MICROBIAL PRODUCTION OF PROTEASES

Complex mixtures of true proteinase and peptidases are usually called proteases. A protease is an enzyme that performs proteolysis; protein catabolism by hydrolysis of peptide bonds. They are relatively unstable and tend to lose their activity during dehydration.

Source: Proteases are produced by bacteria, i.e., Bacillus subtilis and B. licheniformis as well as by fungi, i.e., Aspergillus niger and A. oryzae.

Mode of action of enzyme: Proteases act by digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. Some detach the terminal amino acids from the protein chain (exopeptidases), other attack internal peptide bonds of a protein (endopeptidases).

There are two types of proteases:
A. Alkaline serine proteases:
Type of fermentation: obtained from Bacillus licheniformis by submerged culture method.
Production parameters:
The bacterium is grown on the medium with following composition:
Component.                            Amount (g/L)
Starch hydrolyzate.                   50
Soya bean meal.                       20
Casein.                                      20
Na2HPO4.                                 3.3

Temperature of fermentation is maintained in the range 30-40 C.
pH of the production medium is kept 7.0
Production of enzyme begins when maximum cell growth is reached after 10-20 hours.
At the end of the productive fermentation, protease is the only protein present in the production medium as all other proteins present in the medium are hydrolyzed by protease.

DOWNSTREAM PROCESSING:
⦁ For harvesting of enzyme, culture is filtered or centrifuged.
⦁ Harvested protease product is purified by salt precipitation method. The yield of enzyme increases by the addition of ammonium salts. Acetone precipitation is another method to concentrate the filtrate.
⦁ Further purification is carried out by Column chromatography method.
⦁ The enzyme is marketed primarily in the form of dust-free granules, which contain 1-5% of the enzyme.

B) ACID PROTEASES:

Source: fungi are employed for producing these enzymes such as Mucor miehei, M. pusillus, A.niger etc.

Type of fermentation: acid protease may be produced by either semisolid culture or submerged culture method.
Mucor pusillus is cultivated on a semisolid medium. The medium consists of 60% wheat bran with water.

Production parameters:
⦁ Optimum temperature of fermentation is 30C
⦁ Fermentation requires 3 days for completion
⦁ The yield is 3,200 Soxhlet units per gram of wheat bran.
Mucor miehei is grown by submerged culture method. Production method used for this purpose has the following composition:

Component.                         Amount (%)
Starch.                                    4.0
Soya bean meal.                     3.0
Ground barely.                        10.0
Calcium carbonate.                  0.5

Production parameters:
⦁ Optimum temperature of the fermentation is 30 C.
⦁ Fermentation is completed in 7 days.
⦁ Yield is 3500 soxhlet units per ml of the fermented broth.

DOWNSTREAM PROCESSING:
⦁ After completion of fermentation, fermented substrate is soaked with water for extraction of the enzymes. Firstly mycelium is removed by filtration using a rotary vacuum filter.
⦁ Crude extract of protease is further concentrated and purified by salt precipitation methods. The yield of the enzyme increases by the addition of ammonium salts.
⦁ Finally, purification is carried out by column chromatography.

APPLICATIONS:
⦁ The wide specificity of the hydrolytic action of proteases finds an extensive application in different industries such as food, laundry, detergent, leather, pharmaceutical etc.
⦁ Proteases have a variety of applications in food industry including improving the digestibility and sensory quality of food. The hydrolytic quality of proteases is exploit for degradation of the turbidity complex resulting from protein in fruit juices and alcoholic liquors, gelatin hydrolysis, soy- protein hydrolysis, casein and whey protein hydrolysis, meat protein recovery and meat tenderization.
⦁ The major application of proteases in dairy industry is in the cheese manufacturing, where the primary function of enzymes is to hydrolyze the specific peptide bonds to generate casein and macropeptides. Endo and exoproteinases from A. oryzae have been used to modify wheat gluten in baking processes. The addition of proteases reduces the mixing time of dough and results in increased loaf volumes. Protein hydrolysates from casein, whey protein have applications as constituents of dietic and health products.
⦁ Use of proteases in detergent formulation enhances the detergents ability to remove tough stains by the release of proteinaceous materials and making the detergent environmentally safe.
⦁ In a tannery, a raw hide is subjected to a series of chemical treatments before tanning and finally converted to finished leather. Alkaline proteases play a vital role in these treatments by replacing the hazardous chemicals involved in soaking, dehairing and bating.
⦁ In the pharmaceutical and cosmetic industries, proteases are utilized in the elimination of keratin in acne, depilation degradation of keratinized skin. Enzymes such as papain, bromolain and other proteases have been used on the skin for performing smoothing and peeling.