APPLIED ENZYMOLOGY

1. INTRODUCTION, ENZYME SAFETY AND REGULATORY CONSIDERATIONS

Enzymes have a number of distinct advantages over conventional chemical catalysts.

Foremost amongst these are their specificity and selectivity not only for particular reactions
but also in their discrimination between similar parts of molecules (regioselectivity) or
optical isomers (stereospecificity). They catalyse only the reactions of very narrow ranges
of reactants (substrates), which may consist of a small number of closely related classes of
compounds (e.g. trypsin catalyses the hydrolysis of some peptides and esters in addition to
most proteins), a single class of compounds (e.g. hexokinase catalyses the transfer of a
phosphate group from ATP to several hexoses), or a single compound (e.g. glucose oxidase
oxidises only glucose amongst the naturally occurring sugars). This means that the chosen
reaction can be catalysed to the exclusion of side-reactions, eliminating undesirable by-
products. Thus, higher productivities may be achieved, reducing material costs. As a bonus,
the product is generated in an uncontaminated state so reducing purification costs and the
downstream environmental burden. Often a smaller number of steps may be required to
produce the desired end-product. In addition, certain stereospecific reactions (e.g. the
conversion of glucose into fructose) cannot be achieved by classical chemical methods
without a large expenditure of time and effort. Enzymes work under generally mild
processing conditions of temperature, pressure and pH. This decreases the energy
requirements, reduces the capital costs due to corrosion-resistant process equipment and
further reduces unwanted side-reactions. The high reaction velocities and straightforward
catalytic regulation achieved in enzyme-catalysed reactions allow an increase in
productivity with reduced manufacturing costs due to wages and overheads.

There are some disadvantages in the use of enzymes which cannot be ignored but
which are currently being addressed and overcome. In particular, the high cost of enzyme
isolation and purification still discourages their use, especially in areas which currently
have an established alternative procedure. The generally unstable nature of enzymes, when
removed from their natural environment, is also a major drawback to their more extensive
use.

Enzyme sources

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.

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.

Table I Enzyme sources - extremophiles

Microorganism Conditions Source Enzymes and application

Thermophiles 50 – 110˚C geothermal Amylases, xylanases, proteases, DNA

region polymerases

Psychrophiles 5 – 20 ˚C glacial Proteases, glykosidases, lipases

sediments

Acidophiles pH < 2 Sulphur Sulphur oxidation

springs
Alkalophilesí pH > 9 dregs Lipases, proteases

Halophiles 3 – 20% Salt lake Biotransformations, additives, drugs

salts sediments

Barophiles Higher Submarine

pressure sediments

Producers of industrial enzymes and their customers will share the common aims of
economy, effectiveness and safety. They will wish to have high-yielding strains of
microbes which make the enzyme constitutively and secrete it into their growth medium
(extracellular enzymes). If the enzyme is not produced constitutively, induction must be
rapid and inexpensive. Producers will aim to use strains of microbe that are known to be
generally safe. Users will pay little regard to the way in which the enzyme is produced but
will insist on having preparations that have a known activity and keep that activity for
extended periods, stored at room temperature or with routine refrigeration. They will pay
little attention to the purity of the enzyme preparation provided that it does not contain
materials (enzymes or not) that interfere with their process. Both producers and users will
wish to have the enzymes in forms that present minimal hazard to those handling them or
consuming their product.

The development of commercial enzymes is a specialised business which is usually

undertaken by a handful of companies which have high skills in
1. screening for new and improved enzymes,
2. fermentation for enzyme production,
3. large scale enzyme purifications,
4. formulation of enzymes for sale,
5. customer liaison, and
6. dealing with the regulatory authorities.

Table II Composition of technical enzyme preparations

component Yield (%)
Proteins and amino acids 10 - 15

Active proein 1-5

polysaccharides 5 - 12
sacharides 2 - 40
Anorganic salts 3 - 40
Preservation comp. 0 - 0,3
Form of technical enzyme preparations: liquid, powder, encapsulated
2. DISCOVERY AND DEVELOPMENT OF 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.

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. In some cases, the determination of the potential value of
an enzyme is not easy, for instance when it might be used to produce an entirely novel
substance. In others, for instance when the novel enzyme would be used to improve an
existing process, its potential value can be costed very accurately. In either case, 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. In addition, the probability of potential market
competition and changes in political or legal factors must be considered. Usually the
sensitivity of the project to changes in all of these factors must be estimated, by informed
guesswork, in order to assess the risk factor involved. Financial re-appraisal must be
frequently carried out during the development process to check that it still constitutes an
efficient use of resources.

If agreement is reached, probably after discussions with potential users, that

experimental work would be commercially justifiable, 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.
Some preparations of commercial enzymes are quite rich sources of enzymes other than the
enzyme which is being offered for sale, revealing such preparations as potential
inexpensive sources which are worth investigating.

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. Hence, thermophiles are sought in hot springs,
osmophiles in sugar factories, organisms capable of metabolising wood preservatives in
timber yards and so on. 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 (Km, Vmax),
whether there is substrate or
product inhibition, and the ability
to withstand components of the
expected feedstock other than
substrate. A team of scientists,
engineers and accountants must
then consider the next steps. If
any of these parameters is
unsatisfactory, the screen must
continue until improved enzymes
are located. Now that protein
engineering can be seriously
contemplated, an enzyme with
sufficient potential value could be
improved 'by design' to overcome
one or two shortcomings.
However, this would take a long
time, at the present level of
knowledge and skill, so further
screening of microbes from
selected sources would probably
be considered more worthwhile.

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. The latter approach is becoming increasingly attractive especially as cloning
could also be used to increase the productivity of the fermentation process. 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. However,
cloning is not yet routine and invariably successful so there is still an excellent case to be
made for applying conventional mutation and isolation techniques for the selection of
improved strains. It should be noted that although the technology for cloning glucose
isomerase into 'routine' organisms is known, it has not yet been applied. Several of the
glucose isomerase preparations used commercially consist of whole cells, or cell
fragments, of the selected strains of species originally detected by screening.

The use of immobilised enzymes 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. If this proves possible, the pilot plant will produce samples of the enzyme
preparation to be used by customers who may well also be at the pilot plant stage in the
development of the enzyme-utilizing process. The enzyme pilot plant also produces
samples for safety and toxicological studies provided that the pilot process is exactly
similar to the full scale operation.

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.
Patenting will be initiated as soon as there is evidence that an innovative discovery has
been made.

Retailoring of enzymes
Site-directed mutagenesis is still
a very efficient strategy to elaborate
improved enzymes. Recently, advances
have been made in developing rational
strategies aimed at reshaping enzyme
specificities and mechanisms, and at
engineering biocatalysts through
molecular assembling. These
knowledgebased studies greatly benefit
from the most recent computational
analyses of enzyme structures and
functions. The combination of rational
and combinatorial methods pens up new
vistas in the design of stable and
efficient enzymes.
Schematic representation of the possible
strategies for redesigning
enzyme functions is on the figure:
The upper part of the figure illustrates
the three possible routes to shift a pre-
existing enzyme activity into an original
one. The lower part illustrates an
alternative strategy based on the
assembling of independent enzyme
features, such as a substratebindingsite
and the catalytic machinery, to create a novel biocatalyst. The white symbols (cross, stars)
depict functional amino acid residues that must be either changed or grafted during the
different processes.

Directed evolution is a powerful tool for the creation of commercially useful enzymes,
particularly those approaches that are based on in vitro recombination methods, such as
DNA shuffling. Although these types of search algorithms are extraordinarily efficient
compared with purely random methods, they do not explicity represent or interrogate the
genotype–phenotype relationship and are essentially blind in nature. Recently, however,
researchers have begun to apply multivariate statistical techniques to model protein
sequence–function relationships and guide the evolutionary process by rapidly identifying
beneficial diversity for recombination. In conjunction with state-of-the-art library
generation methods, the statistical approach to sequence optimization is now being used
routinely to create enzymes efficiently for industrial applications (Richard J. Fox and Gjalt
W. Husman (2008) Enzyme optimization: oving from blind evolution to statistical
exploration of sequence–function space Trends in Biotechnology Vol.26 No.3, 132-138)

.
Accelerating impact of biocatalysis. Before 1970 biocatalysis was generally limited to
those applications using enzymes found in nature (e.g. chymosin from calf and sheep
stomach for the manufacture of cheese). With the advent of gene level random and site-
directed mutagenesis in the 1980s, enzyme optimization via rational and directed
evolutionary approaches became possible. Recombination-based approaches, such as DNA
shuffling, were used to accelerate the process of directed evolution in the 1990s. In the
current decade, computational library design coupled with directed evolution has proven to
be a useful strategy along with statistical modeling of sequence–function relationships
(adopted from Richard J. Fox and Gjalt W. Husman (2008) Enzyme optimization: moving
from blind evolution to statistical exploration of sequence–function space.: Trends in
Biotechnology Vol.26 No.3, 132-138).
3. IMMOBILIZED ENZYMES

The use of a relatively expensive catalyst as an enzyme requires, in many instances,

its recovery and reuse to make an economically feasible process. Moreover, the use of an
immobilized enzyme permits to greatly simplify the design of the reactor and the control of
the reaction
Immobilized enzymes have been defined as enzymes that are physically confined or
localized, with retention of their catalytic activity, and which can be used repeatedly and
continuously

Imobilization

Matrix binding Entrapment

a) adsorption b) Covalent c)In the gel d) encapsulation

linkage matrix

Types of matrices
• natural polymers (polysacharides, proteines)
• Synthetic polymers (polystyrene, polyacrylate, hydroxylakyl methacrylate, tec).
• anorganické nosiče ( minerals, active coal, poous glass, porous metal oxides)

Matrices has to have special characteristics:

• Biocompatibility
• Large surface area and high permeability
• Sufficient functional groups for enzyme attachment under nondenaturing
conditions
• Hydrophilic character
• Water insolubility
• Chemical and thermal stability
• Mechanical strength
• High rigidity and suitable particle form
• Resistance to microbial attack
• Regenerability
• Toxicological safety
• Low or justifiable price
Effect of immobilization on the properties:
• Inactivation of enzyme by reactants or products of the immobilization reaction
• reaction conditions of the immobilization reaction
• Enzyme protein molecule can be fixed in the inactive form
• coupling reaction performed via functional groups in active site
• Orientation of the enzyme molecule on the surface of the solid support prevent the
access of substrate
• Influence of the functional groups of the support (Fig. 3)

a) Positively charged
matrix
b) Native enzyme
c) Negatively charged
matrix
4. – 5. CHARACTERISTICS OF ENZYMES FOR INDUSTRIAL AND
BIOTECHNOLOGICAL APPLICATIONS I, II

Up to 80% of enzymes used for technological application are hydrolases from which
dominate proteases and glycosidases. About 12% of total consumption represent glucose
isomerise used in immobilized form for the production of high fructose corn syrup (HFCS).
Thus other types of enzymes are much less used, mainly in special technologies or for
research purposes.
In this chapter we will focus on the representatives of each enzyme class, their
characteristics and, in some unique cases, their applications.

Oxidoreductases:
Oxidoreductases are enzymes catalyzing the transfer of electrons or hydrogens from
one molecule (the reductant, also called the hydrogen or electron donor) to another (the
oxidant, also called the hydrogen or electron acceptor). They are mainly involved in
oxidative reactions in degradation reactions of the metabolism, cellular respiration and
energy transfer. They are in the centre of attention namely in food technologies as they can
affect the processes in positive as well as in the negative manner. Their other applications
are limited mainly by the fact that most of them need coenzymes (such as NAD+) in high
concentrations which negatively influences the economy of the process.

Glucose oxidase
β-D-glucose:O2 1-oxidoreductase (GOD), EC 1.1.3.4 is produced by microorganisms,
above all by fungi Aspergillus and Penicillium. For its antimicrobial effect it was
considered to be an antibiotic shown later to be due to the peroxide formation, then
renamed to notatin. It was isolated also from higher fungi, red algae and citruses.
Catalyzed reaction:
-D-glucose + O2 → -D-gluconolacton + H2O2

GOD is a glycoprotein composed from two identical subunits of about 80 kDa. Mh of the
protein differs according to the origin but is around 160 kDa. There is one molecule of
FAD an one Fe atom per each subunit. Saccharide component consist of 16% of neutral
sugars and 2% of amino sugars. Characteristics of GOD from A.niger: pH optimum 5,5,
with a broad range 4 – 7, temperature stability depends on the redox state of the enzyme,
denaturing temperature for the oxidized form is 76 ºC while for reduced is 10 degrees
higher.

Inhibitors: Ag2+, Hg2+, Cu2+, D-glucal, H2O2 (E-FADH2 100x more)

GOD is highly specific towards β-anomer of D-glucose (see table )

 Substrate specificity of glucose oxidase
Relat. Relat.
Substrate* oxidation Substrate* oxidation
Rate . Rate
(%) (%)
-D-glukosa 100 2-deoxy-D-glukosa 20 – 30
-D-glukosa 0,64 4-O-methyl-D-glukosa 15
L-glukosa 0 6-deoxy-D-glukosa 10
D-mannosa 1,0
D-xylosa 1,0 *about 100 derivatives were tested
D-galaktosa 0,5
maltosa 0,2
melibiosa 0,1
cellobiosa 0,09

Activity of GOD could be assayed in principle by

1. measuring the O2 uptake by Clark’s oxygen sensor
2. measuring the production v hydrogen peroxide
3. titration of gluconic acid spontaneously formel from -D-gluconolacton

Application of GOD:

• Removal of glucose from food products (Maillards reaction)

• Removal of oxygen – (antioxidative agent, preservative, stabilization)
• Production of gluconic acid (food additives, textile industry, detergents, cosmetics,
concrete)
• Production of hydrogen peroxide (preservation)
• Quantitative analysis of glucose or compounds which can be transformed to glucose
• activity assay of enzymes producing glucose or other compounds which can be
transformed to glucose

Peroxidases
Peroxidases (EC 1.11.1.1 -15) are haem proteins and contain iron (III)
protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group. They have a molecular
weight ranging from 30,000 to 150,000 Da. Represent a numerous group of
oxidoreductases that catalyze the reduction of peroxides, such as hydrogen peroxide and
the oxidation of a variety of organic and inorganic compounds:

ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH

Peroxidases could be divided to two superfamilies: 1. mammalian (lactoperoxidase and

myeloperoxidase) and 2. plant and microbial which are further divided to three subgroups:
Class I – intracellular (yeast cytochrom c peroxidise, chloroplastic nad cytosolic ascorbate
peroxidase and bacterial peroxidises)
Class II - extracellular from fungi (lignin peroxidise, Mn peroxidise and other from
basidiomycetes)
Class III - plant extracellular peroxidases (horse raddish peroxidise)

Peroxidases are involved in decomposition of toxic hydrogen peroxide in cells but due
to their ability to oxidize various compounds they posses a substantial potential for various
applications:
1. Analytical application
2. Antibody labelling
3. Biotransformations - hydroxylations
4. Biodegradation of polutants (dayes, polychlorinated biphenyls etc.)
5. biochemical changes in food raw materials

Catalase
EC 1.11.1.6, H2O2:H2O2 oxidoreductase
Common enzyme found in nearly all living organisms which are exposed to oxygen, where
it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen.
Catalase has one of the highest turnover numbers of all enzymes
2 H2O2 → 2 H2O + O2
Catalase is a heme protein, tetramer of Mh 250 kDa

Lipoxygenase
EC 1.13.11.12, linoleát:O2 oxidoreductase, other names lipoxidase, carotenoxidase
Family of non-heme iron-containing enzymes that catalyse the dioxygenation of
polyunsaturated fatty acids in lipids containing a cis,cis-1,4- pentadiene structure (linoleic,
linolenic and arachidonic acid are most common substrates). It catalyses the following
reaction:
fatty acid + O2 → fatty acid hydroperoxide

These enzymes are most common in plants where they may be involved in a number of
diverse aspects of plant physiology including growth and development, pest resistance, and
senescence or responses to wounding. This is due to the following reactions forming
oxylipins.

In mammals a number of
lipoxygenases isozymes
are involved in the
metabolism of
prostaglandins and
leukotrienes.
Polyphenl oxidase
EC 1.10.3.1, o-difenol: O2 oxidoreductase
Polyphenol oxidase catalyses the o-
hydroxylation of monophenols (phenol
molecules in which the benzene ring contains a
single hydroxyl substituent) to o-diphenols
(phenol molecules containing two hydroxyl
substituents). They can also further catalyse the
oxidation of o-diphenols to produce o-
quinones. It is the rapid polymerisation of o-
quinones to produce black, brown or red
pigments (polyphenols) that is the cause of
enzymatic browning.

Tyrosinase, phenol oxidase, EC 1.14.18.1

Laccase EC 1.10.3.2

Transferases
Enzymes that catalyse the transfer of a functional group (e.g. a methyl or phosphate group)
from one molecule (donor) to another (acceptor):
A–X + B → A + B–X
This class of enzymes is not commonly used for technological purposes. Rarely are used in
biotransformation reactions or as a part of bioremediation machinery. We have to take in
an account that many hydrolases posses under specific conditions the transferase activity.

Hydrolases
The most widely used class of enzymes, up to 80% of all enzymes technologically
significant are of this type, most of them being proteases or glycosidases.
Hydrolases catalyse hydrolytic cleavage of a chemical bond:
X-Y + H-OH → H-X + Y-OH
In the enzyme nomenclature, hydrolases are divided to subgroups according to the
character of cleaved bond (ester, peptide bond, ether bond, glycosidic etc.) From
technological point of view it is more practical to classify hydrolases according to their
substrate ( starch degrading enzymes, pectolytic enzymes, proteolytic etc.)

Distribution of enzyme sales. The

contribution of different enzymes to
the total sale of enzymes is indicated.
The shaded portion indicates the total
sale of proteases.
Proteases
Enzymes hydrolyzing peptide bonds in proteins and peptides, often having additional
activities such as coagulation, transpeptidase or esterase activity. They can be divided
according to their origin (animal, plant, microbial), preferential site of cleavage (exo- and
endopeptidases, amino- orcarboxypeptidases), pH-optimum (acid, neutral, alkaline), or
mechanism of catalysis ( serine, aspartic, sulfhydryl or metalloproteases).
Majority of these enzymes is synthetized in the organism in non-active form (zymogen or
proenzyme) and they are activated in the site of their action by proteolytic cleavage of their
molecule.

Major protease producers

Company Country Market share (%)

Novo Industries Denmark 40
Gist-Brocades Netherlands 20
Genencor International United States 10
Miles Laboratories United States 10
Others 20

Animal proteases
Trypsin. Trypsin (Mr 23,300) is the main intestinal digestive enzyme responsible for the
hydrolysis of food proteins. It is a serine protease and hydrolyzes peptide bonds in which
the carboxyl groups are contributed by the lysine and arginine residues (Table 2). Based on
the ability of protease inhibitors to inhibit the enzyme from the insect gut, this enzyme has
received attention as a target for biocontrol of insect pests. Trypsin has limited applications
in the food industry, since the protein hydrolysates generated by its action have a highly
bitter taste. Trypsin is used in the preparation of bacterial media and
in some specialized medical applications.
Chymotrypsin. Chymotrypsin (Mr 23,800) is found in snímal pancreatic extract. Pure
chymotrypsin is an expensive enzyme and is used only for diagnostic and analytical
applications. It is specific for the hydrolysis of peptide bonds in which the carboxyl
groups are provided by one of the three aromatic amino acids, i.e., phenylalanine, tyrosine,
or tryptophan. It is used extensively in the deallergenizing of milk protein hydrolysates. It is
stored in the pancreas in the form of a precursor, chymotrypsinogen,
and is activated by trypsin in a multistep process.
Pepsin. Pepsin (Mr 34,500) is an acidic protease that is found in the stomachs of almost all
vertebrates. The active enzyme is released from its zymogen, i.e., pepsinogen, by
autocatalysis in the presence of hydrochloric acid. Pepsin is an aspartyl protease
and resembles human immunodeficiency virus type 1 (HIV-1) protease, responsible for the
maturation of HIV-1. It exhibits optimal activity between pH 1 and 2, while the optima pH
of the stomach is 2 to 4. Pepsin is inactivated above pH 6.0. The enzyme catalyzes the
hydrolysis of peptide bonds between two hydrophobic amino acids.
Chymosin is a pepsin-like protease (rennin, chymosin; EC 3.4.23.4) that is produced in as
an inactive precursor, prorennin, in the abomasum of all nursing mammals. It is converted
to active rennin (Mr 30,700) by the action of pepsin or by its autocatalysis. It is used
extensively in the dairy industry to produce a stable curd with good flavor. The specialized
nature of the enzyme is due to its specificity in cleaving a single peptide bond in κ -casein
to generate insoluble para-κ-casein and C-terminal glycopeptide.

Plant proteases
The use of plants as a source of proteases is governed by several factors such as the
availability of land for cultivation and the suitability of climatic conditions for growth.
Moreover, production of proteases from plants is a time-consuming process.Papain,
bromelain, keratinases, and ficin represent some of the well-known proteases of plant
origin.
Papain. Papain is a traditional plant protease and has a long history of use. It is extracted
from the latex of Carica papaya fruits, which are grown in subtropical areas of west and
central Africa and India. The crude preparation of the enzymehas a broader specificity due
to the presence of several proteinase and peptidase isozymes. The performance of the
enzyme depends on the plant source, the climatic conditions for growth, and the methods
used for its extraction and purification. The enzyme is active between pH 5 and 9 and is
stable up to 80 or 90°C in the presence of substrates. It is extensively used in industry for
the preparation of highly soluble and flavored protein hydrolysates.
Bromelain. Bromelain is prepared from the stem and juice of pineapples. The major
supplier of the enzyme is Great FoodBiochem., Bangkok, Thailand. The enzyme is
characterized as a cysteine protease and is active from pH 5 to 9. Its inactivation
temperature is 70°C, which is lower than that of papain.
Keratinases. Some of the botanical groups of plants produce proteases which degrade hair.
Digestion of hair and wool is important for the production of essential amino acids such as
lysine and for the prevention of clogging of wastewater systems.

Microbial proteases
The inability of the plant and animal proteases to meet current world demands has
led to an increased interest in microbial proteases. Microorganisms represent an excellent
source of enzymes owing to their broad biochemical diversity and their susceptibility to
genetic manipulation. Microbial proteases account for approximately 40% of the total
worldwide enzyme sales. Proteases from microbial sources are preferred to the enzymes
from plant and animal sources since they possess almost all the characteristics desired for
their biotechnological applications.
Bacteria. Most commercial proteases, mainly neutral and alkaline, are produced by
organisms belonging to the genus Bacillus. Bacterial neutral proteases are active in a
narrow pH range (pH 5 to 8) and have relatively low thermotolerance. Due to their
intermediate rate of reaction, neutral proteases generate less bitterness in hydrolyzed food
proteins than do the animal proteinases and hence are valuable for use in the food industry.
Neutrase, a neutral protease, is insensitive to the natural plant proteinase inhibitors and is
therefore useful in the brewing industry. The bacterial neutral proteases are characterized
by their high affinity for hydrophobic amino acid pairs. Their low thermotolerance is
advantageous for controlling their reactivity during the production of food hydrolysates
with a low degree of hydrolysis. Some of the neutral proteases belong to the
metalloprotease type and require divalent metal ions for their activity, while others are
serine proteinases, which are not affected by chelating agents. Bacterial alkaline proteases
are characterized by their high activity at alkaline pH, e.g., pH 10, and their broad substrate
specificity. Their optimal temperature is around 60°C. Theseproperties of bacterial alkaline
proteases make them suitable for use in the detergent industry.
Fungi. Fungi elaborate a wider variety of enzymes than do bacteria. For example,
Aspergillus oryzae produces acid, neutral, and alkaline proteases. The fungal proteases are
active over a wide pH range (pH 4 to 11) and exhibit broad substrate specificity. However,
they have a lower reaction rate and worse heat tolerance than do the bacterial enzymes.
Fungal enzymes can be conveniently produced in a solid-state fermentation process. Fungal
acid proteases have an optimal pH between 4 and 4.5 and are stable between pH 2.5 and
6.0. They are particularly useful in the cheesemaking industry due to their narrow pH and
temperature specificities. Fungal neutral proteases are metalloproteases that are active at
pH 7.0 and are inhibited by chelating agents. In view of the accompanying peptidase
activity and their specific function in hydrolyzing hydrophobic amino acid bonds, fungal
neutral proteases supplement the action of plant, animal, and bacterial proteases in
reducing the bitterness of food protein hydrolysates. Fungal alkaline proteases are also used
in food protein modification.

Specificity of some proteases

Protease Specificity
Trypsin -Lys (-Arg) -↓
Chymotrypsin, Subtilisin -Trp (tyr, Phe, Leu)- ↓
Papain -Phe (Val, Leu)-Xaa-↓
Thermolysin -↓ Leu (Phe)
Pepsin -Phe(Tyr, Leu) -↓-Trp (phe,Tyr)
Glycosidases
Enzymes hydrolyzing glycosidic linkage in homo- and hetero- oligo- and polysaccharides,
found in essentially all domains of life.
From technological point of view they are usually divided according to the substrate cleved
as follows:

Starch degrading enzymes

Endoamylases - α-amylase (α-1,4)
Exoamylases - β-amylase (α-1,4), glukoamylases (α-1,4 a α-1,6), α-glukosidases (α-1,4 a
α-1,6)
Debranching enzymes- pullulanases (α-1,6) and isoamylases (α-1,6)
Transferases - Cyclodextrin glycosyltransferase (α-1,4), branching enzyme (α-1,6)

Enzymes degrading plant cell wall components

Cellulolytic enzymes
Class of enzymes produced chiefly by
fungi, bacteria, and protozoans that
catalyze hydrolysis of cellulose.
However, there are also cellulases
produced by other types of organisms
such as plants and animals. Several
different kinds of cellulases are known,
which differ structurally and
mechanistically. These enzymes catalyse
hydrolysis of 1,4-β-D-glycosidic
linkages in cellulose, lichenin and cereal
β-D-glucans.
Five general types of cellulases based on the type of reaction catalyzed:
 Endo-cellulase breaks internal bonds to disrupt the crystalline structure of cellulose
and expose individual cellulose polysaccharide chains
 Exo-cellulase cleaves 2-4 units from the ends of the exposed chains produced by
endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose.
There are two main types of exo-cellulases (or cellobiohydrolases, abbreviate CBH)
- one type working processively from the reducing end, and one type working
processively from the non-reducing end of cellulose.
 Cellobiase or β-glucosidase hydrolyses the exo-cellulase product into individual
monosaccharides.
 Oxidative cellulases that depolymerize cellulose by radical reactions, as for instance
cellobiose dehydrogenase (acceptor).
 Cellulose phosphorylases that depolymerize cellulose using phosphates instead of
water.

Hemicellulases
Enzymes degrading heteropolysaccharides of plant cell wall such as xylans, xyloglucans,
glucomannanes

Pectolytic enzymes
Pectin is a linear chain of α-(1-4)-linked D-
galacturonic acid that forms the pectin-
backbone, a homogalacturonan. Into this
backbone, there are regions where
galacturonic acid is replaced by (1-2)-linked
L-rhamnose. From the rhamnose residues,
sidechains of various neutral sugars branch
off. This type of pectin is called
rhamnogalacturonan I. Up to every 25th
galacturonic acid in the main chain is replaced
with rhamnose. Some stretches consist of
alternating galacturonic acid and rhamnose –
“hairy regions”, others with lower density of
rhamnose – “smooth regions”. The neutral
sugars are mainly D-galactose, L-arabinose and D-xylose, the types and proportions of
neutral sugars varying with the origin of pectin.
Rhamnogalacturonans (RGs) are a group of closely related cell wall pectic polysaccharides
that contain a backbone of the repeating disaccharide: 4)-α-D-GalpA-(1,2)-α-L-Rhap-(1,2).
The term rhamnogalacturonan I (RG-I) is typically used to refer to this pectic
polysaccharide.
Another structural type of pectin is rhamnogalacturonan II (RG-II), which is a less frequent
complex, highly branched polysaccharide. In nature, around 80% of carboxyl groups of
galacturonic acid are esterified with methanol.
The pectinolytic enzymes may be divided in three broader groups as follows:
(I) Protopectinases: degrade the insoluble protopectin and give rise to highly polymerized
soluble pectin.
(II) Esterases: catalyze the de-esterification of pectin by the removal of methoxy esters.
(III) Depolymerases: catalyze the hydrolytic cleavage of the α-(1 → 4)-glycosidic bonds in
the D-galacturonic acid moieties of the pectic substances.
Depolymerases act on pectic substances by two different mechanisms, hydrolysis, in which
they catalyze the hydrolytic cleavage with the introduction of water across the oxygen
bridge and trans-elimination lysis, in which they break the glycosidic bond by a trans-
elimination reaction without any participation of water molekule.

Invertase (EC 3.21.26)

Cleaves glycosidic bond in saccharose on the site of fructose. Glycoprotein in which the
saccharides form about 50% of the molecule. Used for the production of invert sugar.

β-galaktosidase (EC 3.2.1.23)

Enzyme which catalyzes hydrolytic cleavage of beta galactosides, i.e. lactose to glucose
and galactose.

Lipases (triacylglycerol acylhydrolases,

E.C. 3.1.1.3) are ubiquitous enzymes of
considerable
physiological significance and industrial
potential. Lipases catalyze the hydrolysis
of triacylglycerols to glycerol and free
fatty acids. In contrast to esterases, lipases
are activated only when adsorbed to an
oil–water interface. Lipases are serine
hydrolases with broad specificity. Lipases
display little activity in aqueous solutions
containing soluble substrates. In contrast,
esterases show normal Michaelis–Menten
kinetics in solution. In eukaryotes, lipases
are involved in various stages of lipid
metabolism including fat digestion,
absorption, reconstitution, and lipoprotein
metabolism. In plants, lipases are found in
energy reserve tissues. For technological
purposes predominantly lipase from
microbial sources are used.
Reactions catalysed by lipases

Phospholipases are degrading phospholipds. They

are divided into groups according to the site of
cleavage of the substrate molecule (scheme).
Products of phospholipase action are signalling
molecules as phosphatidic acid, diacylglycerol or
inositol trisphosphate or lysophospholipids which
have the emulsifying ability.
Phospholipase D catalyses, except the hydrolytic cleavage , in presence of secondary
alcohol the tranphosphatidylation reaction in which phosphatidate moiety is transferred
preferentially to the hydroxyl group of alcohol (instead of water molecule) forming
phosphatidylalcohol.
This reaction could be used for synthesis of various compounds.

H-O-H H-O-R

H R
6. – 7. ENZYMES IN FOOD INDUSTRY I,II

Starch degradation

Starch is the commonest storage carbohydrate in plants. It is used by the plants themselves,
by microbes and by higher organisms so there is a great diversity of enzymes able to
catalyse its hydrolysis. Starch from all plant sources occurs in the form of granules which
differ markedly in size and physical characteristics from species to species. Chemical
differences are less marked. Acid hydrolysis of starch has had widespread use in the past
and it required the use of corrosion resistant materials, gave rise to high colour and saltash
content (after neutralisation), needed more energy for heating and was relatively difficult to
control. It is now largely replaced by enzymatic processes, which was mostly enabled by
the discovery of termostable amylases from microorganisms ( Bacillus amyloliquefaciens,
Bacillus licheniformis )

High-fructose corn syrup (HFCS) – glucose syrup that has undergone enzymatic
processing to convert glucose into fructose. In the United States, HFCS is typically used as
a sugar substitute and is ubiquitous in processed foods and beverages. Xylose isomerase
(glucose isomerase) converts glucose to a mixture of about 42% fructose and 50–52%
glucose with some other sugars mixed in.
Products of starch hydrolysis: maltose and glucose syrups, starch hydrolysis products
(SHP) with DE 5– 8 which maintain the ability to form thermoreversible gels without the
strach flavour, maltodextrines , cyclodextrines.
Modified starches

Progress in understanding
starch biosynthesis and the
cloning of many genes
involved in the prosess has
enabled the genetic
modification of crops in a
rational manner to produce
novel starches with
improved functionality:

Amylose free (waxy) starches gelatinizing easily, waxy starch with short chain amylopectin
possessing high freeze-thaw stability. Alteration of pohosphate level by antisence
inhibition of GWD (α-glucan water dikinase) in amylose could lead to the regulation of the
viscosity of starch slurry.

Dairy industry

Cheese processing –
About 80% of milk protein consists of casein which is hydrophobic in nature. The bovine
caseins are subdivided to four species: αS1-, αS2-, β- and κ-casein occuring in relative
molar ratio 4:1:4:1.6. First three types are highly phosphorylated. Caseins aggregate to
form submicelles which together form micelles the outer surface consisting submicelles
containing relatively high content of κ-casein molecules. Hydrophilic parts of κ-caseins
protrude from the periphery of the
micelle and guarantee its stability due to
electrostatic and entropic reppulsion.
Coagulating anzymes (chymosin,
rennets) specifically split off a distinct
part (casein macro peptide) of κ-casein
hydrolyzing the Phe-Met peptide linkage
and thus unmask the hydrophobic part of
κ-casein calle para- κ-casein. This leads
to the destabilization of casein micelles
which aggregate with each other. Calcium ions facilitate the aggregation due to the
attachmnet to the phosphate groups.
Types of rennets used in cheese production
Rennet source Commercial Comment
preparation
Animal bovine abomasum Stabo 100% pepsin
Bovine + calf Cabo 60 – 100% chymosin
calf

kůzlečí Grandine
Microbial Mucor pusillus Emporase,
Renzyme
Rhizomucor miehei Fromase, Rennilase
Cryphonectria Superen, Switzerland, Italy
parasitica Thermolase
Recombinant Aspergillus niger Chymogen, Not allowed in all countries
Chymostar
Aspergillus oryzae Novoren Used intensively since 1994
Escherichia coli Chymax Not allowed in all countries
Kluyveromyces Maxiren Not allowed in all countries
marxianus
Plant Artyčok kardavý Cardoon Serra de Estrela Portugal

Cheese ripening
The conversion of fresh cheese curd into mature cheese is largely determined by
proteolysis. Other factors, such as lipolysis (essential in hard Italian and in moldripened
cheeses) and lactic and propionic acid fermentation (the latter especially important for
flavor development and eye formation in Swiss-type cheese may also play a role.Natural
ripening process is long lastin and thus costly. Thus there is an attempt to accelerate cheese
flavor development and ripening process. Except the temperature elevation the addition of
proteolytic and lipolytic enzymes by modification of starter cultures is one of the
approaches.

β-galaktosidase
1. milk lactose cleavage
 non lactose milk for the
population intolerant to
lactose
 milk for production of
daity products such as
kondensed milk, yogurt
or ice creams
2. Hydrolysis of lactose in
whey by immoblized
enzyme
3.
 batch (4 h, 37 °C resp. 24 h, 8 °C)
 batch process with ultrafiltration
 continuous
Hydrolysed whey usage:
Sweetener of dairy, confectionery, bakery products and nonalcoholic beverages
Enhancement of fermentation (yogurts, quark, beer and wine)
Controled browning in bakery and confectionery products
Replacement of milk in ice creams
Non-lactose product
Production of alcohol
Pet feed

Protein hydrolysis
Hydrolysis of protein covers broad range of food technologies and the main purposes for
partially hydrolysed protins are:
- Change of physico-chemical properties of proteins such as solubility and
extractability, whipping ability and the ability to bound water.
- Nutritional and senzoric value
- Texture of raw materials
- Lowering of the alergenicity of certain proteins
 Major application of protein hydrolysates
Proteins Properties/changes Application/Advantges
Plant
soya solubility Increase of digestibilty, nutrition value, substitut
of egg white
wheat Taste and flavour Food additives
Hydration/rheology Baking industry
solubility Increased sugestibility and nutrition value
pea Taste and flavour Food additives
solubility
corn solubility Animal feed
Animal
Meat Texture Food additives
solubility animal feed
Taste and flavour
Milk coagulation Cheese production
solubility Increase of nutritional value
Decrease of alergenicity
Blood Solubility, taste and flavour Food additives
Leather texture tanning
Microbial
Yeast Solubility, taste and flavour Food and feed, fermentation broth
Food additives
germ solubility Animal feed
Mixed
wastes solubility Transfer to waste water

Possibility of bitter peptides formation are the major problem in protein hydrolysates
used for food products. Non-terminal hydrophobic amino acid residues in medium-sized
oligopeptides give bitter flavours, the bitterness being less intense with smaller peptides
and disappearing altogether with larger peptides.
Intact food proteins do not display bitterness as their molecular size militates against their
ability to interact extensively with bitterness receptors in the oral cavity. Bitterness in
protein hydrolysates has been classically associated with the release of peptides containing
hydrophobic amino acid residues. Peptide bitterness was quantified on the basis of the
hydrophobicity of the side-chains associated with the residues within a given peptide
sequence. This was calculated from the free energy of transfer of an amino acid side chain
from ethanol to water. From synthetic peptide studies, it was reported that peptides with
hydrophobicity (Q) values>1400 cal per mole and molecular masses <6 kDa display
bitterness. Even though peptides<6 kDa having a high content of Leu, Pro, Phe, Tyr, Ile
and Trp residues are likely to be bitter. the presence of internally sited hydrophobic amino
acid residues lead to greater bitterness than when the hydrophobic residues are located at
either the N- or C-terminus in peptides. The presence of internally sited Pro residues was
shown to be a major and distinct contributor to peptide bitterness due to the unique
conformation associated with this imino acid.
Protein hydrolysate debittering
Numerous options have been investigated in the debittering of food protein
hydrolysates. These include absorption of bitter peptides on activated carbon,
chromatographic removal using different matrices and selective extraction with alcohols.
These procedures, however, lead to the loss of some amino acid residues from
hydrolysates. Bitterness has also been masked in hydrolysates via the addition of
polyphosphates, specific amino acids such as Asp and Glu, a-cyclodextrins and by the
admixture of hydrolysates with intact protein samples. The debittering of hydrolysates via
transpeptidation reactions in the so-called T plastein reaction in addition to cross-linking
using transglutaminase represent other potential routes for hydrolysate bitterness reduction.
However, these latter protocols may not always be suitable when products having high
solubility are required. Much effort has concentrated on the use of peptidases, specifically
exopeptidases including amino- and carboxypeptidases, in food protein hydrolysate
debittering. Significant reductions in bitterness have been observed in proteinase digests of
food proteins during concomitant or subsequent incubation with exopeptidase-rich enzyme
preparations, particularily peptidases which cleave adjacent to hydrophobic amino acid
residues.
Given the unique contribution ofvPro residues to hydrolysate bitterness, much
research has focused on the application of proline specific exopeptidases in hydrolysate
debittering strategies due to the general inability of most general aminopeptidases to
hydrolyse the imino bond.

Baking industry
Amylases for flour standardization
The most widely used enzymes in bread making in terms of amount dosed is fungal
alpha amylase from Aspergillus oryzae (Taka amylase)It is supplemented to flours to
optimize their alpha amylase activity with regard to final volume and crumb structure of
the final product. The advantage of Taka amylase preparation is a very low protease
activity and appropriate thermostability (comparing to bacterial amylases) enabling the
inactivation of an enzyme after initial gelatinization of starch.
Antistaling amylases
Use of amylases as antistalling agents is second role of these enzymes in baking
industry. It is important to keep baked products fresh as long as possibleStaling is a higly
complex process but it is generally accepted that the retrogradation of starch is them main
process influencing this phenomenon. For this purpose, the termostable amylases should be
used as their action is desirable after finishing the baking procedure. Thus the dosage of
these enzymes is a crucial problem to be solved with respect to the influence of the
amylases on the volume and consistence of the products.
Xylanases (pentosanases)
In dough, xylanases, traditionally named pentosanases, reduce problems associated
with variations in flour quality, improve dough handling and dough stability, give
improved crumb structure and bread volume.
Oxidoreductases
Glucose oxidase generated hydrogen peroxide reacts with free SH groups of gluten
forming disulfide bridges and thus increasing the gluten strength. In combination with
xylanases nonsticky, dry dough is obtained.
Application of peroxidases was proven to have an dough improving effect as well.
Lipoxygenase comes with the soybean or bean flour usage. Co-oxidation reactions of
hydroperoxides produced by this enzyme lead to the whitening of bread crumb.

BEVERAGES
Brewing
The major biological changes which occur in the brewing process are
catalyzed by naturally produced enzymes from barley and yeast
respectively.
But in cases when very high levels of unmalted cereals are used the
exogenous enzymes has to be added. Current practise is to replace up to
50% of the malt with barley. Bacterial proteases and alpha- amylases
from Bacillus subtilis are added to enhance the proteolysis and
saccharification. Heat stable beta-glucanase is another enzyme added in this processes.
Low carbohydrate beers “lite” are also produced with the aid of exogenous enzymes –
fungal alpha-amylase or beta-amylase with pullulanase. For the production of low
fermentability beers with low or no content of alcohol heat stable fungal beta-glucanase
and alpha-amylase are used.
Filtration could be enhanced by beta-glucanases which lower the viscosity of wort and
beer.

Beer haze formation

Beer haze may be defined as an insoluble or semisoluble
particulate matter which is small enough to form a
colloidal suspension in beer scattering the light.
It is formed basically by polysaccharides of plant cell
walls, polyphenols and proteins (hydrophobic protein
hordein). The most common beer haze formation in
packaged beers is the agglomeration of proteins and
polyphenolic compounds:
Stabilization of beer could be achieve except by the “classical procedures” also with the aid
of proteolytic enzymes. Most commonly used for this purpose is papain, plant sulfhydryl
protease from Carica papaya.
Beer maturation can be shorten by adding the enzyme alpha-acetolactate decarboxylase
(e.g.Novozymes' Maturex®) at the beginning of the primary fermentation process, it is
possible to bypass the diacetyl step (Figure) and convert alpha-acetolactate directly into
acetoin. Most of the alpha-acetolactate is degraded before it has a chance to oxidise and
less diacetyl is therefore formed. This makes it possible to shorten or completely eliminate
the maturation period.

WINE PRODUCTION
Even in this very traditional manufacture axogenous enzymes are now widely used (20
million hl out of 60 million are now treated with enzymes in France).
Principally the enzymes are used for two purposes. First the enzymes degrading the plant
cell wall components (mainly pectolytic) are used for higher yield of must and in red wines
the addition of pectolytic enzymes allowes the reduction of the skin maceration time.
Second the enzymes are use for the release of wine aromas from aroma precursors
(glycosylated terpenols) by certain glycosidases (alpha-rhamnosidase, alpha-arabinosidase
etc)

FRUIT AND VEGETABLE PROCESSING

Plant material is widely used for the production of valuable food and feed products.
Many ingredients used for beverages, food and feed are produced by the extraction of plant
raw materials.
Various combination of cell wall
degrading enzymes is used for fruit
anf vegetable processing: peeling of
citrus fruits, maceration processes
(fruit and vegetable mashes),
production of fermentable
saccharides.
Enzymatic peeling of citrus fruit is used in the production of fresh peeled fruit, fruit salads
and segments. Enzymatic treatment results in citrus segments with improved freshness as
well as better texture and appearance compared with the traditional process using caustic
soda.

Fruit juice processing

Pectolytic enzyme ( pectin esterase and polygalacturonases) preparations have been

used for more than 60 years in fruit juice production. Today, they play a key role in modern
fruit juice technologies. They are a prerequisite for obtaining clear and stable juices, good
yields, and high-quality concentrates. Enzymes are used for better removal of cloudy haze
in fruit juices, more effective filtration and to improve taste and flavour especially of citrus
juices. The bitterness of these fruits is caused by the presence of naringine, but splitting off
the rhamnose residue by alpha-rhamnosidase leads to thwe substantial decrease of bitter
taste.

OIL PROCESSING
Oil from rapeseed, coconut, maize germ, sunflower seed, palm kernels and olives is
traditionally produced by expeller pressing followed by extraction with organic solvents.
The solvent most commonly used in this process is hexane, which has been identified as a
hazardous air pollutant by recent environmental regulations. Cell wall degrading enzymes
offer a safe and environmentally responsible alternative. They can be used to extract
vegetable oil in an aqueous process by degrading the structural cell wall components. This
concept has already been commercialised in olive oil processing, and it has been
thoroughly investigated for rapeseed oil, coconut oil and maize germ oil. In olive oil
processing, the efficacy of enzymes such as Olivex® has been proved by numerous
independent studies in most olive oil producing countries. Olivex improves both yield and
plant capacity, while no negative effects on the oil have been found. On the contrary, the
quality of the oil is enhanced in many cases.

Enzymatic degumming
Enzymatic degumming is a physical refining process in which a phospholipase
converts non-hydratable phosphatides into fully hydratable lysolecithin. This facilitates
gum removal. In most physical refining methods, a fundamental criterion should be that the
crude oil is degummed as effectively as possible. Traditionally, chemical refining uses
large amounts of caustic soda (NaOH) as a main refining component. The enzymatic
degumming process using phospholipase A2 has many benefits. Lysophospholipids
produced are better soluble in water and have the character of emulsifiers. Enzymatic
degumming works with crude oil as well as water-degummed oil. An overall higher yield is
obtained because the gums contain up to 25% less residual oil, and because no soapstock is
produced, no oil is lost.

RE- TAILORING OF LIPIDS

Interesterification or ester-ester exchange is a process during which the fatty acids
of triglycerides exchange positions from one glyceride to another, thereby altering the
overall chemical composition and physical properties of the interesterified fats. Interesteri-
fication is thus an efficient way for changing and controlling the melting characteristics of
oils and fats and their nutritional value.
Lipase
8. BIOCHEMICAL CHANGES IN FOOD ROW MATERIALS AND PRODUCTS

The quality of food products of plant origin is influenced by various factors and has to be
considered as very complex process. The effects and processes involved in processing of
plant row materials and products are as follows:
a) Natural, in vivo, processes of fruit ripening and tissue senescence
 Changes in cell wall structure an pectin of middle lamellae
 Intracellular processes
b) Stress – water deficit, draught, mechanical damage (wounding, insect feeding),
infection
 Oxidative burst
 Accumulation of phytoalexins, polyamines, phenolic compounds, defence
proteins, calose deposition etc.
 Activation of polyphenol oxidases
c) Postharvest processes
 Mechanical processing (cutting, mashing etc)
 Physiological – postharvest metabolism (up to final stadium – necrosis)

Plant senescence – principal processes:

Metabolism of pigments an phenolic compounds
Metabolism of proteins
Changes in photosynthesis
Oxidative metabolism
Lipids → sacharides (glyoxylate cycle)
Production of reactive oxygen species (ROS)
Degradation of nucleic acids
Regulation of metabolism - endogenous regulators (ethylene)

The most frequently studied model – tomato (Lycopersicon ecsulentum):

Modification of the texture of tomato fruits:
Texture of fruit tissue is mainly influenced by pectolytic enzymes
Polygalacturonase (PG) is not detected in non-ripened tomatoes, it is synthetised de novo
during ripening (1 gene coding for two isoforms mRNA of which represents 1% of total
mRNA in ripening tomato fruit!) Pectin esterase (PE) is constitutively expressed and coded
by multiple genes. Anti sense technology was used for improving the texture by lowering
the expression of these two enzymes. Tomato puree produced from mutant fruits analysed
by Bostwick test showed the same characteristics under low temperature as in case of hot
break treatment.

Modification or retardation of ripening in tomatoes by lowering the ethylene production. It

could be achieved by
a) antisence technology producing antisense amino cyclopropane
carboxylic acid (ACC) synthase or ACC oxidase.
b) Transgenic plants bearing the bacterial enzyme ACC deaminase
degrading the ethylene precursor.

Changes during the food processing:

1. Taste and flavour - lipoxygenase, lipases, fosfolipases, proteases

2. Texture and consistency - pectolytic and cellulolytic enzymes
3. Colour changes – peroxidise, polyphenol oxidase
4. changes in nutritional value – ascorbate oxidase, thiaminase etc.

Enzymatic browning
Half of the world's fruit and vegetable crops is lost due to postharvest deteriorative
reactions. Polyphenol oxidase (PPO), found in most fruit and vegetables, is responsible for
enzymatic browning of fresh horticultural products, following bruising, cuffing or other
damage to the cell.
Physiological function of polyphenol oxidase

Browning results from both enzymatic (PPO) and non-enzymatic oxidation of

phenolic compounds. Browning usually impairs the sensory properties of products because
of the associated changes in colour, flavour and softening (due probably to the action of
pectic enzymes). Once cell walls and cellular membranes lose their integrity, enzymatic
oxidation proceeds much more rapidly. Browning is sometimes desirable, as it can improve
the sensory properties of some products such as dark raisins and fermented tea leaves.
Browning in fruit and in some vegetables, such as lettuce and potato, is initiated by the
enzymatic oxidation of phenolic compounds by PPOs. The formation of shrimp black spot
is another example of browning due to PPO activity. The initial products of oxidation are
quinones, which rapidly condense to produce relatively insoluble brown polymers
melanins). The oxidation product are able to react with many other components such as
proteins, amino acids, ascorbic acid etc. (see below).
CONTROL OF ENZYMATIC BROWNING
1. Breeding of cultivars with lowered kontent of polyphenol oxidace substrates (i.e
phenolic compounds)
2. Inactivation of poyphneol oxidase PPO
a) physical treatment – temperature increase , pH change, limitation of O2 content
b) enzyme inhibition - chelation agents (EDTA), anorganic ionts - halides (NaCl,
NaF) competitive inhibitors (benzoic or cinnamic acid) , reducing agents (sulphites)
3. Combination of the two
9. ENZYMES IN DETERGENCY

BIODETERGENCY
The use of enzymes in detergent formulations is now common in developed countries, with
over half of all detergents presently available containing enzymes. In spite of the fact that
the detergent industry is the largest single market for enzymes at 25 - 30% of total sales.
details of the enzymes used and the ways in which they are used, have rarely been
published.
Detergents has to remove a broad range of complex soils from different fiber surfaces.
Enzymes aid to remove soils of organic origin. Key limitation of the enzyme usage is their
stability under washing condition (alcaline pH, high tmperature, detergents).
Detergent composition (wt%)

Mode of action of alkaline bacterial proteases is based on the cleavage of proteins to

peptides which are better soluble and thus could be removed. Amylases are used for the
removal of the soils of starch, i.e food origin. Lipases act on oil spots but due to the fact
that lipases act only on the interface oil-water the effect is pronounced after several
washing cycles as they are activated during the drying (lower content of water).

Lipase activity during drying process

Cellulases are not
affecting the soil itsef,
but they are capable to
remove cellulose
microfibrils from the
surface of cotton fabrics
and thus facilitate
removal of soil
adsorbed on the surface
and enhance the
brightness of the
material. Recent development in the field is focused on the biodetergents effective to the
certain type of soils. Pectolytic enzymes are added to remove spots from fruits and fruit
jiuces (Pectaway, Pectawash), Mannanase is used for clear away the thickeners
(Mannaway). Oxidoreductases should replace the aggressive bleaching agents.

The mode of action of the enzymes in detergents is komplex.

10. AGRICULTURE

Animal feed industry is an extremely important part of the world agroindustrial

activities. Compound animal feed follows the general trend of global agriculture towards
consolidation and concentration.
Of various additives used by the feed industry, enzymes are relatively novel component
but one which is anticipated to grow rapidly.

NSP-degrading enzymes
Cereals such as wheat, barley and rye are incorporated into animal feeds to provide a major
source of energy. However, much of the energy remains unavailable to monogastrics due
to the presence of non-starch polysaccharides (NSP) which interfere with digestion. As
well as preventing access of the animal’s own digestive enzymes to the nutrients contained
in the cereals, NSP can become solubilised in the gut and cause problems of high gut
viscosity, which further interferes with digestion. The addition of selected carbohydrases
will break down NSP, releasing nutrients (energy and protein) as well as reducing the
viscosity of the gut contents. The overall effect is improved feed utilisation and a more
'healthy' digestive system for monogastric animals. Cellulases and hemicellulases are
widely used for these purposes.

The use of phytases

Around 50-80% of the total phosphorus in pig and poultry diets is
present in the form of phytate (also known as phytic acid), main
storage formo f phosphorus in plants. The phytate-bound
phosphorus is largely unavailable to monogastric animals as they
do not naturally have the enzyme needed to break it down –
phytase. There are two good reasons for supplementing feeds with
phytase. One is to reduce the harmful environmental impact of
phosphorus from animal manure in areas with intensive livestock
production. Phytate in manure is degraded by soil microorganisms, leading to high levels
of free phosphate in the soil and, eventually, in surface water. Optimising phosphorus
intake and digestion with phytase reduces the release of phosphorus by around 30%.
Amount of phosphorus released into the environment could be thus reduced by 2.5 million
tonnes a year worldwide if phytases were used in all feed for monogastric animals. The
second reason is based on the fact that phytate is capable of forming complexes with
proteins and inorganic cations such as calcium, magnesium, iron and zinc. The use of
phytase not only releases the bound phosphorus but also these other essential nutrients to
give the feed a higher nutritional value.

Enzyme flax retting

Because of problems with both water- and dew-retting, a long-term objective for improving the
flax
fiber industry has been development of enzyme-retting. In the 1980s, extensive research was
undertaken in Europe to develop enzyme-retting as a method to replace dew-retting. The strategy
of this research was to replace the anaerobic bacteria with enzyme mixtures in controlled tanks,
thereby producing flax of water-retted quality but without the negative aspects of stench and
pollution. Research resulted in development of the commercial enzyme mixtures containinfg the
plant cell wall degrading enzymes i.e. cellulases, hemicellulases and pectolytic enzymes Enzyme-
retting produced fibers having the fineness, strength, color, and waxiness comparable to the best
water-retted fiber. Advantages of the enzyme method are: (1) time savings of 4–5 days, (2)
increased yield of ca 2% over water-retting, and (3) fiber consistency.

Cross section of flax stem before and after application of enzyme retting

TEXTILE INDUSTRY
Enzymes have found wide application in the textile industry for improving production
methods and fabric finishing. Some procedures being traditional like desizing, some being
a new technology like biostoning:

Desizing of fabrics before bleaching and staining (amylase)

Denim finishing based on the action of cellulases. I tis possible to achieve the
fashionable stonewashed look traditionally achieved through the abrasive action of
pumice
stones.

Bio-Polishing - cellulases are also used to prevent pilling and improve the
smoothness and colour brightness of cotton fabrics in a process called. In addition, a
softer handle is obtained.
Wool and sil finishing (proteases)
Bio-polishing of Lyocell (celulases)
Scouring of cotton fabrics - pectolytic enzymes
11. OTHER NON-FOOD INDUSTRILA APPLICATIONS
LEATHER
Enzymes have always been a part of leather-making, even if this has not always
been recognised. Since the beginning of the last century, when Röhm introduced modern
biotechnology by extracting pancreatin for the bating process, the use of enzymes in this
industry has increased considerably. Nowadays, enzymes are used in all the beamhouse
processes and have even entered the tanhouse. Alkaline proteases and lipases are now
widely used.

PAPERMAKING PROCESSES
Treating pulp with certain enzymes (xylanases) opens up the hemicellulose
structure containing bound lignin and facilitates the removal of residual lignin prior to
bleaching. By using selected xylanases, it is possible to obtain a partial hydrolysis of the
hemicellulose precipitated onto pulp fibre during the alkaline cooking process.
Lipases and phospholipases could be applied for the enhanced removal of the
resins.
Starch modification for paper coatings is another field for enzyme action in paper
industry. In the manufacture of coated papers, a starch-based coating formulation is used to
coat the surface of the paper. Compared with uncoated paper, the coating provides
improved gloss, smoothness and printing properties. Chemically-modified starch with a
low viscosity in solution is used. As an economical alternative to modifying the starch with
aggressive oxidising agents, alpha-amylases can be used to obtain the same reduction in
viscosity. Enzyme-modified starch is available from starch producers or can be produced
on site at the paper mill using a batch or continuous process.
12. ENZYMES AS ANALYTICAL TOOLS, BIOSENSORS
Enzymes as analytical tools
Determination of enzyme activities
 diagnostics (clinical biochemistry)
 indicators of technological and quality changes in food processing
Preparation of samples for chemical analysis
 Disruption of material for analysis
 Extraction of analysed compounds
 Removal of interfering compounds
Other applications
 Structural studies of proteins
 Technologies of genetic and protein engineering

Enzymes as analytical tools

Advantages Disadvantages
Specificity Higher price
Complex samples Interferony compounds
Rapidity Inhibition
Sensitivity Inactivation

Equilibrium methods (end-point)

 Enzymes with high affinity to substrates (low Km value)
 Conditions not strictly kept
 Time consuming
Kinetic methods
 Enzymes with low affinity to the substrates (high Km value)
 Possibility to broaden the range of analyte concentration by addition of competitive
inhibitor in case of enzyme with low Km
 Shorter analysis time
 Condition of analysis has to be strictly kept constant

Compounds which can be directly determined by enzyme analytical methods could be the
substrates or the inhibitors or activators of an enzyme.

Oxidoreductases with NAD+ as cofactor are widely used because of the possibility to
measure changes of absorbance of UV light of the reduced and oxidized form of the
cofactor. It is possible to determine the compounds which are the substrates of
oxidoreductases or or could be transformed by another ecoupled enzyme reaction
producing the substrate for oxidoreductase reaction. The examples of oxidoreductases used
in analysis are lactate dehydrogenase, alcohol dehydrogenase, glucose 6-phosphate
dehydrogenase, malate dehydrogenase .
Determination of glucose
Glucose can be determined by using the non specific hexokinase reaction, producing
glucose 6-phosphate which is then a substrate for highly specific glucose 6-phosphate
dehydrogenase.
In clinical biochemistry glucose is determined in blood serum by automatic analysers based
on immobilised glucose oxidase with Clark’s oxygen sensor detecting the decrease of
oxygen.

An example of the determination of enzyme inhibitors is the reaction of acetylcholine

esterase which is strongly inhibited by organophosphorus compounds such as parathion
which can be determined in the range of 0,5 – 2 mg/ml.

Enzymes with metal ion in their molecule essential for their activity could be used for the
determination of the metal ion in the samples after removal of the ion from the enzyme
molecule. Restore of the enzyme activity by addition of metal ion is proportional to its
concentration. Polyphenol oxidase is suitable for Cu2+ or aminopeptidase for Zn2+
determination, the latter being able to detect Zn ions in the range from 5 to 100 pg/ml.

Immobilized enzymes for analytical purposes could used as

a) Enzyme reactors }with detection system physically separated from the enzyme ,
such as flow injection analysis system (FIA)
b) Biosensors
c) Miscellaneous (dips, strips etc.)

Principle of biosensor:
 a) biocatalyst b) detector c) signal amplification d) signal processing e) signal record

Example of amperometric glucose biosensor:

„Optical“ biosensors:
a) Change of light absorbance
b) Emission of light radiation - luminescence

peroxidase
chromogenic substrate (2H) + H2O2 → dye product + 2H2O

luciferase
ATP + D-luciferin + O2 → oxyluciferin + AMP + PPi + CO2 + hν (562 nm)

Immunosensors:
Electrochemical detection of the immunoreaction:
a) Direct (pulse amperometry, cyclic voltamety etc)
b) Amperometric determination of antibody enzyme label
13. ENZYMES IN CLINICAL BIOCHEMISTRY AND THERAPY

1. Detremination of enzyme activities as diagnostic tools

2. Determination of analytes by enzyme analytycal techniquese
3. Enzymes as labels in immunoassays
4. Enzymes as therapeutics

Enzymes as diagnostic tools

Concentration of enzymes in cells is under normal „healthy“ conditions 103 to 104
higher than in body fluids. Changes of energetic and metabolic state of cells leads to the
deterioration of inner and outer membranes an to the release of enzemes to the surroundigs
and to the body fluids. The velocity of this process depends on many factors such as
molecular mass of the protein, its solubility and concentration.

Cell damage can be cased by:

 Hypoxia - a pathological condition in which the body as a whole or region of
the body is deprived of adequate oxygen supply - anemia, cardiovascular
deseases
 Chemicals, drugs, polutants ( haevy metals, PCBs etc.), alcohol, narcotics
 Physical – cold, heat, radiation
 Microbial infections, protozoa,
 Alergens
 Genetic defects
 Nutritional deficiency
 other – proliferation of certain type of cells

„Clearance“ of enzymes is the rate at which an enzyme is removed or cleared from the
body fluids. It depends on the stability of the enzyme and on the rate of removal by
reticuloendothelial and renal system. The half-life time of certain enzyme is of an
important diagnostic value
Important diagnostic enzymes

Transaminases – Aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase(EC

2.6.1.2)
Increased levels of these enzyme could be observed in liver deseases (virus hepatitis,
mononucleosis, cirrhosis ), muscle dystrophy, muscle injuries, pancreatitis

Oxaloacetate and pyruvate could be determined spectrophotometrically using the malate

dehydrogenase or pyruvate dehydrogenase reaction

Creatine kinase (EC 2.7.3.2, ATP: creatin N-phosphotransferase)

Protein dimer from 2 different subunits M and B forming isoforms CK-1 (BB), CK-2
(MB), CK-3 (MM), CK-Mt. Diagnostic tool for myocard infarction.

Determination of creatin kinase activity

Cr + ATP  CrP + ADP (creatine kinase) pH 9

ADP + PPyr  ATP + Pyr (pyruvate kinase)
Pyr + NADH + H+  lactate + NAD+ (lactatet dehydrogenase)

Cr + ATP  CrP + ADP (creatine kinase)

ATP + luciferin + O2  APM + oxiluciferin + PPi + CO2 + h (luciferase)

CrP + ADP  Cr + ATP (creatine kinase) pH 6,7

ATP + Glc  Glc-6-P + ADP (hexokinase)
GlC-6-P + NADP+  6-P-gluconate + NADPH + H+ (Glc-6-P dehydrogenase)

Lactate dehydrogenase (EC 1.1.1.27

Tetramer of two differen subunits M and H:

 LDH-1 (4H) - in the heart

 LDH-2 (3H1M) - in the reticuloendothelial system
 LDH-3 (2H2M) - in the lungs
 LDH-4 (1H3M) - in the kidneys placenta, and pancreas
 LDH-5 (4M) - in the liver and striated muscle

Alkaline phosphatase, ALP (EC 3.1.3.1),

pH optimum ≈ 10, with unknown physiological function, it is present in all tissues, higher
concentration can be found in epithelial cells of intestine, in bones, liver and placenta.
Diagnosis of liver deseases and bone deformations, osteoporisis

Acid phosphatase ACP (EC 3.1.3.2),

pH optimum < 7, found in lysosomes, spleen, milk, bone marrow, platlets and prostate
diagnosis of prostate cancer
γ-glutamyltransferase (EC 2.3.2.2), γ-glutamyl-peptid:aminokyselina γ-
glutamyltransferasa
Amino acid transport across the mebmranes, metabolism of glutathione
Diagnosis of liver diseases and gall bladder

Acetylcholin esterase (EC 3.1.1.7)

Liver diseases, intoxicatin by pesticides, lung embolism, infections

Enzymes of digestive trakt

Amylases - pancreas
Proteolytic enzymes trypsin, chymotrpsin, pepsin
Lipase - pancreas, lipoproteinlipase - triacylglycerole metabolism, arteriosclerosis

Determination of analytes by enzymatic methosds

Uric acid
Oxidative stress marker, increased levels in cardiovascular diseases, renal insuficiencies,
gout
Lower levels are found at pacients with sclerosis multiplex
Urate oxidase (uricase) is used for determination of uric acid:

Uric acid + O2 + H2O → 5-hydroxyisourate + H2O2→ allantoin + CO2

Enzymes as therapeutics
1. replacement of enzymes which are missing or defective as a consequence of
inherited genetic disease
2. Replenishment of enzymes which are deficient ot present only in inadequate
quantity as a consequence of of acquired disease in the organ where they are
normally synthetized
3. Enzymes with specific biological effect

Many of the requirements are not met by the older enzyme preparations of
non/human sources approved prior to 1980, but they are still in use as generics , some of
them also as nutritional additives, oral hygiene products, cosmetics etc. on the other hand
only a few recombinant enzymes have been developed until now.
Following overview includes older enzymes still in use.
In the development of therapeutic enzymes, as with other biopharmaceuticals, special
attention must be paid to regulatory requirements and to aspects of quality assurance, assay
methods and standardization.
Oxidases – urate oxidase for hyperuricemia treatment, superoxidedismutase for
inflammatory arthrosis, asthma, prevention of bronchopulmonary diseases.
From class of hydrolases many coagulating factors are used for thr treatment of blood
coagulation disorders. Hydrolases of digestive tract are used as digestion aids and there are
many preparations on the market.
14. ENZYMES IN BIOTRANSFORMATIONS

Biotransformation are defined as production of chemical individuals by transformation of

precursors. Reaction could be catalyzed by enzymes or by whole cells with desired activity.
Here we are not taking in the account the biotransformation of pollutants by
microorganisms and plants or combination of both. This process called also bioremediation
represents the separate subject field.

Whooel cells:
Influence of organic solvents on biocatalysis - advantages and disadvantages

The advantages of enzymes in synthesis include that:

(i) they are effcient catalysts; the rates of enzyme-mediated processes are accelerated
compared with chemical catalysts and enzymes can be effective at very low mole fractions
of catalyst;
(ii) they act under mild conditions; the moderate operating temperature range of enzymes
(20±40°C) minimises undesired side-reactions such as rearrangement;
(iii) they catalyse a broad range of reactions; enzyme-catalysed processes exist for a wide
range of reactions and can often promote reactions at ostensibly non-activated sites in a
substrate;
(iv) they display electivity; such as (a) hemoselectivity (enzymes can act on a single type
of functional group in the presence of other sensitive functional groups), (b)
regioselectivity and diastereoselectivity (enzymes can distinguish between functional
groups with diferent chemical environments, (c) enantioselectivity (enzymes are chiral
catalysts and their specificity can be exploited for selective and asymmetric conversions
(v) they are not restricted to their natural substrates; the majority of enzymes display high
specificity for a specific type of reaction while generally accepting a wide (although
sometimes narrow) variety of substrates;
(vi) they can work outside an aqueous environment; although some loss of activity is
usually observed some enzymes can operate in organic solvents .
The main disadvantages of enzymes in synthesis are that enzymes are usually made from
L-amino acids and thus it is impossible to invert their chiral induction on a reaction.
However, with the isolation of new enzymes and with the progress of modern molecular
biology techniques for creating modi®ed enzymes this may eventually be overcome.
Enzymes are prone to substrate or product inhibition. Substrate inhibition can be overcome
by keeping the substrate concentration low. Product inhibition can be overcome by tandem
in situ reactions in which the product of one reaction becomes the substrate of the next
reaction. They display their highest catalytic activity in aqueous solvents. However, for
most organic reactions the solvents of choice are non-aqueous solvents that help promote
substrate solubility. Enzymes require a narrow operation range; elevated temperatures and
extremes in pH, or high salt concentrations all lead to deactivation of the enzyme.
Immobilization of an enzyme can
overcome the problems associated with
the altered conditions imposed on that
enzyme when promoting the solubility of
an organic substrate.
Reversed micelles could be also a helpuf
instrument.
Examples of enzymes used in organic synthesis