MODULE 1 IN

ENZYME TECHNOLOGY

CHE EL 62

Department of Chemical Engineering

SCHOOL OF ENGINEERING AND ARCHITECTURE

 REF SEA-BSCHE-CHEEL62-2021

MODULE 1:
INTRODUCTION TO ENZYME TECHNOLOGY

At the end of this module you should be able to:

 Define an enzyme, its structure, nomenclature, and major function/s. And
for each type of enzyme, their specific function and classification
according to the type of chemical reaction.
 Discuss the mechanisms in enzyme preparations as well as their sources
and forms which they are used for.
 Explain the techniques involved in Enzyme Immobilization.
 Define a mathematical model for the kinetics of immobilized enzymes
and to be able to express this rate in terms of Michaelis-Menten's
equation.

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 Unit 1:
Enzymes and their Functions

Engage

Biotechnology offers an increasing potential for the production of goods to meet

various human needs. In enzyme technology – a subfield of biotechnology – new processes
have been and are being developed to manufacture both bulk and high-added value
products utilizing enzymes as biocatalysts, in order to meet needs such as food and
pharmaceuticals. Enzymes are also used to provide services, as in washing and
environmental purposes, or for analytical and diagnostic purposes.

The driving force in the development of enzyme technology, both in academia and
in industry, has been and will continue to be the development of new and better products,
processes, and services to meet these needs, and/or the improvements of processes to
produce existing products from new raw materials such as biomass.

Explore

Enzymes and Life Processes

The living cell is the site of tremendous biochemical activity called metabolism. This is
the process of chemical and physical change which goes on continually in the living
organism. Build-up of new tissue, replacement of old tissue, conversion of food to energy,
disposal of waste materials, reproduction – all activities that we characterize as “life”.

This building up and tearing down takes place in the face of an apparent paradox.
The greatest majority of these biochemical reactions do not take place spontaneously. The
phenomenon of catalysis makes possible biochemical reactions necessary for all life
processes. Catalysis is defined as the acceleration of a chemical reaction by some
substance which itself undergoes no permanent chemical change. The catalysts of
biochemical reactions are enzymes and are responsible for bringing about almost all of the
chemical reactions in living organisms. Without enzymes, these reactions take place at a
rate far too slow for the pace of metabolism.

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 Why Enzymes?

Catalysts increase the rate of otherwise slow or imperceptible reactions without

undergoing any net change in their structure. The early development of the concept of
catalysis in the 19th century went hand in hand with the discovery of powerful catalysts from
biological sources. These were called enzymes.

An enzyme is a biological catalyst that speeds up essential biochemical

reactions. Without enzymes, biological reactions would occur at only a
very slow rate.

Enzymes mediate all synthetic and degradative reactions carried out by living
organisms. They are very efficient catalysts, often far superior to conventional chemical
catalysts, for which reason they are being employed increasingly in today’s high-
technological society, as a highly significant a wide diversity of industrial processes,
consumer products, and the burgeoning of field biosensors. Further applications are being
discovered significantly.

Enzymes have a number of distinct advantages over conventional chemical

catalysts.
1. Specificity and Selectivity
2. The product generated is in an uncontaminated state
3. Works at mild processing conditions
4. High reaction velocities

There are some disadvantages in the use of enzymes which cannot be ignored but
which are currently being addressed and overcome.
1. High cost of enzyme isolation
2. Unstable nature

Historical Background and Scientific Foundations

The existence of enzymes has been known for well over a century.
 Some of the earliest studies were performed in 1835 by the Swedish chemist Jon Jakob
Berzelius who termed their chemical action catalytic.
 Enzymes in wild yeasts have been used for thousands of year in brewing and wine
making.

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  Modern enzyme technology began with Danish chemist Christian Hansen with his
isolation of rennet from dried calves’ stomachs in 1874. Rennet is actually a mixture of
enzymes that is applied in the coagulation of milk proteins to make cheese.

Molecular Structure of Protease Enzyme in Rennet

Digital Image. May 2012, https://www.indiamart.com/proddetail/proteases-enzyme-6710533033.html

 Buchner first extracted active enzymes from living cells in 1897. The experiment for
which Buchner won the Nobel Prize consisted of producing a cell-free extract of yeast
cells and showing that this “press juice” could ferment sugar. The cell-free extract was
produced by combining dry yeast cells, quartz and kieselguhr and then pulverizing
the yeast cells with a pestle and mortar. This mixture would then become moist as the
yeast cells’ contents would come out of the cells. Once this step was done, the moist
mixture would be out through a press and the resulting “press juice” had glucose,
fructose, or maltose added and carbon dioxide was seen to evolve. Microscopic
investigation revealed no living yeast cells in the extract.
 In the 20th century, increasing application of enzymes in the food industry emerged.
In Japan, mold fungi are used to make a range of products based on soy protein,
such as miso and tempeh.
 Enzymes also proved to be useful for desizing textiles. Bacterial amylase, derived from
Bacillus subtilis, was derived for this purpose in 1917.

Bacillus Subtilis
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development-size-share-and-covid-19-impact-analysis-2025/amp/

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  German industrialist Otto Röhm studied the role of enzymes in the bating of leather,
which traditionally was carried out with dog and pigeon excrement. He argued that
it was the digestive enzymes in the excrement that helped prepare the leather and
began to use pancreatic extracts instead, with successful results. He began marketing
it as Oropon in the early 1900s.
 In 1920, Sumner was able to successfully isolate a pure enzyme. The enzyme he
worked with was urease, which he isolated from jack beans. He accomplished this by
mixing purified urease with acetone and then chilling it; the chilled solution produced
crystallized urease. He was also able to show by chemical tests that his pure urease
was a protein. This was the first experimental proof that an enzyme is a protein, a
controversial question at the time.
 John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical
Research shared the 1947 Nobel Prize with Sumner. They discovered a complex
procedure for isolating pepsin. This precipitation technique devised by Northrop and
Stanley has been used to crystallize several enzymes.
 The first purified industrial enzyme was a bacterial protease, first marketed in 1959 and
employed detergent manufacturers beginning in 1965.

Enzyme Nomenclature

All enzymes contain a protein backbone. In some enzymes this is the only component
in the structure. However, there are additional non-protein moieties usually present which
may or may not participate in the catalytic activity of the enzyme. Covalently attached
carbohydrate groups are commonly encountered structural features which often have no
direct bearing on the catalytic activity, although they may well effects an enzyme’s stability
and solubility. Other factors often found are metal ions (cofactors) and low molecular weight
organic molecules (coenzymes). These may be loosely or tightly bound by noncovalent or
covalent forces. They are often important constituents contributing to both the activity and
stability of the enzymes. This requirement for cofactors and coenzymes must be recognized
if the enzymes are to be used efficiently and is particularly relevant in continuous processes
where there may be a tendency for them to become separated from an enzyme’s protein
moiety.

Enzymes are classified according to the report of Nomenclature Committee

appointed by International Union of Biochemistry (1984). The enzyme commission assigned
each enzyme a recommended name and a 4-part distinguishing number. It should be
appreciated that some alternative names remain in such common usage. The enzyme
commission (EC) numbers divide enzymes into six main groups according to the type of
reaction catalyzed:

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 (1) Oxidoreductases which involve redox reactions in which hydrogen or oxygen atoms
or electrons are transferred between molecules. This extensive class includes the
dehydrogenases (hybride transfer), oxidases (electron transfer to molecular oxygen),
oxygenases (oxygen transfer from molecular oxygen) and peroxidases (electron
transfer to peroxide).
Example: Glucose oxidase
EC 1.1.3.4
b-D-glucose:oxygen 1-oxidoreductase

(2) Transferases which catalyse the transfer of an atom or group of atoms (e.g. acyl-,
alkyl- and glycosyl-), between two molecules, but excluding such transfers as are
classified in the other groups (e.g. oxidoreductases and hydrolases).
For example: Aspartate aminotransferase
EC 2.6.1.1
L-aspartate:2-oxoglutarate aminotransferase; glutamic-
oxaloacetic transaminase (GOT)

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 (3) Hydrolases which involve hydrolytic reactions and their reversal. This is presently the
most commonly encountered class of enzymes within the field of enzyme
technology and includes the esterases, glycosidases, lipases and proteases.
For example: Chymosin
EC 3.4.23.4
Rennin

(4) Lyases which involve elimination reactions in which a group of atoms is removed
from the substrate. This includes the aldolases, decarboxylases, dehydratases and
some pectinases but does not include hydrolases.
For example: Histidine ammonia-lyase
EC 4.3.1.3
L-histidine ammonia-lyase; histidase

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 (5) Isomerases which catalyse molecular isomerisations and include the epimerases,
racemases and intramolecular transferases.
For example: Xylose isomerase
EC 5.3.1.5
D-xylose ketol-isomerase ; glucose isomerase

(6) Ligases, also known as synthetases, form a relatively group of enzymes which involve
the formation of a covalent bond joining two molecules together, coupled with the
hydrolysis of a nucleoside triphosphate.
For example: Glutathione synthase
EC 6.3.2.3
g-L-glutamyl-L-cysteine:glycine ligase ; glutathione synthetase

Chemical Nature of Enzymes

All known enzymes are proteins. They are high molecular weight compounds made
up principally of chains of amino acids linked together by peptide bonds.

Enzymes can be denatured and precipitated with salts, solvents and other reagents.
They have molecular weights ranging from 10,000 to 2,000,000.

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 Typical Protein Structure – two amino acids joined by a peptide bond

Many enzymes require the presence of other compounds – cofactors – before their
catalytic activity can be exerted. This entire active complex is referred to as holoenzyme;
i.e., apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-
ion-activator) is called holoenzyme.

Holoenzymes – apoenzymes plus various types of cofactors

The cofactor may be

1. A coenzyme – a non-protein organic substance which is dialyzable , thermostable
and loosely attached to the protein part.
2. A prosthetic group – an organic substance which is dialyzable and thermostable
which is firmly attached to the protein or apoenzyme portion.
3. A metal-ion-activator – these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++,
Ca++, and Mo++.

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 The catalytic ability of enzymes is due to its particular protein structure. A specific
chemical reaction is catalyzed at a small portion of the surface of an enzyme, which is
known as the active site.

The Active Site

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Specificity of Enzymes

One of the properties of enzymes that makes them so important as diagnostic and
research tools is the specificity they exhibit relative to the reactions they catalyze. A few
enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction.
Other enzymes will be specific for a particular type of chemical bond or functional group. In
general, there are four distinct types of specificity:

1. Absolute specificity - the enzyme will catalyze only one reaction.

2. Group specificity - the enzyme will act only on molecules that have specific functional
groups, such as amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

Though enzymes exhibit great degrees of specificity, cofactors may serve many
apoenzymes. For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a
great number of dehydrogenase reactions in which it acts as a hydrogen acceptor. Among
them are the alcohol dehydrogenase, malate dehydrogenase and lactate dehydrogenase
reactions.

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 The Structure of Enzymes

Enzymes always act as catalysts and small quantities compared to their substrate are
required to considerably increase the rate of chemical reactions, wherein the enzymes
themselves experience overall change. In contrast to all true catalysts, an enzyme does not
alter the ultimate equilibrium position of a reaction, which is thermodynamically determined,
thus merely the rate of completion of equilibrium of a feasible reaction is augmented. In
addition to catalytic properties, enzymes exhibit the physic-chemical behaviour of proteins:
their solubility, electrophoretic properties, electrolytic behaviours and chemical reactivity.
The primary structural configuration and catalytic action of enzymes is determined by the
linear chain of amino acid residues linked via peptide bonds, which constitute a protein
molecule. Localized folding of the primary structure is called a secondary structure, whereas
the complete folding of the molecule is known as a tertiary structure. In contrast to these
structural configurations, a quaternary structure is the agglomeration of several folded
chains. The structural features of enzymes are shown in the figure below.

Digital Image. Biologydictionary, https://biologydictionary.net/ap-biology/3-1-enzyme-structure/

Enzyme Structure
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 In contrast to traditional chemical catalysts, e.g. hydrogen ions, heavy metals or
metal oxides, which are most effective in organic solvents, at very high temperatures or at
extreme pH values, enzymes operate most efficiently under very mild conditions. When using
enzymes, there are certain issues that require attention, such as deviation from
homogeneous aqueous solutions, physiological pH and temperature, which can rapidly
destroy enzyme activity. However, under normal conditions the increase in reaction rate is
rarely matched by their non-protein counterparts.

Explain

Impact and Issues

Because the natural environment of enzymes is the living cell, they catalyze reactions
in physiological conditions, which usually means near neutral pH, aqueous solution, and at
temperatures in a narrow range around 98.6°F (37°C). Replacing more conventional
processes with one based on a biocatalyst often makes it more environmentally friendly by
reducing energy inputs and waste. For instance, use of biological detergent enables laundry
to be washed at a much lower temperature than with a soap-based detergent. However, it
is in the chemical and pharmaceutical industries that biocatalysts have most to offer over
traditional catalysts. Production processes requiring several steps often can be streamlined,
which cuts down on raw materials and waste. This may go hand in hand with the elimination
of one or more reactions that require a toxic solvent, replacing it with one that can be utilized
in aqueous solution.

In drug manufacturing, the use of biocatalysts has the advantage that it can produce
a chiral drug molecule, in which all the carbon atoms are in the correct orientation in space.
The three-dimensional structure of an enzyme drives its specificity in a way that does not
apply to simple metal catalysts. The enzyme will produce the correct chiral form of the drug
from intermediate molecules in a very selective way. The importance of having the correct
chiral form of a drug was revealed by the thalidomide tragedy of the 1960s. Babies born to
mothers taking this drug suffered from congenital birth defects, and the drug had to be
withdrawn. It was later discovered that one of the chiral forms of thalidomide caused the
damage, while the other form was the one that had the therapeutic impact on the patient.
Increasingly, regulators demand that manufacturers minimize harm to the patient by making
the correct chiral form of the drug only.

The introduction of enzymes into industrial processes is limited by their availability.

Around 3,000 enzymes are known, but only a tiny proportion is available in amounts large
enough and cheap enough to allow them to be used on a large scale. Genetic engineering
processes are complex and costly to develop, so most enzymes have not yet been exploited

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 in this way. Even when an enzyme is available, it may not be appropriate for an industrial
process. Enzymes are proteins and depend upon their three-dimensional structure for correct
function. They rapidly unfold if subject to harsh conditions such as high temperatures or high
salt concentration, both of which are prevalent in industrial processes. It is not always easy
to replace a conventional process with one that can be carried out in mild conditions
suitable for enzymes.

The biodiversity of nature, however, may provide many more industrial enzymes in the
future. There are thousands of enzyme genes in genome databases just waiting to be
exploited commercially. Some are more able to tolerate harsh conditions than enzymes
currently available. A particularly rich source of enzymes is microbial life existing in extreme
environments. For example, researchers at the Idaho National Laboratory have isolated an
enzyme from a microbe that exists in the hot springs of Yellowstone National Park. The
temperature range of this catalyst is 86°F to more than 212°F (30°C to more than 94°C), and
its pH range is 6–10. Experiments show that this catalyst is very stable compared to other
catalysts at higher temperatures and pH. Its half life was 15 days at 176° (80°C) and pH 10,
whereas a conventional enzyme from Aspergillus niger had a half life of just 15 seconds under
the same conditions. This novel enzyme has potential application for the removal of
hydrogen peroxide in processes involving bleach, such as those used in the paper and pulp,
textiles, and food pasteurization industries. The advantage would be cheaper treatment of
wastewater and lower energy costs.

Elaborate and Evaluate

Evaluative Assessment:

Research further on the history of one enzyme. Discuss its discovery,

development, and application today. You may choose one enzyme discussed
in this module or an enzyme not discussed.

Output should be computerized. Creativity will be graded.

References

Chaplin, M.F. and C. Bucke. (1990). Enzyme Technology. Cambridge University Press.

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 Unit 2:
Enzyme Preparation and Use

Engage

Enzyme Units

The amount of enzyme present or used in a process is difficult to determine in absolute

terms (e.g. grams), as its purity is often low and a proportion may be in an inactive, or partially
active, state. More relevant parameters are the activity of the enzyme preparation and the
activities of any contaminating enzymes. These activities are usually measured in terms of
the activity unit (U) which is defined as the amount which will catalyse the transformation of
1 micromole of the substrate per minute under standard conditions. Typically, this represents
10-6 – 10-11 Kg for pure enzymes and 10-4 – 10-7 kg for industrial enzyme preparations.

Another unit of enzyme activity is the katal (kat). This is defined as the amount which
will catalyse the transformation of one mole of substance per second (1 kat = 60 000 000 U).
It is an impracticable unit and has not yet received widespread acceptance. Sometimes
non-standard activity units are used, such as Soxhet, Anson and Kilo Novo units, which are
based on physical changes such as lowering viscosity and supposedly better understood by
industry.

Rightfully, such units are gradually falling into disuse. The activity is a measure of
enzyme content that is clearly of major interest when the enzyme is to be used in a process.
For this reason, enzymes are usually marketed in terms of activity rather than weight. The
specific activity (e.g. U/kg) is a parameter of interest, some utility as an index of purity but
lesser importance. There is a major problem with these definitions of activity; the rather vague
notion of "standard conditions". These are meant to refer to optimal conditions, especially
with regard to pH, ionic strength, temperature, substrate concentration and the presence
and concentration of cofactors and coenzymes. However, these so-termed optimal
conditions vary both between laboratories and between suppliers. They also depend on the
particular application in which the enzyme is to be used. Additionally, preparations of the
same notional specific activity may differ with respect to stability and be capable of very
different total catalytic productivity (this is the total substrate converted to product during
the lifetime of the catalyst, under specified conditions). Conditions for maximum initial
activity are not necessarily those for maximum stability. Great care has to be taken over the
consideration of these factors when the most efficient catalyst for a particular purpose is to
be chosen.

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 Explore

Sources of Enzymes

Biologically active enzymes may be extracted from any living organism. A very wide
range of sources are used for commercial enzyme production from Actinoplanes to
Zymomonas, from spinach to snake venom. Of the hundred or so enzymes being used
industrially, over a half are from fungi and yeast and over a third are from bacteria with the
remainder divided between animal (8%) and plant (4%) sources. A very much larger number
of enzymes find use in chemical analysis and clinical diagnosis. Non-microbial sources
provide a larger proportion of these, at the present time. Microbes are preferred to plants
and animals as sources of enzymes because:

1. they are generally cheaper to produce.

2. their enzyme contents are more predictable and controllable,
3. reliable supplies of raw material of constant composition are more easily arranged,
and
4. plant and animal tissues contain more potentially harmful materials than microbes,
including phenolic compounds (from plants), endogenous enzyme inhibitors and
proteases.

Attempts are being made to overcome some of these difficulties by the use of animal
and plant cell culture.

Enzyme Source Scale of Industrial Use

Production
Animal Enzymes
Catalase Liver - Food
Chymotrypsin Pancreas - Leather
Lipase Pancreas - Food
Rennet Abomasum + Cheese
Trypsin Pancreas - Leather
Plant Enzymes
Acitinidin Kiwi fruit - Food
Α-amylase Malted barley +++ Brewing
b-amylase Malted barley +++ Brewing
Bromelain Pineapple latex - Brewing
b-glucanase Malted barley ++ Brewing
Ficin Fig latex - Food
Lipoxygenase Soybeans - Food
Papain Pawpaw latex ++ Meat
Bacterial enzymes
a-amylase Bacillus +++ Starch
b-amylase Bacillus + Starch
Asparaginase Escherichia coli - Health
Glucose isomerase Bacillus + Fructose syrup

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 Penicillin amidase Bacillus - Pharmaceutical
Protease Bacillus +++ Detergent
Pullulanase Klebsiella - Starch
Fungal enzymes
a-amylase Aspergillus ++ Baking
Aminoacylase Aspergillus - Pharmaceutical
Glucoamylase Aspergillus +++ Starch
Catalase Aspergillus - Food
Cellulase Trichoderma - Waste
Dextranase Penicillum - Food
Glucose oxidase Aspergillus - Food
Lactase Aspergillus - Dairy
Lipase Rhizopus - Food
Rennet Mucor miehei ++ Cheese
Pectinase Aspergillus ++ Drinks
Pectin lyase Aspergillus - Drinks
Protease Aspergillus + Baking
Raffinase Mortierella - Food
Yeast enzymes
Invertase Saccharomyces - Confectionary
Lactase Kluyveromyces - Dairy
Lipase Candida - Food
Raffinase Saccharomyces - Food
Note: +++ >100 tons/ year; ++ >10 tons/year; + >1 ton/ year; - <1 ton/ year

In practice, the great majority of microbial enzymes come from a very limited number
of genera, of which Aspergillus species, Bacillus species and Kluyveromyces (also called
Saccharomyces) species predominate. Most of the strains used have either been employed
by the food industry for many years or have been derived from such strains by mutation and
selection. There are very few examples of the industrial use of enzymes having been
developed for one task. Shining examples of such developments are the production of high
fructose syrup using glucose isomerase and the use of pullulanase in starch hydrolysis.

Screening for Novel Enzymes

If a reaction is thermodynamically possible, it is likely that an enzyme exists which is

capable of catalysing it. One of the major skills of enzyme companies and suitably funded
academic laboratories is the rapid and cost-effective screening of microbial cultures for
enzyme activities. Natural samples, usually soil or compost material found near high
concentrations of likely substrates, are used as sources of cultures. It is not unusual at
international congresses of enzyme technologists to see representatives of enzyme
companies collecting samples of soil to be screened later when they return to their
laboratories.

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 The first stage of the screening procedure for commercial enzymes is to screen ideas,
i.e. to determine the potential commercial need for a new enzyme, to estimate the size of
the market and to decide, approximately, how much potential users of the enzyme will be
able to afford to pay for it. A cumulative cash flow must be estimated, balancing the initial
screening and investment capital costs including interest, tax liability and depreciation,
against the expected long term profits. Full account must be taken of inflation, projected
variation in feedstock price and source, publicity and other costs. Financial re-appraisal must
be frequently carried out during the development process to check that it still constitutes an
efficient use of resources.

The next stage involves the location of a source of the required enzyme. Laboratory
work is expensive in manpower so clearly it is worthwhile using all available databases to
search for mention of the enzyme in the academic and patents literature. Cultures may then
be sought from any sources so revealed. If these first searches are unsuccessful, it is probably
necessary to screen for new microbial strains capable of performing the transformation
required. This should not be a 'blind' screen: there will usually be some source of microbes
that could have been exposed for countless generations to the conditions that the new
enzyme should withstand or to chemicals which it is required to modify. A classic example of
the detection of an enzyme by intelligent screening was the discovery of a commercially
useful cyanide-degrading enzyme in the microbial pathogens of plants that contain
cyanogenic glycosides.

The identification of a microbial source of an enzyme is by no means the end of the

story. The properties of the enzyme must be determined; i.e. temperature for optimum
productivity, temperature stability profile, pH optimum and stability, kinetic constants,
whether there is substrate or product inhibition, and the ability to withstand components of
the expected feedstock other than substrate. If any of these parameters is unsatisfactory,
the screen must continue until improved enzymes are located.

Once an enzyme with suitable properties has been located, various decisions must
be made concerning the acceptability of the organism to the regulatory authorities, the
productivity of the organism, and the way in which the enzyme is to be isolated, utilised (free
or immobilised) and, if necessary, purified.

Once an enzyme with suitable properties has been located, various decisions must
be made concerning the acceptability of the organism to the regulatory authorities, the
productivity of the organism, and the way in which the enzyme is to be isolated, utilised (free
or immobilised) and, if necessary, purified. If the organism is unacceptable from a regulatory
viewpoint two options exist; to eliminate that organism altogether and continue the
screening operation, or to clone the enzyme into an acceptable organism. Cloning may
also be attractive when the organism originally producing the enzyme is acceptable from
the health and safety point of view but whose productivity is unacceptable.

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 The use of immobilised enzymes (Unit 3) is now familiar to industry and their
advantages are well recognised so the practicality of using the new enzymes in an
immobilised form will be determined early in the screening procedure. If the enzyme is
produced intracellularly, the feasibility of using it without isolation and purification will be
considered very seriously and strains selected for their amenability to use in this way.

It should be emphasised that there will be a constant dialogue between laboratory

scientists and biochemical process engineers from the earliest stages of the screening
process. Once the biochemical engineers are satisfied that their initial criteria of productivity,
activity and stability can be met, the selected strain(s) of microbe will be grown in pilot plant
conditions. It is only by applying the type of equipment used in full scale plants that accurate
costing of processes can be achieved. Pilot studies will probably reveal imperfections, or at
least areas of ignorance, that must be corrected at the laboratory scale.

Screening for new enzymes is expensive so that the intellectual property generated
must be protected against copying by competitors. This is usually done by patenting the
enzyme or its production method or, most usefully, the process in which it is to be used.

Media for Enzyme Production

Details of components used in industrial scale fermentation broths for enzyme

production are not readily obtained. This is not unexpected as manufacturers have no wish
to reveal information that may be of technical or commercial value to their competitors.
Also some components of media may be changed from batch to batch as availability and
cost of, for instance, carbohydrate feedstocks change. Such changes reveal themselves in
often quite profound differences in appearance from batch to batch of a single enzyme
from a single producer. The effects of changing feedstocks must be considered in relation
to downstream processing. If such variability is likely to significantly reduce the efficiency of
the standard methodology, it may be economical to use a more expensive defined medium
of easily reproducible composition.

Clearly defined media are usually out of the question for large scale use on cost
grounds but may be perfectly acceptable when enzymes are to be produced for high value
uses, such as analysis or medical therapy where very pure preparations are essential. Less-
defined complex media are composed of ingredients selected on the basis of cost and
availability as well as composition. Waste materials and byproducts from the food and
agricultural industries are often major ingredients. Thus molasses, corn steep liquor, distillers
solubles and wheat bran are important components of fermentation media providing
carbohydrate, minerals, nitrogen and some vitamins. Extra carbohydrate is usually supplied
as starch, sometimes refined but often simply as ground cereal grains. Soybean meal and
ammonium salts are frequently used sources of additional nitrogen. Most of these materials
will vary in quality and composition from batch to batch causing changes in enzyme
productivity.

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 Preparation of Enzymes

Often the enzyme may be purified several hundred-fold but the yield of the enzyme
may be very poor, frequently below 10% of the activity of the original material. In contrast,
industrial enzymes will be purified as little as possible, only other enzymes and material likely
to interfere with the process which the enzyme is to catalyse, will be removed. Unnecessary
purification will be avoided as each additional stage is costly in terms of equipment,
manpower and loss of enzyme activity. As a result, some commercial enzyme preparations
consist essentially of concentrated fermentation broth, plus additives to stabilise the
enzyme's activity.

The content of the required enzyme should be as high as possible (e.g. 10% w/w of
the protein) in order to ease the downstream processing task. This may be achieved by
developing the fermentation conditions or, often more dramatically, by genetic
engineering.

It is important that the maximum activity is retained during the preparation of

enzymes. Enzyme inactivation can be caused by heat, proteolysis, sub-optimal pH,
oxidation, denaturants, irreversible inhibitors and loss of cofactors or coenzymes. Of these
heat inactivation, which together with associated pH effects, is probably the most
significant. It is likely to occur during enzyme extraction and purification if insufficient cooling
is available, but the problem is less when preparing thermophilic enzymes. Proteolysis is most
likely to occur in the early stages of extraction and purification when the proteases
responsible for protein turnover in living cells are still present. It is also the major reason for
enzyme inactivation by microbial contamination. In their native conformations, enzymes
have highly structured domains which are resistant to attack by proteases because many of
the peptide bonds are mechanically inaccessible and because many proteases are highly
specific. The chances of a susceptible peptide bond in a structured domain being available
for protease attack are low.

Some intracellular enzymes are used commercially without isolation and purification
but the majority of commercial enzymes are either produced extracellularly by the microbe
or plant or must be released from the cells into solution and further processed. Solid/liquid
separation is generally required for the initial separation of cell mass, the removal of cell
debris after cell breakage and the collection of precipitates.

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 Flow Diagram for the Preparation of Enzymes

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 Elaborate and Evaluate

Evaluative Assessment:

Discussion Forum via Google Classroom

What do you think is the most important factor that need to be considered
upon preparing an enzyme for industrial use? Why?

References
Chaplin, M.F. and C. Bucke. (1990). Enzyme Technology. Cambridge University Press.

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 Unit 2:
Enzyme Kinetics

Engage

Basic Enzyme Reactions

Enzymes are catalysts and increase the speed of a chemical reaction without
themselves undergoing any permanent chemical change. They are neither used up in the
reaction nor do they appear as reaction products

The basic enzymatic reaction can be represented as follows

𝑆+𝐸 →𝑃+𝐸
Where E represents the enzyme catalysing the reaction, S the substrate, the substance being
changed, and P the product of the reaction.

Energy Levels

Chemist have known for almost a century that for most chemical reactions to
proceed, some form of energy is needed. They have termed this quantity of energy, “the
energy of activation.” It is the magnitude of the activation energy which determines just how
fast the reaction will proceed. It is believed that enzymes lower the activation energy for the
reaction they are catalyzing.

Energy of Activation ΔEa is less than ΔE’a due to the effect of the enzyme on the substrate

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 The enzyme is thought to reduce the "path" of the reaction. This shortened path would
require less energy for each molecule of substrate converted to product. Given a total
amount of available energy, more molecules of substrate would be converted when the
enzyme is present (the shortened "path") than when it is absent. Hence, the reaction is said
to go faster in a given period of time.

Explore

The Enzyme Substrate Complex

A theory to explain the catalytic action of enzymes wa proposed by the Swedish

chemist Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some
intermediate substance which is known as the enzyme substrate complex. The reaction can
be represented as:
𝑘1
𝑆 + 𝐸 ↔ 𝐸𝑆
𝑘2
𝑘3
𝐸𝑆 → 𝑃 + 𝐸
The existence of an intermediate enzyme-substrate complex has been demonstrated in the
laboratory, for example, using catalase and a hydrogen peroxide derivative. The rate of
reaction is the rate at which the product is formed.
𝑑𝐶𝑝
𝑟= = 𝑘3 𝐶𝐸𝑆
𝑑𝑡
Where Ces indicates the molar concentration of the enzyme-substrate complex.

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 The graph above is generated from a computer simulation of the progress curve
dCES/dt, CS and CP for a reaction obeying simple Michaelis-Menten kinetics, with k1 = 106 /M.s,
k2 = 1000/s, k3 = 10/s, CEo = 10-7 M and CSo = 0.01 M. The

The rate of an enzyme-catalyzed reaction is expressed in terms of the Michaelis-

Menten equation
𝑟𝑚𝑎𝑥 𝐶𝑠
𝑟=
𝐾𝑚 + 𝐶𝑠
Where rmax and Km are kinetic parameters that are dependent on the enzyme used. Rmax is
the maximum rate of reaction, which occurs when the enzyme is completely saturated with
substrate. Km, also known as the Michaelis constant has values typically in the range of 10 1
– 10-5 M. It is the dissociation constant of the enzyme substrate complex, k2/k1.

The substrate concentration in these equations is the actual concentration at the time
and, in a closed system, will only be approximately equal to the initial substrate
concentration during the early phase of the reaction. Hence, it is usual to use these
equations to relate the initial rate of reaction to the initial, and easily predetermined,
substrate concentration as shown in the figure below.

It has been established that few enzymes follow the Michaelis-Menten equation over
a wide range of experimental conditions. However, it remains by far the most generally
applicable equation for describing enzymic reactions. Indeed it can be realistically applied
to a number of reactions which have a far more complex mechanism than the one
described here. In these cases Km remains an important quantity, characteristic of the

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 enzyme and substrate, corresponding to the substrate concentration needed for half the
enzyme molecules to bind to the substrate (and, therefore, causing the reaction to proceed
at half its maximum rate) but the precise kinetic meaning derived earlier may not hold and
may be misleading. In these cases the Km is likely to equal a much more complex relationship
between the many rate constants involved in the reaction scheme. It remains independent
of the enzyme and substrate concentrations and indicates the extent of binding between
the enzyme and its substrate for a given substrate concentration, a lower Km indicating a
greater extent of binding. Rmax clearly depends on the enzyme concentration and for some,
but not all, enzymes may be largely independent of the specific substrate used. Km and rmax
may both be influenced by the charge and conformation of the protein and substrate(s)
which are determined by pH, temperature, ionic strength and other factors.

Many applications of enzymes involve open systems, where the substrate

concentration remains constant, due to replenishment, throughout the reaction time-
course. This is, of course, the situation that often prevails in vivo. Under these circumstances,
the Michaelis-Menten equation is obeyed over an even wider range of enzyme
concentrations than allowed in closed systems, and is commonly used to model immobilised
enzyme kinetic systems.

Enzyme Inhibition

A number of substances may cause a reduction in the rate of an enzyme catalyzed-

reaction. Some of these are non-specific protein denaturants. Others, which generally act
in a fairly specific manner, are known as inhibitors. Loss of activity may be either reversible,
where activity may be restored by the removal of the inhibitor, or irreversible, where the loss
of activity is time dependent and cannot be recovered during the timescale of interest. If
the inhibited enzyme is totally inactive, irreversible inhibition behaves as a time-dependent
loss of enzyme concentration. In other cases, involving incomplete inactivation, there may
be time-dependent changes in both Km and rmax. Heavy metal ions should generally be
prevented from coming into contact with enzymes as they usually cause such irreversible
inhibition by binding strongly to the amino acid backbone.

Competitive Inhibition

This occurs when both the substrate and inhibitor compete for binding to the active
site of the enzyme. The inhibition is most noticeable at low substrate concentrations but
can be overcome at sufficiently high substrate concentrations as the rmax remains
unaffected. The rate equation is given by
𝑟𝑚𝑎𝑥 𝐶𝑠
𝑟=
𝐾𝑚𝐼 + 𝐶𝑠

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 Where
𝐶𝐼
) 𝐾𝑚𝐼 = 𝐾𝑚 (1 +
𝐾𝐼
Normally the competitive inhibitor bears some structural similarity to the substrate,
and often is a reaction product (product inhibition, e.g. inhibition of lactase by galactose),
which may cause a substantial loss of productivity when high degrees of conversion are
required.

Uncompetitive Inhibition

This occurs when the inhibitor binds to the site which only becomes available after
the substrate has bound to the active site of the enzyme. This inhibition is most commonly
encountered in multi-substrate reactions where the inhibitor is competitive with respect to
one substrate but uncompetitive with respect to another.

The inhibition is most noticeable at high substrate concentrations and cannot be

overcome as both rmax and Km are equally reduced.
𝑟𝑚𝑎𝑥,𝐼 𝐶𝑠
𝑟=
𝐾𝑚𝐼 + 𝐶𝑠
Where
𝑟𝑚𝑎𝑥
𝑟𝑚𝑎𝑥,𝐼 =
𝐶
1+ 𝐼
𝐾𝐼
𝐾𝑚
𝐾𝑚𝐼 =
𝐶
1+ 𝐼
𝐾𝐼
In this case the specificity constant remains unaffected by the inhibition. Normally
the uncompetitive inhibitor also bears some structural similarity to one of the substrates
and, again, is often a reaction product.

Noncompetitive Inhibition

This occurs when the inhibitor binds at a site away from the substrate binding site,
causing a reduction in the catalytic rate. It is quite rarely found as a special case of mixed
inhibition. The fractional inhibition is identical at all substrate concentrations and cannot be
overcome by increasing substrate concentration due to the reduction in rmax. The rate
equation is given by

𝑟𝑚𝑎𝑥,𝐼 𝐶𝑠
𝑟=
𝐾𝑚 + 𝐶𝑠
Where
𝑟𝑚𝑎𝑥
𝑟𝑚𝑎𝑥,𝐼 =
𝐶
1 + 𝐾𝐼
𝐼

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 Immobilized Enzyme Technology

Immobilization of an enzyme means that it has been confined or localized so that it

can be reused continuously. There are several reason why immobilization may be desirable:
for processing with isolated enzymes, an immobilized form can be retained in the reactor.
With enzymes in solution, on the other hand, some enzymes will leave the reactor with the
final product. Not only must new enzymes be introduced to replace the lost ones, but
enzymes in the product may be undesirable impurities which must be removed. Immobilized
enzymes may also retain their activity longer than those in solution. Finally, an immobilized
enzyme may be fixed in position near other enzymes participating in a catalytic sequence,
thereby increasing the catalyst efficiency for the multistep conversion.

These characteristics make immobilized enzymes attractive if a very large throughput

of substrate is required and/or the enzymes involved are expensive. Moreover, the ability to
confine an enzyme in a well-defined, predetermined space provides opportunities for
applications unique to immobilized enzymes.

Many methods are available for enzyme immobilization. As seen in the figures below,
the immobilization method used greatly influences the properties of the resulting biocatalyst.
Thus, the selection of an immobilization strategy derives from process specifications for the
catalyst including such parameters as overall catalytic activity, effectiveness of catalyst
utilization, deactivation and regeneration characteristics and, of course, cost.

The methods used for immobilization may be subdivided into two general classes:
chemical methods, where covalent bonds are formed with the enzyme, and physical
methods, where weaker interactions or containment of the enzymes are involved.

Chemical Methods

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 Physical Methods

Chemical Methods

The two major type of immobilization of enzymes on the surfaces of support materials
are adsorption and covalent binding.

Adsorption is the attachment of enzymes on the surfaces of support particles by weak

physical forces, such as van der Waals or dispersion forces. The active site of the adsorbed
enzyme is usually unaffected, and nearly full activity is retained upon adsorption. However,
desorption of enzymes is a common problem especially in the presence of strong
hydrodynamic forces, since binding forces are weak.

Enzymes may be adsorbed in a variety of carriers, shown in the table below, offering
in some cases the practical convenience of simple regeneration by removal of deactivated
enzyme and reloading with fresh, active catalyst. If a support or entrapping material is used,
its properties combined with those of the enzyme and the immobilization procedure dictate
overall catalyst properties.

Interactions and carriers used for enzyme immobilization by adsorption

Interaction Adsorbents
Physical adsorption Activated carbon, silica gel, alumina. Starch, clay, glass

Modified materials: tannin-aminohexyl cellulose, Concanavalin A-

Sepharose

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 Ionic binding Cation exhangers
Carboxymethyl-cellulose, Aberlite, CG-50, Dowex 50

Anion Exchangers
Diethylaminoethyl-cellulose, DEAE-Sephadex,
polyaminopolystyrene, Amberlite IR-45

Covalent binding is the retention of enzyme on support surfaces by covalent bond

formation. Enzyme molecules bind to support material via certain functional groups, such as
amino, carboxyl, hydroxyl, and sulfhydryl groups. These functional groups must not be in the
active site. One common trick is to block the active site by flooding the enzyme solution with
a competitive inhibitor prior to covalent binding. Functional groups on support material are
usually activated by using chemical reagents, such as cyanogen bromide, carbodiimide,
and glutaraldehyde.

Selecting a support for chemical or adsorption immobilization of enzyme depends first

upon its surface properties. The table below lists several materials which have been
employed for covalent enzyme immobilization and some of their interesting surface
functional groups.

Natural supports Synthetic supports

Cellulose (- OH) Polyacrylamide derivatives
CM-Cellulose (- COOH) (Bio-Gel, Enzacryl) (- aromatic amino)
Agarose (Sepharose) (- OH) Polyaminopolystyrene (- NH2)
Dextran (Sephadex) (- OH) Maleic anhydride copolymers

Protocols for covalent enzyme immobilization often begin with a surface modification
or activation step. Silanization, coating the surface with organic functional groups using an
organofunctional silane reagent is a widely used strategy for initial surface modification of
inorganic supports. Another commonly studied surface modification is attachment of flexible
spacer arm moieties to the support, to which enzyme is subsequently linked. This may render
the surface more “flexible” so it can conform to the enzyme’s structure, thereby retaining to
a greater extent and potentially stabilizing native catalytic activity.

Typical examples of immobilization chemistries which utilize these surface

functionalities and those appearing in the table above are summarized in the figure in the
next page. Taking advantage of the available repertoire of surface modification techniques,
typically there are several options for immobilizing a particular enzyme to a particular
support.

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 When considering selection of a support and adjustment of its surface properties, one
must also consider interaction between the support surface and the reaction mixture. The
hydrophobicity or hydrophilicity of the support will influence the local concentrations of
solutes and solvents according to their hydrophobicity/hydrophilicity.

Another important role of the support surface is in defining, directly and indirectly, the
molecular environment of the enzyme. That is, to some degree the enzyme will contact, at
the molecular level, the support surface. The remainder of the immobilized enzyme will be in
contact with the local fluid environment which is influenced by the support as just outlined.
Efforts to understand enzyme-environment interactions at the molecular level and to apply
them for improved understanding of enzymes in nature and for optimizing process
biocatalysts are active topics of current research and development.

The physical and mechanical properties of the support are also important. The pore
size distribution and porosity of a solid material determines the quantity of enzyme which can
be immobilized in the support and the accessibility of substrate to the enzyme attached to
the internal surfaces. The mechanical strength of support influences significantly catalyst
suitability for different reactor configurations. The swelling properties of the support are also
important.

Enzymes may be cross-linked with several bi- or multifunctional reagents. The most
widely used method employs glutaraldehyde which establishes inter-molecular cross-links at
amino groups as follows

Corss-linking can be achieved in several different ways: enzymes can be cross-linked,

or cross-linking may take place following the impregnation of porous support material with
enzyme solution. cross-linking my cause significant changes in the active site of enzymes,
and also sever diffusion limitations may result.

Other cross-linking reagents include bisbiazobenzidine, cyanuric chloride and

hexamethylene-diisocyanate. Particles of crosslinked enzyme alone are gelatinous and lack
the mechanical properties required in many applications.

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 Physical Methods

Entrapment is the physical enclosure of enzymes in a small space. Matrix entrapment

and membrane entrapment, including microencapsulation, are the two major methods of
entrapment.

Matrices used for enzyme immobilization are usually polymeric materials such as Ca-
alginate, agar, k-carrageenin, polyacrylamide, and collagen. However, some solid matrices
such as activated carbon, porous ceramic, and diatomaceous earth can also be used for
this purpose. The matrix can be a particle, a membrane, or a fiber. When immobilizing in a
polymer matrix, enzyme solution is mixed with polymer solution before polymerization takes
place. Polymerized gel-containing enzyme is either extruded or a template is used to shape
the particles from a liquid polymereenzyme mixture.

Membrane entrapment of enzymes is possible; for example, hollow-fiber units have

been used to entrap an enzyme solution between thin, semipermeable membranes.
Membranes of nylon, cellulose, polysulfone, and polyacrylate are commonly used.
Configurations, other than hollow fibers, are possible, but in all cases, a semipermeable
membrane is used to retain high molecular weight compounds (enzyme), while allowing
small molecular weight compounds (substrate or products) access to the enzyme.

Despite the aforementioned advantages, enzyme entrapment may have its inherent
problems, such as enzyme leakage into solution, significant diffusion limitations, reduced
enzyme activity and stability, and lack of control of microenvironmental conditions.

Enzyme entrapment on a macroscopic scale

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 Diffusion limitations can be eliminated by reducing the particle size of matrices and/or
capsules. Reduced enzyme activity and stability are due to unfavourable
microenvironmental conditions, which are difficult to control. However, by using different
matrices and chemical ingredients, by changing processing conditions, and by reducing
particle or capsule size, more favorable microenvironmental conditions can be obtained.

Important characteristics of different immobilization techniques are summarized in the

table below. Here “enzyme activity” denotes the intrinsic catalytic activity of the immobilized
enzyme molecule relative to that enzyme’s activity in solution.

Carrier – binding Method

Characteristic Physical Ionic Binding Covalent Cross-linking Entrapping
Adsorption Binding Method Method
Preparation Easy Easy Difficult Difficult Difficult
Enzyme Low High High Moderate High
activity
Substrate Unchangeable Unchangeable Changeable Changeable Unchangeable
specificity
Binding force Weak Moderate Strong Strong Strong
Regeneration Possible Possible Impossible Impossible Impossible
General Low Moderate Moderate Low High
applicability
Cost of Low Low High Moderate Low
immobilization

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 Explain

Physical Methods

Several large-scale industrial processes already in operation employ immobilized-

enzyme catalysts at some point. Two notable examples are production of high-fructose
syrups from corn starch and manufacture of L-amino acids by resolution of racemic amino
acid mixtures. Also, immobilized penicillin acylase has been used commercially in
manufacture of semisynthetic penicillins. The first application mentioned is the most
important economically at present.

Further Reading:

Chapter 4, Section 4.3.2, Biochemical Engineering

Fundamentals by Bailey and Ollis

Elaborate and Evaluate

Evaluative Assessment:

Research on a SPECIFIC application of Immobilized Enzymes on Medical and

Analytical Application.
The method of immobilization used should be present.

References

Bailey, J. E. and D. Ollis (1986). Biochemical Engineering Fundamentals. McGraw-Hill Book

Co.
Chaplin, M.F. and C. Bucke. (1990). Enzyme Technology. Cambridge University Press.
Liu, S. (2013). Bioprocess Engineering. Elsevier.

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