||Article by Prashantkumar Kudli Shrinivas
Most of the reactions in living organisms are catalyzed by protein molecules called enzymes. Enzymes can rightly be called the catalytic machinery of living systems. The real break through of enzymes occurred with the introduction of microbial proteases into washing powders. The first commercial bacterial Bacillus protease was marketed in 1959 and major detergent manufactures started to use it around 1965.
The industrial enzyme producers sell enzymes for a wide variety of applications. The estimated value of world market is presently about US$ 2 billion. Detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of industrially produced enzymes.
Presently more than 3000 different enzymes have been isolated and classified. The enzymes are classified into six major categories based on the nature of the chemical reaction they catalyze:
1. Oxidoreductases catalyze oxidation or reduction of their substrates.
2. Transferases catalyze group transfer.
3. Hydrolases catalyze bond breakage with the addition of water.
4. Lyases remove groups from their substrates.
5. Isomerases catalyze intramolecular rearrangements.
6. Ligases catalyze the joining of two molecules at the expense of chemical energy.
Only a limited number of all the known enzymes are commercially available . More than 75 % of industrial enzymes are hydrolases. Protein-degrading enzymes constitute about 40 % of all enzyme sales. More than fifty commercial industrial enzymes are available and their number is increasing steadily.
Some enzymes still extracted from animal and plant tissues. Enzymes such as papain, bromelain and ficin and other speciallity enzymes like lipoxygenase are derived from plants and enzymes pepsin and rennin are derived from animal. Most of the enzymes are produced by microorganisms in submerged cultures in large reactors called fermentors. The enzyme production process can be divided into following phases:
1. Selection of an enzyme.
2. Selection of production strain.
3. Construction of an overproducing stain by genetic engineering.
4. Optimization of culture medium and production condition.
5. Optimization of recovery process.
6. Formulation of a stable enzyme product.
Criteria used in the selection of an industrial enzyme include specificity, reaction rate, pH and temperature optima and stability, effect of inhibitors and affinity to substrates. Enzymes used in the industrial applications must usually tolerant against various heavy metals and have no need for cofactors.
Microbial production strains
In choosing the production strain several aspects have to be considered. Ideally the enzyme is secreted from the cell. Secondly, the production host should have a GRAS-status. Thirdly, the organism should be able to produce high amount of the desired enzyme in a reasonable life time frame. Most of the industrially used microorganism have been genetically modified to overproduce the desired activity and not to produce undesired side activities.
Enzyme production by microbial fermentation
Once the biological production organism has been genetically engineered to overproduce the desired products, a production process has to be developed. The optimization of a fermentation process includes media composition, cultivation type and process conditions. The large volume industrial enzymes are produced in 50 -500 m3 fermentors. The extracellular enzymes are often recovered after cell removal (by vacuum drum filtration, separators or microfiltration) by ultrafiltration.
Often enzymes do not have the desired properties for an industrial application. One option is find a better enzyme from nature. Another option is to engineer a commercially available enzyme to be a better industrial catalyst. Another option is to engineer a commercially available enzyme to be a better industrial catalyst. Two different methods are presently available: a random method called directed evaluation and a protein engineering method called rational design.
This field deals with how are the enzymes used and applied in practical processes. The simplest way is to use enzymes is to add them into a process stream where they catalyze the desired reaction and are gradually inactivated during the process. This happens in many bulk enzyme applications and the price of the enzymes must be low to take their use economical.
An alternative way to use enzymes is to immobilize them so that they can be reused. Enzyme can be immobilized by using ultra filtration membranes in the reactor system. The large enzyme molecule cannot pass through the membrane but the small molecular reaction products can. Many different laboratory methods for enzyme immobilization based on chemical reaction, entrapment, specific binding or absorption have been developed.
Large scale Enzyme applications
Bacterial proteinases are still the most important detergent enzymes. Lipases decompose fats into more water-soluble compounds. Amylases are used in detergents to remove starch based stains.
2] Starch hydrolysis and fructose production
The use of starch degrading enzymes was the first large scale application of microbial enzymes in food industry. Mainly two enzymes carry out conversion of starch to glucose: alpha-amylase and fungal enzymes. Fructose produced from sucrose as a starting material. Sucrose is split by invertase into glucose and fructose, fructose separated and crystallized.
Enzymes have many applications in drink industry. Lactase splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers. Addition of pectinase, xylanase and cellulase improve the liberation of the juice from pulp. Similarly enzymes are widely used in wine production.
The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology. The enzymes used in the textile field are amylases, catalase, and lactases which are used to remove the starch, degrade excess hydrogen peroxide, bleach textiles and degrade lignin.
5] Animal feed
Addition of xylanase to wheat-based broiler feed has increased the available metabolizable energy 7-10% in various studies. Enzyme addition reduces viscosity, which increases absorption of nutrients, liberates nutrients either by hydrolysis of non-degradable fibers or by liberating nutrients blocked by these fibers, and reduces the amount of faeces.
Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life. Use of xylanases decreases the water absorption and thus reduces the amount of added water needed in baking. This leads to more stable dough. Proteinases can be added to improve dough-handling properties; glucose oxidase has been used to replace chemical oxidants and lipases to strengthen gluten, which leads to more stable dough and better bread quality.
7] Pulp and Paper
The major application is the use of xylanases in pulp bleaching. This reduces considerably the need for chlorine based bleaching chemicals. In paper making amylase enzymes are used especially in modification of starch. Pitch is a sticky substance present mainly in softwoods. Pitch causes problems in paper machines and can be removed by lipases.
Leather industry uses proteolytic and lipolytic enzymes in leather processing. Enzymes are used to remove unwanted parts. In dehairing and dewooling phases bacterial proteases enzymes are used to assist the alkaline chemical process. This results in a more environmentally friendly process and improves the quality of the leather . Bacterial and fungal enzymes are used to make the leather soft and easier to dye.
9] Speciality enzymes
There are a large number of specialty applications for enzymes. These include use of enzymes in analytical applications, flavour production, protein modification, and personal care products, DNA-technology and in fine chemical production.
10] Enzymes in analytics
Enzymes are widely used in the clinical analytical methodology. Contrary to bulk industrial enzymes these enzymes need to be free from side activities. This means that elaborate purification processes are needed.
An important development in analytical chemistry is biosensors. The most widely used application is a glucose biosensor involving glucose oxidase catalysed reaction.
Several commercial instruments are available which apply this principle for measurement of molecules like glucose, lactate, lactose, sucrose, ethanol, methanol, cholesterol and some amino acids.
11] Enzymes in personal care products
Personal care products are a relatively new area for enzymes. Proteinase and lipase containing enzyme solutions are used for contact lens cleaning. Hydrogen peroxide is used in disinfections of contact lenses. The residual hydrogen peroxide after disinfections can be removed by catalase enzyme. Some toothpaste contains glucoamylase and glucose oxidase. Enzymes are also studied for applications in skin and hair care products.
12] Enzymes in DNA-technology
DNA-technology is an important tool in enzyme industry. Most traditional enzymes are produced by organisms, which have been genetically modified to overproduce the desired enzyme. The specific order of the organic bases in the chain of DNA constitutes the genetic language. Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process.
13] Enzymes in fine chemical production
In spite of some successes, commercial production of chemicals by living cells using pathway engineering is still in many cases the best alternative to apply biocatalysis. Isolated enzymes have, however, been successfully used in fine chemical synthesis. Some of the most important examples are represented here.
13 A] Chirally pure amino acids and aspartame
Natural amino acids are usually produced by microbial fermentation. Novel enzymatic resolution methods have been developed for the production of L- as well as for D-amino acids. Aspartame, the intensive non-calorie sweetener, is synthesized in non-aqueous conditions by thermolysin, a proteolytic enzyme.
13 B] Rare sugars
Recently enzymatic methods have been developed to manufacture practically all D- and L-forms of simple sugars. Glucose isomerase is one of the important industrial enzymes used in fructose manufacturing.
13 C] Semisynthetic penicillins
Penicillin is produced by genetically modified strains of Penicillium strains. Most of the penicillin is converted by immobilised acylase enzyme to 6-aminopenicillanic acid, which serves as a backbone for many semisynthetic penicillins.
13 D] Lipase based reactions
In addition to detergent applications lipases can be used in versatile chemical reactions since they are active in organic solvents. Lipases used in transesterification and also used for enantiomeric separation of alcohols and separate racemic amine mixtures. Lipases have also been used to form aromatic and aliphatic polymers.
13 E] Enzymatic oligosaccharide synthesis
The chemical synthesis of oligosaccharides is a complicated multi-step effort. Biocatalytic syntheses with isolated enzymes like glycosyltransferases and glycosidases or engineered whole cells are powerful alternatives to chemical methods. Oligosaccharides have found applications in cosmetics, medicines and as functional foods.
Future trends in industrial enzymology
Industrial enzyme market is growing steadily. The reason for this lies in improved production efficiency resulting in cheaper enzymes, in new application fields. Tailoring enzymes for specific applications will be a future trend with continuously improving tools and understanding of structure-function relationships and increased search for enzymes from exotic environments.
New technical tools to use enzymes as crystalline catalysts, ability to recycle cofactors, and engineering enzymes to function in various solvents with multiple activities are important technological developments, which will steadily create new applications.
Dr. Prashantkumar Kudli Shrinivas has a vast industrial experience of Aroma chemicals, Essential oils, Perfumery and Flavour formulations. He is a permanent member of Indian Institute of Chemical Engineers & NMR Association of India.
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