Healthy growth is forecast for enzymes, and new developments in

TABLE4 Detergent Enzyme Markets by Type and Region: 1995 (million U.S.
dollars)

Total Protease Lipase Cellulase Amylase

United States 140 80 10 40 10

Western Europe 185 100 20 45 20
Japan 65 30 15 20
Others 125 110 10 5
Total 515 320 55 110 30
Source: Colin A. Houston & Associates estimates.
Detergent Enzymes' Market 21

enzymes are anticipated to continue to drive periods of rapid growth as detergent

producers rely more heavily on the remarkable effectiveness of enzymes to
release stains and certain soils under increasingly difficult wash conditions.
The development of new enzymes will be well received by consumers as they
continue to favor easy-care developments, which allow increased time for friends
and family.

REFERENCES
1. P. Brodeur, in Asbestos and Enzymes, Ballantine Books, New York, 1971.
2. L. H. Dalgaard, et al., Dishwashing-a new area for enzymes. Inform 2:37 ( 1991 ).
3. P. Layman, Promising new markets emerging for commercial enzymes. Chern. Eng.
News Sept. 24:17 (1990).
4. New! improved! no kidding! Forbes, Mar. 7:154 (1988).
5. C. A. Starace, Detergent enzymes: developments during the last decade. J. Am. Oil
Chern. Feb:165 (1981).
6. C. A. Starace, Detergent enzymes: Past, present and future. HAPPI, Mar:49 (1983).
7. Genencor to buy solvay enzyme business. Chern. Market. Rep. Dec 4:5 ( 1995).
8. U. Hotz, Verwendung von Cellulasen in Waschmitteln. Tenside 30:388 (1993).
9. D. Kochavi, Enzymes for household detergents. Inform, Sept:990 (1993).
10. E. D'Amico, Technological advances help market penetration. Chern. Week, Oct.
25:34 (1995).
11. Gist-Brocades opens new enzyme plant. Inform Feb:200 (1995).
12. M. Ward, Expanding role of enzyme culture. Eur. Chern. News Feb. 20:24 (1995).
13. J. DiCorpo, Global visions. Chern. Market. Rep. Jan. 16:18 (1995).
3
Enzymes: Their Applications and
Biochemical Characterization
ERIK J. BAAS Business Innovation Partners B.V., Amsterdam, The Netherlands
MARC M.P. BOLLIER Genencor International B.V., Delft, The Netherlands
PETER F. PLANK and DEBORAH S. WINETZKY Applications
Development-Enzyme Cleaning Products, Genencor International, Inc.,
Palo Alto, California

I. Enzyme Biochemistry 24
A. Enzymes are proteins 24
B. Enzyme-substrate interaction: mechanism and kinetics 25
C. Biochemical assays: enzyme efficacy determination 29
D. Enzyme development 33
II. Areas of Enzyme Applications 34
A. Enzymes used in the detergent industry 35
B. Commercial detergent enzymes and their applications 39
ill. Detergent Market Information 42
A. Geographic washing conditions 42
B. Detergent formulation trends 42
C. Enzymatic stability considerations 44

IV. Enzyme Wash Performance Testing 46

A. Laboratory-controlled detergent evaluations 47
B. Enzyme-cleaning performance evaluations 48
C. Consumer testing 52
V. Conclusions 54
A. Trends in the detergent industry 54
B. Enzymatic activity versus performance evaluation and the
formulator 54

23
24 Baas et al.

Appendix l. Enzyme Safety Considerations or Product Stewardship 55

A. Possible health effects 55
B. Control technology 56

References 57

I. ENZYME BIOCHEMISTRY

A. Enzymes Are Proteins

Proteins are one of the most versatile and diverse group of biopolymers found in
nature. Composed from a limited set of building blocks-the 20 amino acids-a
wide variety of proteins can be made. Proteins have numerous functions within
living organisms, ranging from maintenance of cell structure, to nutrient transport
and communication, to control of the biochemical processes that are essential
for life.
Enzymes are a class of proteins that function as biocatalysts. They are involved
in the formation and degradation of all biological substances. The biocatalytic
process will be described in more detail in the sections that follow.
The basic structure of all proteins, including enzymes, is the peptide chain,
which is a liner array of amino acids. The amino acids are linked together by a
peptide bond, coupling the carboxylic acid (COOH) and amino (NH2) residues
between two peptides. The peptide bond (-C(O)-NH-) and its subsequent protease
hydrolysis products are depicted in Fig. 1.

0 0
II II
-N-CH -N CH-c-o-
1 I I H

I H20
#
H

FIG. 1 Peptide bond and protease hydrolysis products. (Courtesy of Dr. Raj Lad,
Genencor International, Inc., Palo Alto, CA.)
Enzymes: Applications and Biochemistry 25

Each amino acid has specific chemical characteristics, determined by its

appended side chain. Protein characteristics are determined not only by the amino
acid side chains, but also by the three-dimensional structure of the polypeptide
chain. For a protein to fulfill its biological function, it is essential that it has a
specific, well-defined structure. Any disturbances in this structure may result in
denaturation, which can lead to reduced activity.
Microbial enzymes vary in length from 100 to 500 amino acids, and typically
have a molecular weight ranging from 25,000 to 50,000 Da. The active site of the
enzyme is a specific three-dimensional area, within the folded enzyme, involved
in substrate binding and the biocatalytic process. In certain cellulases, there is
another distinct region involved specifically in substrate binding, which is
referred to as the binding domain.
Enzymes are characterized by the biochemical reactions they catalyze (e.g.,
proteases catalyze the hydrolysis of the peptide bonds in proteins and lipases
catalyze the hydrolysis of ester bonds in triglycerides and fats). Table 1 depicts
these enzyme classifications [1] as specified by the International Union of
Biochemistry (IUB).

B. Enzyme-Substrate Interaction: Mechanism and

Kinetics
Enzymes are biocatalysts that facilitate biochemical transformations. They allow
unfavorable biochemical reactions to proceed more efficiently. Enzymes carry
out highly specific biochemical transformations, yet remain unchanged during

TABLE 1 Enzyme Classification

IUB classification Mechanism of action

Oxidoreductases Catalyze oxidation/reduction reactions (e.g., glucose oxidase)

Transferases Catalyze transfer of a functional group between molecules
Hydro lases Catalyze the addition of water across a bond (e.g., protease,
lipase, a-amy lase, and cellulase)
Lyases Catalyze the addition of a functional group to a double bond
or generate double bonds (e.g., pectin lyase [transelirninase])
Isomerases Catalyze the isomerization or rearrangement of a molecule
(e.g., glucose isomerase)
Ligases (synthetases) Catalyze the breaking or formation of two molecules
concomitant with cleavage of a nucleoside triphosphate
(e.g., glutamine synthetase)
Source: Ref. 1.
26 Baas et al.

Uncatalyzed Reaction
Catalyzed Reaction

Substrate
Product

Reaction time

FIG. 2 Thermodynamic reaction profile. (From Ref. 1.)

this process. Enzymes increase the rate of biochemical conversion of substrate [S]
to product [P] by decreasing the free energy of activation barrier (AG*); Fig. 2).
'I}'pically, enzymes can increase the rate of reaction 1-millionfold or more. In the
absence of enzymes, few biochemical reactions would proceed at all.
The molecular basis of catalysis is dependent on the stabilization of the
enzyme-substrate complex [ES] formed at the enzyme's active site during the
catalytic reaction (Figs. 2 and 3). Figure 4 depicts the tetrahedral intermediate
formed by the enzyme-substrate interaction.
The [ES] complex is stabilized both electrostatically and through van der Waals
attractive forces attributable to the amino acid side chains present at the enzyme's
active site. The formation of the enzyme-substrate complex helps overcome the
thermodynamic barrier depicted in Fig. 2.

1. Protease-Hydrolase Reaction Mechanism

The overall hydrolysis reaction for a protein by a serine endopeptidase is depicted
in Figure I. The serine endopeptidase contains a catalytic triad of amino acids at
the active site: an aspartyl residue containing a ~-Coo-: a histidine containing
the imidazole group; and a serine residue with a ~-OH as a functional group. The
serine hydroxyl group functions as a potential nucleophile, whereas both the

E+S [ES] p
k.{
FIG. 3 Simple enzyme-substrate reaction mode. (From Ref. 1.)