Enzyme Technology

Applications of Enzymes
The biocatalysts (enzymes and cells) are used in multifarious ways in different field. Trevan (1987) has grouped the applications into four broad categories: (i) therapeutic uses, (ii) analytical uses, (iii) manipulative uses, and (iv) industrial uses.
 
Therapeutic Uses

Enzymes are used for this purpose where some inborn errors of metabolism occur due to missing of enzyme (see Monoclonal antibodies) where specific genes are introduced to encode specific missing enzymes. However, in most of cases certain diseases are treated by administering the appropriate enzyme. For example, virilization of a disease developed due to loss of an hydroxylase enzyme from adrenal cortex and introduction of hydroxyl group (-OH) on 21-carbon of ring structure of steroid hormone. Steroids are compounds having a common skeleton in the form of perhydro-1, 2-cyclo-pentano-phenanthrene (Fig. 17.4). The missing enzyme synthesizes aldosterone (male hormone) in excess leading to masculinization of female baby and precocious sexual activity in males in about 5-7 years.
 
Similarly, treatment of leukaemia (a disease in which leukaemic cells require exogenous asparagine for their growth) could be done by administering asparaginase of bacterial origin.
 
Analytical Uses
Use of enzymes for analytical purposes is also important. Gen­erally end point and kinetic analysis are pos­sible. End point analysis refers to total conver­sion of substrates into products in the presence of enzymes in a few minutes while kinetic analysis involves the rate of reaction and sub­strate/product concentration. Moreover, analy­sis of antibodies, immunoglobins, necessary for human use poses a great promise. The usable enzymes are alkaline phosphatase, b-galactosidase, b-lactamase, etc.

A common skeleton of the steroids

Fig. 17.4. A common skeleton of the steroids

  Content
Microorganisms
Properties of enzymes
 
Presence of species specificity
 
Variation in activity and stability
 
Substrate specificity
 
Activation and inhibition
Methods of enzyme production
 
Isolation of microorganisms, strain development and preparation of inoculum 
 
Medium formulation and preparation
 
Sterilization and inoculation of medium, maintenance of culture and fluid filtration
 
Purification of enzymes
Immobilization of enzymes
 
Advantages of using immobilized enzymes
 
Methods of enzyme immobilization
 
 
Adsorption
 
 
Covalent bonding (Ionic bonding)
 
 
Entrapping
 
 
Cross linking
 
 
Encapsulation
 
Effects of enzyme immobilization on enzyme stability
Enzyme engineering
⇒ Application of enzymes
 
Therapeutic uses
 
Analytical uses
 
Manipulative uses
 
Industrial uses
 
 
In dairy industry
 
 
In detergent industry
 
 
In starch industry
 
 
In brewing industry
 
 
In wine industry
 
 
In pharmaceutical industry
Biosensor
 
Types of biosensor
 
Applications of biosensor
Biochips
 
Principles of Biochips
 
Application of Biochips
Another use of enzyme is in biosensor, device of biologically active material displaying characteristic specificity with chemical or electronic sensor to convert a biological compound into an electronic signal. It is constructed to measure almost anything from blood glucose. A simple carbon electrode, an ion sensitive electrode, oxygen electrode or a photocell, may be a sensor (Trevan, 1987).

 

Manipulative Uses

A variety of enzymes isolated from different sources are now-a-days applied in genetic engineering as one of the biological tools. Some of them are available in market (Table 3.1). Examples and brief discussion of these enzymes are given in Example of some enzymes.

 

Industrial Uses

Enzymes are used in industries in different ways.

 

In dairy industry

For a long time calf rennet has been used in dairy industry. In recent years, calf rennets are replaced by microbial rennets (e.g. Mucor michei). They are acid aspartate proteases. They slightly differ from calf rennets as they depend for reaction with casein on Ca++, temperature, pH, etc.

 

Lactase (produced by Bacillus stearothermophilus) is used for hydrolysis of lactose in whey or milk, and lipase for flavor development in special cheeses.

In detergent industry

During normal washing proteinaceous dirt often precipitates on solid cloths and proteins facilitate to adhere the dirt on textile fibers and make stains on cloths. These stains are difficult to remove from clothes. Nevertheless, it can be easily removed by adding proteolytic enzymes to the detergent. It attacks on peptide bonds and therefore, dissolves protein. The alkaline serine protease obtained from B. licheniformis is most widely preferred to use in detergent. In addition, the serine protease of B. amyloliquefaciens is also used for this purpose. It contains a-amylase, hence to some extent it may be advantageous (Aunstrup et al., 1979).



In starch industry
It has been mentioned earlier that hydrolysis of starch began in early 1960s to prepare dextrose and glucose syrups. Furthermore, for complete acid hydrolysis of starch to dextrose glucoamylase was coupled with bacterial a-amylase. Currently, various enzymatic processes are applied for various products (Fig. 17.5).

Glucose isomerase is an important enzyme used commercially in conversion of glucose to fructose via isomerization. Fructose is used in the preparation of fructose syrup.
  Enzymatic process applied in starch industry
 

Fig. 17.5. Enzymatic process applied in starch industry



The reaction mixture at the end contains 42% fructose, 52% glucose and 6% dextrins. The mixture is sweeter than glucose and as sweet as sucrose. Now, techniques have been developed to obtain 55% fructose concentration in syrup (Singh, 1987).
 
In brewing industry
Enzymes used in brewing industry are a-amylase, b-glucanase and protease which are required for malt in substitution of barley. Source of these enzymes is B. amyloliquefaciens. a-amylase is not required for liquefaction or brewing adjuncts and b-glucanase alleviates filtration problems due to poor malt quality and neutral protease helps in the inhibition of alkaline protease by an inhibitor.
 
In wine industry
Pectic enzymes are used in wine industry for high yield of products of improved quality. The pectic enzymes are pectin transeliminase (PTE), polymethyl galacturonase (PMG), polygalacturonase (PG),pectine esterase (PE), etc. However, pectic enzymes give a good result when combined with other enzymes e.g. protease glucoamylase, etc.
  glucose isomerase.....fructose

In pharmaceutical industry
Penicillin G/V acylase, glucose isomerase, etc. are widely used in Pharmaceuticals for the production of semisynthetic penicillins and fructose syrup, respectively. All penicillins consist of an active beta lactam ring i.e. 6-amine penillanic acid (6 APA) group combined with different side chains (R group) (Fig. 16.2) Penicillin G/V acylase removes G/V group from penicillin G/V resulting in separation of 6 APA and R groups.


Finally, new synthetic side chains are coupled with 6 APA to synthesize new semisynthetic penicillins. Enzyme reaction are as below:

 
penicillin G acylase
 
Penicillin G
 arrow
6APA + G side chain
 
penicillin V acylase
 
Penicillin V
Arrow 
6APA + V side chain
 
side chain addition
 
6APA
 arow
semisynthetic penicillins

Among penicillin G acylase producing microorganisms, E. coli strains are the most explored and exploited ones. The biosynthesis of penicillin acylase in E. coli is controlled by altering the nutrients and culture conditions. Sudhakaran and Berkar (1989) investigated the effect of growth substrate, inducers and regulators on enzyme formation. E. coli NCJM-2400 produced penicillin G acylase intracellularly when grown in nutrient broth containing phenyl acetic acid (PAA). PAA (20 mM) stimulatd enzyme synthesis by 8-10 fold. Glucose, lactose, sorbitol, acetate and lactate (all 0.1%) catabolically repressed the enzyme formation. Phosphate and yeast extract were found essential for both the growth and enzyme biosynthesis. Penicillin V acylase occurs in fungal and actinomycetes sources. However, its activity has been found in many bacteria such as Bacillus sphaericus, Erwinia aroideae, and Pseudomonas acidovorans. Lowe et al (1986) have identified a strain of Fusarium oxysporum which showed intracellular penicillin V acylase activity. Activity was induced by phenoxyacetic acid in culture. Enzyme was partially purified and concentrated from disrupted cells (cells hydrolyzed with 5% penicillin V solutions) by fractional precipitation with miscible solvents.