Industrial Production Process of Enzymes

Introduction;

Commercial production of an enzyme has been growing day by day due to the demand of novel biocatalyst and to expand the market of an enzyme ,enzymes are produced in large volume and the large number of products. Enzyme production is the most powerful successor central to the modern biotechnology industry. The enzyme industries are expanding and they are applying the new method,new technology to improve the traditional production . For the new application and the recognition of potential to use biocatalyst in various sectors and improve the stability of enzymes are produced.Nowadays enzymes are being common in every sector. Most of the human beings use enzymes for various purpose.Enzymes are used in brewing ,baking,fermentation,cheese production etc.The enzymatic action and the conversion are carried out in the only 19th century. This enzyme is first isolated by Payen and person in 1833 from malt termed as 'diastase' . Diastase converts gelatinized starch into sugars ,ie maltose.

The commercial production of enzymes use different technology and combines the techniques of microbiology ,genetics,biochemistry , The production of the enzyme and the applied technology is increasing day by day and is demand is also increasing due to the broad application field of enzymes.The industrial production of enzymes includes protease,amylase,pectinase,chitinase. etc.The growing demand d and to fulfill the demand ,process to manufacture enzymes are also being developed. The new technologies and the sophisticated materials are being used . The recombinant gene technology plays the role in the present production of the enzyme and it has the great impact on enzyme industry. Genetic engineering techniques are also being prominent in the enzyme production to produce sufficient quantities of enzyme. In this chapter, we study the whole process from the first step ie; selection of enzyme source to the last step ie; purification of the enzyme .

The production of the enzyme is comparable to the methods used for the manufacture of other industrial products. The general steps in enzyme production can be described in following steps:

1) Selection of source of enzyme:

Primary steps in the production of the enzyme are the selection of the best source from which we can easily produce an enzyme in the short period of time. Several sources are being applied in the production of enzymes. Animal,plant,fungal and microbial sources are mostly used as strain. Biological active enzymes can be extracted from the above living organisms . Half of the enzymes sources used fungi and yeast as the main source and over 1/3 use bacteria while remaining use animal and plants. But microorganisms are preferred as the best source of the enzyme as they can be cultured easily enlarged quantities in relatively short period of time. The microorganisms can be grown easily and they can be easily manipulated to produce enzyme of need.They can produce the large variety of enzymes so they are mostly used as the main source of enzyme .

2) Isolation and improvement of strain:

Microorganisms producing desired enzymes are screened from their natural habitat. The aim for isolating suitable organisms is for:

• Production of an enzyme in high amount and other metabolites in low amount.
• Completion of fermentation in the short time.
• Utilization of low-cost culture medium by the microorganism.

The selected microorganisms are then improved for the enzyme production by appropriate methods. Mutagenesis such as chemical mutagens and UV rays are used to induce mutations .The strain can also be genetically engineered to produce a large amount of enzyme and this selected and improved strain is preserved for future use.

3) Preparation of inoculum:

The main objective of the preparation of inoculum is to obtain organisms in an optimal state which is more compactable with inoculation into cell culture ,media ,tissue culture . The aim is to achieve a high biomass in a large quantity for the use as an inoculum.This process has many advantages in the industrial microbiology to produce enzymes ,beverages, toxins, drugs,proteins,vitamins ,food etc. The inoculum should be free from contamination and must be in an active stage of growth and size. The selected and improved organisms are multiplied in broth medium to obtain inoculum of spores,mycelium, bacterial cells to be used in an industrial process.

4) Formulation and preparation of media:

The culture medium is formulated in such a way that it contains all the nutrients to support adequate growth of microorganisms that will ultimately result in a good quality of enzyme production. The ingredients of the medium should be readily available at low cost and are nutritionally safe. An ideal medium should contain a chief source of carbon,nitrogen, amino acids , growth promoters,trace elements and little amount of salt . A typical medium should have following constituents;

a) Carbohydrates: Molasses,barley,corn,wheat, etc

b) Protein: Soybean,cottonseed ,peanut,whey,etc.

The pH of the medium should be kept optimum for good microbial growth and enzyme production. The constituents are mixed in appropriate proportion ,autoclave or filtered for optimum production yield.

5) Production process:

It involves the sterilization of medium,inoculation of culture and the fermentation process. Industrial production of an enzyme is mostly carried out by submerged state fermentation and to the lesser extent by solid state fermentation .Submerged culture technique yields more with less contamination and is thus a preferred method. However, fungal enzymes such as amylase ,cellulase,protease, and pectinase are produced by solid state fermentation.

The medium is sterilized by using suitable sterilization technique . The fermentation is started by inoculating the medium with sufficient amount inoculum.

The growth conditions( temperature,pH, oxygen supply ,nutrient supply) are maintained at the optimum level. The foam formed is minimized by using the antifoaming agent.

The production of an enzyme is mostly carried out by batch fermentation rather than by continuous fermentation process. The bioreactor is left for 2-7 days in most production cases .

Most of the enzymes are produced when the exponential phase is complete but in few cases, they are produced during exponential phase.

6) Recovery and purification of enzymes:

The enzyme produced may be extracellular or intracellular. The enzyme may be obtained in crude or purified forms,solid or liquid form. The steps involved during down streaming process depends on upon the nature of enzyme and degree of purity desire. The recovery of all extracellular enzyme is easy compared to intracellular. The recovery of intracellular enzymes requires additional steps of the disruption . The various method employed for extraction of intracellular enzymes are:

a) Physical method:

It includes ultrasonication,osmotic shock ,heat shock ,high pressure ,grinding with glass and others.

b) Chemical method:

It includes treating with alkalies, organic solvents, and detergents etc.

c) Enzymatic method:

It includes treatment with lysozyme and others.

The steps after extraction of intracellular enzyme are similar with both types of enzymes and includes

• Removal of cell debris by filtration or centrifugation,
• Removal of nucleic acid by their precipitation
• Precipitation of enzyme using salts (eg; ammonium sulfate), organic solvents eg; ethanol, isopropanol, acetone etc.
• Concentration of enzyme by liquid -liquid partition.
• Separation by the chromatographic technique such as ion exchange ,size exclusion, and hydrophobic interaction chromatography.
• Drying by evaporation freeze dryers and packing of the enzyme to obtain powdered enzyme.

For obtaining liquid enzyme ,the enzyme obtained after separation of biomass and removal of nucleic acid is preserved and stabilized by using preservatives and stabilizers.