Microbial Protease enzyme:

• Proteases are the second most important industrial enzymes after amylase.
• About 500 tons of the enzymes are produced per year.
• This can be produced commercially from bacteria and fungi.
• The proteases on the market include-alkaline, neutral and acidic proteases.

i. Alkaline protease:

• 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.

ii. Neutral protease:

• 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).

iii. Acidic protease:

• 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.

Production process of protease enzyme:

step I: Isolation of proteolytic microbes:

• 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.

Step II: Media formulation:

• 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.

step III: Fermentation:

• 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.

step IV: Purification:

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