Industrial Production of Protease

There are different important enzymes that play the key and vital role in the industry as well in the metabolism of different living organisms .The production of the enzyme is comparable to the methods that are used for the other products that are prepared industrially.Enzyme production can be carried out in different steps. Firstly we have to choose or select the organisms ,isolate and improvement of strains, preparation of inoculum, preparation of medium,production process and at last recovery and purification of the enzyme.The selection of the source of the enzyme is the first and key steps in the production of enzymes .There are different kinds of enzymes that are prepared industrially.In this part, we are going to describe the industrial production of protease enzyme;

Industrial production of Protease enzyme

Protease is one of the major group of an enzyme that plays the important role in the regulation and nutritional value in the living system.Due to the diverse application field of protease enzyme, they are in great demand and are being produced in high amount.Protease can be used in the detergents, food industries,cheese production, meat processing, the medical field so the demand is also increasing day by day. In recent time protease accounts for the 60% of the total enzyme consumed in the market.As we know the proteases are the enzyme that breaks down protein or hydrolyzed protein and they are also degradative in nature. Protease can be obtained by the different kinds of sources that why it is widely used. This enzyme can be obtained from fungi and microorganism but the microbial protease is most predominantly produced by fermentation methods. This enzyme can be produced either intracellularly as well as extracellularly. But the extracellularly produced protease are commercially more important . Protease producing enzyme are ; Alcaligens , Proteus, Pseudomonas , Serratia, Penicillium, Bacillus, Aspergillus , Mucor, Rhizopus etc

The industrially produced protease is marked as

a) Acid protease:

Optimum pH for acidic protease range from 2-3

Organism includes ;Rhizopus niveus, Mucor pusillus

b) Neutral Proteas:e

Optimum pH for neutral protease ranges from 5-8

Organisms include = Bacillus cerevs, B. substilis, B. megaterium,Aspergillus oryzae

c) Basic protease:

Optimum PH for basic protease ranges from 8.5- 12

Organisms include B. substilis, B. licheniformis, B. alcalophilus, B. formis , A. oryzae.

Production process of Protease enzyme:

The production of protease in industrial level is carried out by using improved and preserved strains . In industries, the protease is produced by two methods;ie, Solid state fermentation and Submerged state fermentation. Among these two process, solid state fermentation is more preferred than submerged fermentation due to the various advantages of SSF like high productivity, simple processing process etc.

1) Solid State Fermentation (SSF)

Solid state fermentation takes place on the surface of the solid substrate without any water and is a heterogeneous medium. It Consumes low energy in compared to submerged state fermentation .The Protease enzyme is mostly produced by filamentous fungi than bacteria because the fungi can grow in variable environmental condition. This is widely applied the technique for production of protease, especially in Japan. The production process by this method is described below;

A) Producing organisms:

a) Acidic:Mucor meihei

b) Basic: Aspergillus oryzae

c) Neutral: Aspergillus oryzae

B) Media:

Media is the complex type and includes rice brands,wheat brands and does not require optimization. The media is sterilized before inoculation.

C) Inoculum development:

The fungal spores are produced in liquid/broth culture before the start of fermentation to obtain fungal spores of around 3/ 10⁵ spores per litre.

D) Fermenter: Tray fermenters are used as fermentation tank.

E) Fermentation process:

• pH range from 3-7 depending upon the nature of enzyme to be produced
• Temperature ranges from 28-30 degree.
• Aeration is done at the rate of 0.8 -1 vvm ( volume of air / volume of liquid /m³ )
• Moisture content should be greater than 15%
• The fermentation is carried out for 7-10 days after the inoculation into the fermenter.
• Product recovery; The whole substrate is extracted in water and further purification steps are adopted .

2) Submerged State Fermentation;

A) Producing organisms:

Bacillus sps, Aspergillus sps

B) Media:

The media in submerged state fermentation is mostly organic in nature and is optimize with the source of carbon and nitrogen.

1. Carbon source; Glucose in fed-batch fermentor
2. Nitrogen source; Ammonium ions are used
3. Another source; Potassium hydrogen phosphate ,magnesium sulphate ,sodium carbonate etc.

C) Inoculum preparation:

It is prepared by shake flask culture method for preparation of bacterial inoculum ,while the fungi are grown in solid media for spore production. Bacterial inoculum is produced in 24 hrs while fungal inoculum requires 5-7 days .Sometimes for the production of large amount of inoculum seed tank of 120 m³ is used . The temperature for bacterial inoculum is 30-35 degree.

D) Fermenter: The fermenter used is stirred type fermenter having a capacity of 4200m³.

E) Fermentation process;

• Fed-batch fermenter
• pH is maintained at 3 ,7, 10 depending upon the nature of enzyme to be produced.
• Temperature ranges from 30-35 degree celsius
• Aeration is done in the rate of 1vvm
• Inoculum size for bacteria is 6- 10% v/v
• Inoculum size for fungi is 3*10⁵ spores litre of suspension
• Duration; For bacteria, 3 days after the inoculum is inoculated into the sterile media
• For fungi ,5 days after the inoculum is inoculated into the sterile media,

Product Recovery:

Both solid state and submerged state ,the extracellular enzyme produced by either of the methods is subjected to recovery and purification by various means or methods. For recovery of enzyme produced by solid state fermentation, the whole substrate is extracted in water . After this , the steps involved are common in both types of fermentation . The product recovery is outlined below:

Fig:Recovery and purification of enzyme

Application of protease enzyme:

Protease can be widely used in various field some of the applications are given below:

1) In the medical field:

• Protease is used in the treatment of blood clots in ischemic stroke,
• It is also used in the wound debridement,
• Sometimes protease is used as antibiotics therapy and enzyme therapy.
• Protease sometimes applicable as an anti-inflammatory activity,

2) In Food industries:

• For the production of quality and nutritional food products protease enzyme are utilized,
• Protease is useful in the baking, brewing, and production of various food such as sauce, meat,cheese etc,
• In the preparation of milk products protease plays key role,
• Processing and storage of food juice and to increase the self-life of juice protease is important,

3) Protease is also used in the isolation of nucleic acids and removal of cellular debris ,