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

region

Amylases, xylanases, proteases, DNA

polymerases

Psychrophiles 5 – 20 ˚C glacial

sediments

Proteases, glykosidases, lipases

Acidophiles pH < 2 Sulphur

springs Sulphur oxidation

Alkalophilesí pH > 9 dregs Lipases, proteases

Halophiles 3 – 20%

salts

Salt lake

sediments Biotransformations, additives, drugs

Barophiles Higher

pressure

Submarine

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

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

Imobilization Matrix binding Entrapment

a) adsorption b) Covalent linkage

c)In the gel matrix

d) encapsulation

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

Substrate*

Relat.

oxidation

Rate

(%)

2-deoxy-D-glukosa 20 – 30

4-O-methyl-D-glukosa 15

6-deoxy-D-glukosa 10

*about 100 derivatives were tested

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)

Substrate*

Relat.

oxidation

Rate .

(%)

-D-glukosa 100

-D-glukosa 0,64

L-glukosa 0

D-mannosa 1,0

D-xylosa 1,0

D-galaktosa 0,5

maltosa 0,2

melibiosa 0,1

cellobiosa 0,09

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.

H-O-H H-O-RH-O-H H-O-R

HH RR

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.

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

preparation

Comment

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

parasitica

Superen,

Thermolase

Switzerland, Italy

Recombinant Aspergillus niger Chymogen,

Chymostar

Not allowed in all countries

Aspergillus oryzae Novoren Used intensively since 1994

Escherichia coli Chymax Not allowed in all countries

Kluyveromyces

marxianus

Maxiren Not allowed in all countries

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

Hydration/rheology

solubility

Food additives

Baking industry

Increased sugestibility and nutrition value

pea Taste and flavour

solubility

Food additives

corn solubility Animal feed

Animal

Meat Texture

solubility

Taste and flavour

Food additives

animal feed

Milk coagulation

solubility

Cheese production

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 10

4

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