Microbial Proteases: industrial application and production process

Enzymes- Introduction

Enzymes are proteins which catalyzes specific biochemical reactions is a very efficient manner.
Enzymes can be produced by different types of micro-organisms, plant cells and animal cells.
Microbial enzymes have gained much popularity.
They can produce two different types of enzymes:
- Extracellular enzymes
- Intracellular enzymes
Microbial enzymes have two advantages over plant and animal enzymes.
They are economical and can be produced on large scale within the limited space and time.
It can be easily produced and purified.
There are technical advantages in producing enzymes by using micro-organisms like:
- They have ability to produce wide variety of enzymes.
- Can grow in wide range of environmental condition.
- They show genetic flexibility so can be genetically manipulated to increase the yield of enzymes.
- They have short generation time.
The industrial production of enzyme date back to 1894.
Initially, enzymes were produced by solid state fermentation.
But most current production processes are based on submerged cultures which can be effectively aerated.
Most important enzyme are extracellular which make their recovery from broth relatively easier.
A number of enzymes are produced on large scale and used in commercial operations.

Many bacteria and fungi excrete alkaline protease and the most important producer are Bacillus strains like Bacillus licheniformis, Bacillus amylotiquefaciens, B. megaterium, B. purilis and Streptomyces strains like Streptomyces fradiae, S. griseus, S. rectus and fungi like Aspergillus niger, A. sojae, A. oryzae, A. flavus
Alkaline proteases are commonly used in detergents.
Mainly proteases from bacillus licheniformis.
Alkaline proteases have some features which makes its applications in industrial scales like stability at high temperature, stability in alkaline range (pH 9-11), stability in association with chelating agents.

Neutral proteases are produced by bacteria and fungi. E.g. Bacillus subtilis, B. coreus, B. megaterium, Pseudomonas aeruginosa, Streptomyces griseus, Aspergillus oryzae, Aspergillus sojae.
Neutral proteases are relatively unstable and calcium, sodium and chloride must be added for maximal stability.
Its pH range for activity is narrow and sensitive to increased temperature.
These are quickly inactivated by alkaline proteases.
Because of these limitations, they have restricted industrial applications but can be used in leather industries and in food industries (For manufacture of breads and rolls).

Acidic proteases are found in animal cells, yeast and molds but never in bacteria.
These microbial renin-like enzymes are derived from Mucor michei, Mucor pusillus, Mucor racemaeus, Mucor bacilliformis.
Pepsin like acid proteases are derived from Aspergillus species and Rhizopus spp.
Renin like proteases are commonly used in cheese production in optimum pH 2-4.

Proteolytic microbes can be isolated by observing hydrolysis in casein agar.
After isolating the suitable strain, it necessarily increases enzyme production by optimizing process parameter like media composition, pH, volume, moisture content (in-case of solid-state fermentation), concentration of mineral salts, age and size of inoculum, fermentation time and temperature, organic and inorganic supplements.
Among the various proteases, bacterial proteases are the most significant as compared with animal and fungal proteases.
Among bacteria, Bacillus spp are specific producer of extracellular proteases.
For industrial utilization, the genes for formation of several proteases have been cloned- protein engineering has been used to develop, modify Bacillus subtilapeptideases with altered amino-acid sequences.
Corresponding changes in enzymatic properties such as substrate specificity, pH optimum and stability to bleaching agent.

Media rich in nitrogen sources such as soyabean milk, casein, gelatin and carbohydrate sources such as starch, or lactose are generally used for protease production.

The nature of fermentation, solid or submerged influences the growth of moss as well as enzyme production.
For the production of alkaline proteases by using B. subtilopeptidase cultured are stored in lyophilized state or under liquid nitrogen (for sterility).
Initial growth is carried out in shaken flask and small fermenter at 30-37^oC in 40-100 mm³.
Process:
- Fed batch process (to keep down the concentration of NH₄⁺ ions and aminoacids).
- Sometimes continuous process can also be used but it is not so common.
- Aeration-1vvm, time 48-72 hrs.

Different methods can be applied for purification of enzyme like ultrafiltration.
Chromatographic technique (ion exchange) purification by treatment with activated charcoal and H₂O_2.