APPLIED ENZYME CATALYSIS

All enzymes used in applications are derived from living sources. Although all living

cells produce enzymes, one of the three sources – plant, animal, or microbial – may be favored

for a given enzyme or utilization.

While all enzymes used today are derived from living organisms, considering only

enzymes which are utilized in the absence of life, such biological catalysts include extracellular

enzymes, secreted by cells in order to degrade polymeric nutrients into molecules small enough

to permeate cell walls. Grinding, mashing, lysing, or otherwise killing and splitting whole cells

open frees intracellular enzymes, which are normally confined within individual cells.

Application of hydrolytic enzymes

The action of hydrolytic enzymes is important not only in obvious macroscopic

degradations such as food spoilage, starch thinning, and waste treatment, but also in the

chemistry of ripening picked green fruit, autolysis, preventing beer haze, desirable aging of meat.

A general classification of the major hydrolytic enzymes is given below:

The three groups of enzymes are those involved in the hydrolysis of ester, glycosidic, and

various nitrogen bonds, respectively. Some enzymes catalyze the hydrolysis of a large variety of

glucose linkages whereas other enzymes may catalyze the hydrolysis of only one glucose

oligomer. Thus, the name of the enzymes by itself is not necessarily indicative of the precise

substrate specificity. Enzymes from different plant or animal sources which catalyze a given

reaction will not always have the same molecular structure or necessarily the same kinetics.

Consequently, maximum reaction rate, Michaelis constant, pH of optimum stability or activity,

and other properties will depend on the particular enzyme source used.

Many hydrolases are directed to specific compartments separated from cyto plasm by

membranes. This serves the obvious purpose of protecting essential cytoplasmic biopolymers
from degradation. However, a few hydrolases are found in the cytoplasm. They serve important

recycle functions in the cell’s use of chemical resources. Most hydrolytic enzymes used

commercially are extracellular microbial products.

Hydrolysis of Starch and Cellulose

Amylases are extensively applied enzymes which can hydrolyze the glycosidic bonds in

starch and related glucose-containing compounds. To differentiate between the two major types

of amylases, recall that starch contains straight-chain glucose polymers called amylose and a

branched component known as amylopectin. The branched structure is relative more soluble than

the linear amylose and is also effective in rapidly raising the viscosity of starch solution. The

action of α- amylase reduces the solution viscosity by acting randomly along the glucose chain at

α-1,4 glycosidic bonds: α- amylase is often called the the starch-liquefying enzyme for this

reason. Β-amylase can attack starch α-1,4 bonds only on the non-reducung ends of the polymer

and always produces maltose when a linear chain is hydrolyzed. Β-amylase is also called a

saccharifying enzyme because of the characteristic production of sugar maltose. A soluble

mixture of starch and Β-amylase yields maltose and a remainder of dextrins, starch remnants

with 1,6 linkages on the end. Β-amylase cannot hydrolyze these bonds.

Another saccharifying enzyme, amyloglucosidase (also called glucoamylase, among

other names) attacks primarily the non-reducung α-1,4 linkages at the ends of starch, glycogen,

dextrins and maltose. Sequential treatment with α-amylase and glucoamylase or enzyme

mixtures are utilized where pure glucose rather than maltose is desired: in distilleries, in the

manufacturing of glucose syrups and crystalline glucose. The relative proprotions of α- and β-

amylase selected in various applications depend on the result desired. These enzymes are one of
the most important groups in commercial use because of this and other applications given in the

table below

The sources of amylases are very numerous since starch is a common form of carbon fuel

for many life forms. Amylases are produced by a number of bacteria and molds. Commercial

amylase preparations used in human foods are normally obtained from grains, notably barley,

wheat, rye, oats, maize sorghum and rice. The ratio of saccharifying to liquefying enzyme

activity depends not only on the particular grain but also upon whether the grain is germinated.

Cellulase, the name usually used to describe the enzyme material active in

depolymerizing cellulose, is a complex mixture of several different of several different enzymes.

Furthermore, the different enzymes present and their relative quantities depend upon the

microorganism used for cellulose production and, in some situations, on the enzyme production
process. Hydrolysis rates and yields achieved in a particular process depend upon the interactions

of substrate properties, pretreatment effects, and multiple enzyme activities and modes of attack.

Proteolytic Enzymes

The varieties and uses of enzymes which attack nitrogen-carrying compounds selectively,

especially proteins, are quite large. As with the amylases, the mode of attack on polyamino acids

is either on terminal groups or internal linkages.

Since enzymes, the essential catalysts of living organisms, are themselves protein, it is

not surprising that protein splitting enzymes are often initially synthesized in an inactive form.

The enzyme is synthesized in an inactive form suitable either for storage or for transport from the

site of synthesis to the desired site of activity, as in the case of pepsin, trypsin, chymotrypsin, and

carboxypeptidase. Activation of these proteolytic enzymes is then accomplished in one of the

two ways shown below:

 It is important to note that the activation of pepsin and trypsin is autocatalytic: the

inactive enzyme precursor is a substrate for the active form of the enzyme, the reaction product

being more of the activate enzyme.

The commercial sources of proteases include animals (pancreas) and large plants (sap,

juices) as well as yeasts, molds, and bacteria. The major uses of free proteases occur in dry

cleaning, detergents, meat processing (tenderization), cheesemaking (rennin only), tanning, silver

recovery from photographic film (pepsin), production of digestive aids etc.

Esterase Application

This group of enzyme carries out the cleavage or synthesis of various ester bonds to yield

an acid and an alcohol.

 The most important subgroups of these enzymes are the lipases, which hydrolyze fats into

glycerol and fatty acids:

Pancreatic lipase, secreted into the digestive tract following neutralization of stomach-imparted

acidity, splits ingested fats to fatty acids as well as the intermediate products of mono- and

diglycerides. The specificity of lipases and a second group of esterases known as aliesterases is

not extreme. Lipases are active toward hydrolysis of high molecular weight fats and inactive

toward the hydrolysis of fats formed from short-chain fatty acids: the reverse applies to

aliesterases.

Enzyme Mixtures, Pectic Enzymes, and Additional Applications

Mixture of enzymes, either of the same general type, for example, α- and β- amylase, or

trypsin and chymotrypsin, or of different types such as found in pancreas extracts e.g trypsin,

lipase, and amylase, are often used more conveniently than single enzyme preparations. Thus,

blends of different amylases achieve large yields of saccharified starch suitable for yeast

fermentation yielding alcohol; a combination with less β- amylase achieves the desired thinning

of starches without too great a saccharification.

Oxidation of galactose to galacturonic acid followed by dehydration and resulting

polymerization yields polygalacturonic acid molecules. Naturally occurring plant molecules

containing such polygalacturonic acid species as a major fraction are collectively termed pectins.

The two major pectin-hydrolysing enzymes are pectin methylesterase and polygalacturonase,

which hydrolyze the methyl ester and the glycosidic linkages, respectively. Major sources are

fruits for the former and fungi for the latter.

There are two main application for pectic enzymes. Crushing fruits and vegetables yields

juices which have high viscosities, desirable in the production of tomato and citrus juices but not

so often for apple cider and other fruit juices. A controlled partial pectin hydrolysis of these

juices yields a free-flowing product which retains enough viscosity to prevent undesirable

settling of particulate matter. A greater hydrolysis is effected with apple juice: the hydrolysed

product is much more easily filtered to yield a clear juice.

The second important application of pectic enzyme is wine production. Addition of the

pectic enzyme mixture to the crushed grapes tends to increase the weight yields of juices, allows

extraction of greater colour from the grape skin, and permits faster filtering and pressing. Later

addition to the fermented product again gives a faster subsequent separation of the wine from the

yeast and grape sediment and yields a clear wine with an increased stability. In both these cases,

a major use of pectic enzymes is thus the development of a process stream with a desirable

viscosity and filterability.

Other Application and uses of enzymes in Solution

While the hydrolytic-enzyme applications considered above dominate enzyme

technology, other enzyme processes currently serve important functions in the food,

pharmaceutical, and biochemical industries.

1. Medical Application of Enzymes

2. Nonhydrolytic Enzymes in industrial Technology
Immobilized enzyme Technology

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

be reused continuously. There are several reasons why immobilization may be desirable: for

processing with isolated enzymes, an immobilized form can be retained in the reactor. A main

advantage of immobilized enzyme is that it can be reused since it can be easily separated from

the reaction solution and can be easily retained in a continuous-flow reactor. Furthermore,

immobilized enzyme may show selectively altered chemical or physical properties and it may

simulate the realistic natural environment where the enzyme came from, the cell.

Immobilization techniques can be classified by basically two methods, the chemical and

the physical method. The former is covalent bond formation dependent and the latter is non-

covalent bond formation dependent.

Chemical Method

Covalent Attachment: The covalent attachment of enzyme molecules via nonessential amino

acid residues (that is, amino acids minus water) to water-insoluble, functionalized supports are

the most widely used method for immobilizing enzymes. Functional groups of the nonessential

amino acid residues that are suitable for the immobilization process are free α-,β- or γ-carboxyl

groups, α- or β amino groups, and phenyl, hydroxyl, sulfhydryl, or imidazole groups.

Anothervariation of immobilization by covalent attachment is the copolymerization of the

enzyme with a reactive monomer (M) such as

where MnE may have the following structure:

Commonly employed water-insoluble supports for the covalent attachment of enzymes

include: synthetic supports such as acrylamide-based polymers, maleic anhydride-based

polymers, methacrylic acid-based polymers, styrene-based polymers, and polypeptides, and

natural supports such as agarose (Sepharose), cellulose, dextran (Sephadex), glass, and starch

(Zaborsky, 1973).

Already active polymers such as maleic anhydride copolymers will be simply mixed with

enzymes to produce immobilized enzymes. Normally, natural or synthetic polymers need to be

activated by treating them with reagents before adding the enzyme. The activation involves the

chemical conversion of a functional group of the polymer. The enzyme's active site should not be

involved in the attachment, in which case the enzyme would lose its activity upon

immobilization.

Cross-linking Using Multifunctional Reagents: Water-insoluble enzymes can be prepared by

using multifunctional agents that are all bifunctional in nature and have low molecular weight,

such as glutaraldehyde.

There are several different methods for producing immobilized enzymes with

multifunctional reagents, as illustrated below.

Enzymes can be reacted with multifunctional reagent alone so that they are cross-linked

intermolecularly by the reagent to form a water insoluble derivative. Another method is to adsorb

enzymes on a water-insoluble, surface-active support followed by intermolecular cross-linking

with multifunctional reagents to strengthen the attachment. Multifunctional reagents can be also
used to introduce functional groups into water-insoluble polymers, which then react covalently

with water-soluble enzymes.

Physical Method

Adsorption: This method is the simplest way to immobilize enzymes. Enzymes can be adsorbed

physically on a surface-active adsorbent by contacting an aqueous solution of enzyme with an

adsorbent. Commonly employed adsorbents are (Zaborsky, 1973): alumina, amon-exchange

resins, calcium carbonate, carbon, cation-exchange resins, celluloses, clays, collagen, colloid-

ion, conditioned metal, glass plates, diatomaceous earth, and hydroxyapatite. The advantages of

adsorption techniques are as follows:

1. The procedure of immobilization is simple.

2. It is possible to separate and purify the enzymes while being immobilized.

3. The enzymes are not usually deactivated by adsorption.

4. The adsorption is a reversible process.

However, adsorption techniques also have several disadvantages:

1. The bonding strength is weak.

2. The state of immobilization is very sensitive to solution pH, ionic strength, and temperature.

3. The amount of enzymes loaded on a unit amount of support is usually low.

Entrapment: Enzymes can be entrapped within cross-linked polymers by forming a highly

cross-linked network of polymer in the presence of an enzyme. This method has a major
advantage in the fact that there is no chemical modification of the enzyme, therefore, the intrinsic

properties of an enzyme are not altered. However, the enzyme may be deactivated during the gel

formation. Enzyme leakage is also a problem. The most commonly employed crosslinked

polymer is the polyacrylamide gel system. This has been used to immobilize alcohol

dehydrogenase, glucose oxidase, amino acid oxidase, hexokmase, glucose isomerase, urease, and

many other enzymes.

Microencapsulation: Enzymes can be immobilized within semipermeable membrane

microcapsules. This can be done by the interfacial polymerization technique. Organic solvent

containing one component of copolymer with surfactant is agitated in a vessel and aqueous

enzyme solution is introduced. The polymer membrane is formed at the liquid-liquid interface

while the aqueous phase is dispersed as small droplets. One example of this process is the

polyamide nylon system, in which 1, 6-diaminohexane is the water-soluble diamine and 1,10-

decanoyl chloride is the organic-soluble diacid halide. The organic solvent for this system is a

chloroformcyclohexane mixture containing usually 1 percent Span85 surfactant (Zaborsky,

1973). The immobilized enzyme produced by this technique provides an extremely large surface

area.

Effect of mass-transfer resistance

The immobilization of enzymes may introduce a new problem which is absent in free soluble

enzymes. It is the mass-transfer resistance due to the large particle size of immobilized enzyme

or due to the inclusion of enzymes in polymeric matrix. If we follow the hypothetical path of a

substrate from the liquid to the reaction site in an immobilized enzyme, it can be divided into

several steps.
1. Transfer from the bulk liquid to a relatively unmixed liquid layer surrounding the immobilized

enzyme;

2. Diffusion through the relatively unmixed liquid layer; and

3. Diffusion from the surface of the particle to the active site of the enzyme in an inert support.

Steps 1 and 2 are the external mass-transfer resistance. Step 3 is the intraparticle mass-transfer

resistance.

Rajiv Dutta Fundamentals of Biochemical Engineering (2008)