Okafor N. Modern Industrial Microbiology and Biotechnology

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" Modern Industrial Microbiology and Biotechnology

Nucleases split nucleic acids (DNA and RNA). Based on the substrate type, they are

divided into RNase and DNase. RNase catalyzes the hydrolysis of RNA and DNase acts

on DNA. They may also be divided into exonuclease and endonuclease. The

exonuclease progressively splits off single nucleotides from one end of DNA or RNA.

The endonuclease splits DNA or RNA at internal sites.

Phosphatase catalyzes dephosphorylation (removal of phosphate groups).

4. Lyases catalyze the cleavage of C-C, C-O, C-S and C-N bonds by means other than

hydrolysis or oxidation. Common names include decarboxylase and aldolase.

5. Isomerases catalyze atomic rearrangements within a molecule. Examples include

rotamase, protein disulfide isomerase (PDI), epimerase and racemase.

6. Ligases catalyze the reaction which joins two molecules. Examples include peptide

synthase, aminoacyl-tRNA synthetase, DNA ligase and RNA ligase.

22.3 USES OF ENZYMES IN INDUSTRY

Most of the enzymes used in industry are hydrolases (i.e., those which hydrolyze large

molecules). In particular amylases, proteases, pectinases, and to a lesser extents lipases

have been most commonly used. Enzymes are used in a wide range of industries and

some uses are discussed below.

(i) Production of nutritive sweeteners from starch: Enzymic hydrolysis has now almost

completely replaced the use of acid in starch hydrolysis (Chapter 4). The sweeteners

which have been produced from starch are high conversion (or high DE) syrup, high

maltose syrup, glucose syrup, dextrose crystals and high fructose syrup. These

sweeteners are often called corn syrups because they are produced from maize, although

starch from any source (e.g. cassava, sorghum, or potatoes) may be used. The processes of

production of sweeteners from corn consists of the gelatinization of starch production of

water-soluble dextrins with a-amylase, the subsequent application of a de-branching

enzyme (e.g. pullulanase) and, depending on the sugar sought, the application of a third

enzyme. a-Amylase from B. licheniformis is particularly suitable for dextrinization

because its optimum temperature is 110°C, a convenient temperature on account of the

need to boil starch to gelatinize it. If high maltose syrup is sought a-amylase is applied,

while gluco-amylase is applied if glucose syrup is sought (Fig. 22.1). Dextrose crystals are

usually produced by removing minerals with ion exchange resins and then crystallizing

the liquid after concentration.

Nowadays, most sweeteners produced from starch are in the form of high fructose

corn syrup (HFCS), whose production is discussed below. Glucose has a rather bland

taste and is not as sweet as sucrose. Fructose, on the other hand, is about 1.7 as sweet as

sucrose. In the confectionary industry therefore glucose resulting from the hydrolysis of

starch is converted to fructose by the enzyme glucose isomerase which rearranges the

glucose molecule to yield fructose and the syrup itself into a glucose/fructose syrup.

Glucose isomerase is completely specific for monomeric D-glucose. The maltose,

maltotriose and higher maltooligosaccharides present in glucose syrup are untouched

by the enzyme. An acceptable composition of high-fructose glucose syrups in commerce

is: fructose 42%, glucose, 50%, maltose, 6%, and maltotriose, 2%. These high fructose

Biocatalysts: Immobilized Enzymes and Immobilized Cells "

mixtures are used in place of sucrose and invert sugar in the baking and beverages

industries. HFCS production indeed represents one of the major uses of enzymes.

Besides its role as a sweetener, fructose has other qualities which make it superior to

glucose. It is regarded as a low-calorie sweetener, since it is so much sweeter than

sucrose. Furthermore, fructose is favored for intravenous infusions or drip over glucose

because the body tolerates it better. Twice as much fructose as of glucose can therefore be

permitted in drips, representing a greater caloric intake in as much fluid. It is therefore the

preferred sugar for patients in a state of shock. Finally it is more quickly absorbed than

glucose and does not need the enzyme and hormonal system required for absorbing the

former.

STARCH

Heat “solution” of starch

GELATINIZATION

a-Amylase (sometimes with prior acid treatment)

DEXTRINIZATION

Fungal Amylase Amyloglucosidase

pH ADJUSTMENT

PULLULANASE

b - AMYLASE

HIGH MALTOSE

GLUCOSE SYRUP

HIGH CONVERSION

SYRUP

De-ash with

ion exchange,

concentrate,

and crystallize

Ph ADJUSTMENT

CRYSTALLINE

GLUCOSE

Pass through column of immobilized

glucose isomerase

HIGH FRUCTOSE

SYRUP

Fig. 22.1 Hydrolysis of Starch for High Fructose Corn Syrup Production

" Modern Industrial Microbiology and Biotechnology

Organisms which have served as sources for glucose isomerase include more than two

dozen strains of Streptomyces, several of Arthobacter, Nocardia, Micromonospora as well as

Lactobacillus brevis and Pseudomonas hydrophilla.

(ii) Proteolytic enzymes in the detergent industry: The detergent industry is at present one

of the greatest consumers of enzymes, and uses mostly proteases. Blood and pus stains

from hospital linen and other protein dirt precipitate and coagulate on clothes and are

ordinarily difficult to remove. The inclusion of proteolytic enzymes in a detergent or

washing soap greatly facilitates the removal of such stains. The proteolytic enzymes used

for this purpose should have a high pH optimum of 9-11, which is the pH of detergents,

and a high temperature optimum of 65-70°C since hot water facilitates laundering.

Furthermore, the enzyme should be able to cleave peptide bonds randomly and facilitate

the dissolution of the protein. Such proteolytic enzymes have been produced mainly by

alkalophilic and aerobic spore-formers such as strains of Bacillus licheniforms and Bacillus

amyloliquifaciencs. The latter has the advantage of producing a-amylases as well.

It is worth mentioning that the history of the use of enzymes in detergents has not

always been smooth. Soon after their introduction in the early 1960s, some factory

workers handling the enzymes suffered from allergic reactions. Strong public protests led

to the withdrawal of enzymes in detergents and, although a commission of inquiry

showed that there was no danger to the user, the use of enzymes in detergents suffered a

temporary setback. Subsequently, enzymes are added in dust-free encapsulated

preparations, to avoid inhalation by producers and users.

(iii) Microbial rennets: Rennin is an acid protease found in gastric juice of young

mammals where it helps to digest milk. It is used in the manufacture of cheese and

functions by hydrolyzing a polypeptide fragment from milk protein-kappa casein to

leave paracasein; this then forms an insoluble complex with cations to give a firm curd.

The commercial form of rennin known as rennets is obtained from the fourth stomachs of

young calves. It is therefore expensive and tedious to produce since it involves the

maturation, gestation and delivery of cows. Due to this, a search for substitutes ensued.

Strains of Mucor miehi, M. pusillus, Endothia parasitica, Bacillus polymyxa, B. subtilis and

Aspergilus are used to produce acid proteases which successfully substitute for rennin.

Indeed, microbial rennets constitute about the third largest use of microbial enzymes.

(iv) Lactase: Lactase hydrolyzes the disaccharide lactose into its component galactose

and glucose, both of which are sweeter than lactose and correspond to the addition of

0.9% sucrose. Thus, dairy products containing lactose, such as yoghurt, and ice cream,

can be sweeter and more acceptable to consumers without the extra expense of

extraneously added sugar. Galactose and glucose are also metabolized by a far wider

range or organisms than can attack lactose. The result is that lactase-hydrolyzed whey

can be used to produce alcohol or soft drinks. Furthermore, milk in which lactose is

hydrolyzed is preferred by individuals in some parts of the world where intestinal

lactase is low. Finally, when lactose occurs in high concentrations, such as is in ice

cream, it tends to crystallize out giving the impression during consumption that grains of

sand are present in the product. The addition of lactase prevents such crystallization.

Lactase is now produced commercially from Kluyveromyces fragilis, Saccharomyces lactis or

Aspergillus niger.

Biocatalysts: Immobilized Enzymes and Immobilized Cells "!

(v) The textile industry: In the textile industry large amounts of starch, gelatin and their

derivatives such as glue are used to strengthen threads (yarns) of synthetic and natural

materials (e.g. cotton) to enable them to stand abrasion during weaving and also to polish

sewing thread. Starch and its derivatives are furthermore used to restrict dye stuffs and

prevent their diffusion to other portions of the fabric.

At the end of the manufacturing the starch is removed with thermostable a-amylases

from Bacillus licheniformis. The cloth is passed through hot water to remove inorganic

salts and to raise the temperature. It is then passed into an enzyme solution where it is

allowed to remain from 15 minutes to several hours depending on the enzyme

concentration and other factors.

Natural silk threads are proteinaceous in nature and consist of a highly resistant

protein known as fibroin, but they are held together by a semi-soluble protein gum known

as sericin. Sericin is removed with a neutral proteolytic enzyme; the silk threads are then

sized and woven into cloth after which they are de-sized in an amylase or protease

depending on the sizing material.

(vi) Pectinases for use in fruit juice and wine manufacture: Pectinases are enzymes which

attack pectic substances, a group of complex acidic polysaccarides. Pectic substances are

high molecular weight substances made up of poly – D – galacturonic acid. As the

carboxylic acid groups of the sugar units are partially esterified with methanol, they are

regarded as poly-uronides. They are the cementing material holding plant cells together.

The Agricultural and Food Chemistry section of the American Chemical Society has

proposed the following nomenclature for the various pectic substances:

Pectic Substances: a group designation for those complex carbohydrate derivatives which

occur in or are prepared from plants and contain a large proportion of

anhydrogalacturonic acid units. The carboxyl groups of polygalactoronic acids may be

partly esterified by methyl groups and partly or completely neutralized by one or more

bases.

Protepectin: The water-insoluble parent pectic substance which occurs in plants and

which, upon restricted hydrolysis, yields pectin or pectinic acids.

Pectinic acids: are colloidal polygalacturonic acids containing more than a negligible

proportion of methyl ester groups.

Pectic substances may be regarded as polygalacturonides composed of unbranched

a–1, 4 galacturonic acid residues (Fig. 22.2) but with other non-uronides bound to the

chain. However, pectic materials are not uniform because of the great variations which

have been observed by many authors in the molecular weights, degree of esterification

and acetylation, amount and type of neutral sugars and non-uronide residues found in

various preparations of pectic substances.

Pectinolytic enzymes or pectinases are widely distributed in plants and among

microorganisms. These enzymes vary greatly in their mechanisms of action, but may be

grouped into two: esterases which de-esterify pectin to pectic acid and depolymerases

which depolymerize pectin, pectin acid or short-chain galacturonic acid (ligo-D-

galacturonates) derived from pectin and pectic acids (Table 22.1). Aspergilli (A.niger, A.

oryzae, A. wentii, and A. flavus) and other fungi (Table 22.1) are used for industrial enzyme

production. The industrial enzymes themselves are a mixture of various pectinolytic

enzymes.

"" Modern Industrial Microbiology and Biotechnology

COOCH

H OH COOCH

HOHHH

OH H H H H OH H

H OH COOCH

HOH

Fig. 22.2 Structure of Pectin

Table 22.1 Distribution of pectinolytic enzymes in some microorganisms

Source PMGE PG PGL PMG PMGL OG OGL

Bacillus sp. + +

Erwinia aroideae ++ +

Pseudomonas sp. + +

Ps. Marginalis +++

Xanthomonas campestris ++

Clostridium multifermentans ++

Aspergillus niger ++ + +

Penicillium digitatum +++

Key:

PMGE = Polymethylgalacturonase esterase (de-esterify pectin to pectic acid by

removal of methoxy residues)

PG = Polygalacturonases (hydrolyze pectic acids randomly, successive bonds or

alternate bonds)

PGL = Polygalactorunate lyase (hydrolyzes pectic acids by transelimination)

PMG = Polymethylgalacturonase (hydrolyzes pectin in random or

sequential fashion)

PMGL = Polymethylgalacturanate lyase (cleaves pectic acid randomly or

sequentially)

OG = Oligogalacturonase (hydrolyzes oligo-galactosiduronates i.e. breakdown

products of pectin and pectic acids)

OGL = Oligogalacturonate lyase (acts as OG but by transelimination)

Pectinolytic enzymes are used principally in the fruit juice, fruit processing and the

wine industries. In the fruit juice and fruit industry, pectinases are used to disintegrate

the fruits, and to clarify the resulting juices to give a clear sparkling liquid after the

filtration of the debris.

Another application of pectinases which does not involve the isolation of the enzymes

but which deserves mention is the retting of plants for flax (linen) from (Linum

usitatissimum) and hemp (Cannabis sativa) and of jute from Corchorus sp. Retting has not

been studied intensively probably because of the advent of man-made fibres. Pectinolytic

enzymes are produced anaerobically by Clostridium spp. when the plants are immersed

in water. When aerobic conditions prevail as in ‘dew-retting’ the organisms which have

been isolated include Bacillus comesii, and the fungi, Cladosporium, Aspergillus, and

Penicillium.

Biocatalysts: Immobilized Enzymes and Immobilized Cells "#

(vii) Naringinase is used for removing the bitter tasting substance from citrus fruits,

especially grape fruits. Naringin is a flavonoid found in grapefruits, and gives grapefruit

its characteristic bitter flavor. Flavonoids are a group of polyphenolic secondary

metabolites secreted by plants and found widely among plants. They are present in many

plant-based foods such as tea and soybeans, and are generally believed to be beneficial to

health. Although naringin is supposed to have some beneficial effect such as stimulating

our perception of taste by stimulating the taste buds (for which reason some people eat

grape fruits before a meal), the bitter taste is undesirable in fruit juices. Therefore,

grapefruit processors attempt to select fruits with a low naringin content, or use

naralginase produced by strains of Aspergillus spp. to remove it.

Fig. 22.3 Structure of Naringin

(viii) Enzymes in the baking industry: Flour contains 72-75% starch, 11-13% protein, and

0.04-0.4% minerals. It also contains amylases and proteases derived from wheat. These

enzymes play major roles in the nature of the final baked product. When the flour is

deficient in amylases, unusually low amounts of sugar and intermediate products are

produced, giving rise to low volume, dry texture and other undesirable characteristics in

the finished bread. Similarly, while wheat brands rich in gluten (or ‘hard’ wheat) are

suitable for bread making because they are highly elastic, they are not suitable for cakes.

For the latter, ‘soft’ wheat with low gluten contents is required. ‘Hard’ wheat is rendered

‘soft’ however by the hydrolysis of some of the gluten using proteases. Bakery amylases

and proteases are derived from fungi.

(ix) Enzymes in the alcoholic beverages industry: Amylases derived from fungi or bacilli

may be employed in the distilled alcoholic beverages to hydrolyze starch to sugars prior

to fermentation by yeasts. The enzymes may also be used to hydrolyze unmalted barley

and other starchy adjuncts converting them to maltose and thus reducing the cost of beer

brewing. Although it is unusual, turbidities due to starch may arise in beer due to the

destruction of the amylases following the use of too high a temperature during malt

kilning. Amylases are used to remove turbidities due to starch. In beer, chill-hazes are due

mainly to protein-tannin (protein-polyphenol) precipitates. Chill-hazes may be removed

in several ways, one of which is the addition of proteases. Proteases from Aspergillus niger

are often used (Chapter 12).

(x) Leather baiting: After animal skin has been trimmed of flesh, it is de-haired with lime

or a proteolytic enzyme and it is then baited. The purpose of baiting, is to prepare the de-

haired skins and hides for tanning. Baiting used to be done by keeping the skin in a warm

suspension of chicken and dog dung, which probably yielded proteolytic bacteria.

Nowadays proteolytic enzymes from Bacillus spp. and Aspergillus spp. are used. The

"$ Modern Industrial Microbiology and Biotechnology

effect of baiting is to make the fibrous protein collagen of which leather is composed more

amenable to the subsequent processes of leather manufacture.

(xi) Some medical uses of microbial enzymes: At present, the most successful medical

applications of enzymes are the use of proteolytic enzymes from Bacillus spp. and other

bacteria for the treatment of burns and skin cancers, and the treatment of life-threatening

disorders within the blood circulation using hemolytic enzymes produced from B-

hemolytic streptococci. Other uses are given below:

(a) Fungal acid proteases may be used to treat alimentary dyspepsia, because of the

acid resistance of the enzyme. Fungal amylases may also be used to help digestion.

(b) Dextrans deposited on the teeth by Streptococcus mutans may be removed with the

use of fungal dextranase often introduced into the toothpaste, thus helping to fight

dental decay.

used in the treatment of certain kinds of leukemia.

(d) Penicillinases produced by many organisms are sometimes used in emergency

cases of penicillin hypersensitivity.

(e) Rhodanase which catalyses the reaction, S

2–

+ CN

–

+ SCN

–

has been used to combat cyanide poisoning. Rhodanase is produced by the

thermoacidophilic bacterium Sulfobacillus sibiricus.

The above are only a few of some of the uses to which microbial enzymes have been put

in the medical area.

22.4 PRODUCTION OF ENZYMES

22.4.1 Fermentation for Enzyme Production

Most enzyme production is carried out in deep submerged fermentation; a few are best

produced in semi-solid media.

22.4.1.1 Semi solid medium

This system, also known as the ‘Koji’ or ‘moldy bran’ method of ‘solid state’ fermentation

is still widely used in Japan. The medium consists of moist sterile wheat or rice bran

acidified with HCl; mineral salts including trace minerals are added. An inducer is also

usually added; 10% starch is used for amylase, and gelatin and pectin for protein and

pectinase production respectively. The organisms used are fungi, which appear

amenable to high enzyme production because of the low moisture condition and high

degree of aeration of the semi-soluble medium.

The moist bran, inoculated with spores of the appropriate fungi, is distributed either in

flat trays or placed in a revolving drum. Moisture (about 8%) is maintained by

occasionally spraying water on the trays and by circulating moist air over the

preparation. The temperature of the bran is kept at about 30°C by the circulating cool air.

The production period is usually 30-40 hours, but could be as long as seven days. The

optimum production is determined by withdrawing the growth from time to time and

assaying for enzyme. The material is dried with hot air at about 37°C–40°C and ground.

The enzyme is usually preserved in this manner. If it is desired, the enzyme can be

Biocatalysts: Immobilized Enzymes and Immobilized Cells "%

extracted. Growth in a semi-solid medium seems sometimes to encourage an enzyme

range different from that produced in submerged growth. Thus, Aspergillus oryzae on

semi-solid medium will produce a large number of enzymes, primarily amylase,

glucoamylose, and protease. In submerged culture amylase production rises at the

expense of the other enzymes. Similarly, if Aspergillus oryzae producing takadiastase (a

commercial powder containing amylase and some protease) is grown in submerged

culture four protease components are formed whereas on semi-solid medium not only are

two proteases formed, but these are less heat resistant than those produced in submerged

fermentation.

22.4.1.2 Submerged production

Most enzyme production is in fact by submerged cultivation in a deep fermentor (Chapter

9). Submerged production has replaced semi-solid production wherever possible

because the latter is labor intensive and therefore expensive where labor is scarce, and

because of the risk of infection and the generally greater ease of controlling temperature,

pH and other environmental factors in a fermentor.

The medium must contain all the requirements for growth, including adequate

sources of carbon, nitrogen, various metals, trace elements, growth substances, etc.

However, a medium adequate for growth may not be satisfactory for enzyme production.

For the production of inducible enzymes, the inducers must be present. Thus, pectic

substances need to be in the medium when pectinolytoc enzymes are being sought.

Similarly, in the production of microbial rennets soy bean proteins are added into the

medium to induce protease production by most fungi. The inducer may not always be the

substrate but sometimes a breakdown or end-product may serve. For example, cellobiose

may stimulate cellulose production.

Sometimes some easily metabolizable components of the medium may repress enzyme

production by catabolite repression. Strong repression is often seen in media containing

glucose. Thus, a-amylase synthesis is repressed by glucose in Bacillus licheniformis and B.

subtilis. Fructose on the other hand represses the synthesis of the enzyme in B.

stearothermophilus. In many organisms protease synthesis is repressed by amino acids as

well as by glucose. It is therefore usual to replace glucose by more slowly metabolized

carbohydrates such as partly hydrolyzed starch. High enzyme yield may also be

obtained by adding constantly, low amounts of the inducer.

End-product inhibition has also been widely observed. Some specific amino acids

inhibit protease production in some organisms. Thus, isoleucine and proline are

involved in the case of B. megaterium while sulphur amino acids inhibit protease

formation in Aspergillus niger.

Temperature and pH requirements have to be worked out for each organism and each

desired product. The temperature and pH requirements for optimum growth, enzyme

production, and stability of the enzyme once it is produced are not necessarily the same

for all enzymes. The temperature adopted for the fermentation is usually a compromise

taking all three requirements into account.

The oxygen requirement is usually high as most of the organisms employed in enzyme

production are aerobic. Vigorous aeration and agitation are therefore done in the

submerged fermentations for enzyme production. Batch fermentation is usually

employed in commercial enzyme fermentation and lasts from one to seven days.

"& Modern Industrial Microbiology and Biotechnology

Continuous fermentation, while successful experimentally, does not appear to have been

used in industry.

In a few cases the enzyme production is highest during the exponential phase of

growth. In most others, however, it occurs post-exponentially. Furthermore, different

enzymes are produced at different stages of the growth cycle. Thus Asp. niger produces

mostly a–amylases in the first 72 hours but mainly maltase thereafter.

22.4.2 Enzyme Extraction

The procedures for the extraction of fermentation products described in Chapter 10 are

applicable to enzyme extraction. Care is taken to avoid contamination. In order to limit

contamination and degradation of the enzyme the broth is cooled to about 20°C as soon

as the fermentation is over. Stabilizers such as calcium salts, proteins, sugar, and starch

hydrolysates may be added and destabilizing metals may be removed with EDTA. Anti-

microbials if used at all are those that are normally allowed in food such as benzoates

and sorbate. Most industrial enzymes are extra-cellular in nature. In the case of cell

bound enzymes, the cells are disrupted before centrifugation and/or vacuum filtration.

The extent of the purification after the clarification depends on the purpose for which

the enzyme is to be used. Sometimes enzymes may be precipitated using a variety of

chemicals such as methanol, acetone, ethyl alcohol or ammonium sulfate. The precipitate

may be further purified by dialysis, chromatography, etc., before being dried in a drum

drier or a low temperature vacuum drier depending on the stability of the enzymes to

high temperature. Ultra-filtration separation technique based on molecular size may be

used.

22.4.3 Packaging and Finishing

The packing of enzymes has become extremely important since the experience of the

allergic effect of enzyme dust inhalation by detergent works. Nowadays, enzymes are

packaged preferably in liquid form but where solids are used, the enzyme is mixed with

a filler and it is now common practice to coat the particles with wax so that enzyme dusts

are not formed.

22.4.4 Toxicity Testing and Standardization

The enzyme preparation should be tested by animal feeding to show that it is not toxic.

This test not only assays the enzyme itself but any toxic side-product released by the

microorganisms. For a new product extensive testing should be undertaken, but only

spot checks need to done for a proven non-toxic enzyme in production. The potency of the

enzyme preparation, based on tests carried out with the substrate should be determined.

The shelf life and conditions of storage for optimal activity should also be determined.

22.5 IMMOBILIZED BIOCATALYSTS: ENZYMES AND CELLS

The major handicap in the traditional use of enzymes is that they are used but once. This

is mainly because the enzymes are unstable in the soluble form in which they are used

and because recovery would be expensive, even if it were possible. It is not surprising that

Biocatalysts: Immobilized Enzymes and Immobilized Cells "'

influenced by the idea of the catalyst in the chemical industry ways should be sought to

re-use biological catalysts. The immobilization of enzymes and cells provides a basis for

the re-use of enzymes and cells. Interest in immobilized enzymes has grown since the

1960s and numerous conferences and papers have been held and given on them.

Immobilized cells have received a great deal but perhaps slightly less attention judging

from the literature, since their study came a little later than that of immobilized enzymes.

(i) Imobilized enzymes: An immobilized enzyme may be defined as an isolated or

purified enzyme confined or localized in a defined volume of space.

(ii) Immobilized cells: Immobilized cells, also referred to as controlled biological

catalysts, may be defined as a high density of cells physically confined on a solid

phase or in pellets or clumps and in which cell movement is restricted for the

period of their use as biological agents. This definition excludes cells in a

chemostat, or cells which are recovered by centrifugation in a batch culture and

returned to the fermentation. It is not a completely satisfactory definition as the

term ‘high density’ can be elastic. Cell immobilization has existed or been

exploited long before it became recognized as potentially valuable in industry.

Thus, microorganisms in natural habitats such as soil, marine, alimentary canal,

dental plaque or in the ‘Orleans’ process of vinegar production where cells are

immobilized on wood shavings, the activated and trickling filter treatment of

wastewater, may be seen as examples of immobilized cells.

22.5.1 Advantages of Immobilized Biocatalysts in General

The advantages of immobilized enzymes beside reuse are as follows:

(i) They can be easily separated from the reaction mixture containing any residual

reactants and reused in subsequent conservations.

(ii) Immobilized enzymes are more stable over broad ranges of pH and temperature.

(iii) Enzymes are absent in the waste-stream

(iv) Immobilized systems specially lend themselves to continuous processes.

(v) Reduced costs in industrial production.

(vi) Greater control of the catalytic effect.

(vii) Greater ease of new applications for industrial and medical purposes.

(viii) Immobilized enzymes permit the use of enzymes from organisms which would not

normally be regarded as safe (i.e. non-GRAS).

22.5.2 Methods of Immobilizing Enzymes

Immobilized enzymes have been classified in a number of ways. The classification

method adopted here is the one published in 1995 by the International Union of Pure and

Applied Chemistry (IUPAC), and which divides methods of immobilized enzymes into

four broad groups, based on:

(a) covalent bonding of the enzyme to a derivatized water-insoluble matrix,

(b) intermolecular cross-linking of enzyme molecules using multifunctional reagents,