Production of protease enzyme from different sources.

Contains information about protease productioin from different sources such as microbes, plants and animals.

1. PRODUCTION OF PROTEASE ENZYME FROM DIFFERENT SOURCES L. THARRUN DANIEL PAUL , DEPARTMENT OF BIOTECHNOLOGY,

2. INTRODUCTION WHAT ARE ENZYMES ? Enzymes are type of proteins that act as biocatalyst in our body. One of the most important roles of protease is the hydrolysis activity (breaking down). WHAT ARE PROTEASES ? Proteases (also called as proteolytic enzymes, peptidases) are a group of proteins that hydrolyze the long chain protein molecules into shorter fragments and eventually into amino acids. Proteases denature proteins into their primary structure. Proteases have a wide range of industrial applications.

3. HISTORY 1833- French chemist Anselme Payen-  Diastase. 1860s- Louis Pasteur- fermentation techniques with sugar. 1897- Eduard Buchner- paper on ‘Study of yeast extracts’. 1926- James B. Sumner- proved urease and catalase(1937) were proteins. 1 965-  David Chilton Phillips- structure of lysozyme by x-ray crystallography.

4. TYPES OF PROTEASE Based on catalytic residue Serine proteases  Cysteine proteases  Threonine proteases  Aspartic proteases Glutamic proteases Metalloproteases  Asparagine peptide lyases  Peptide lyases Classification based on optimal pH Acid proteases Neutral proteases  Basic proteases (or alkaline proteases)

5. PROTEASE PRODUCTION IN LABORATORIES STAGES: Screening of enzyme activity- plate assay-well diffusion Substrate quantification using Folin ’ s phenol Protein estimation- Bradford or Lowry’ s assay Optimization of substrate, temperature and pH. Precipitation using ammonium sulphate (salting out). Dialysis Qualitative test- SDS- PAGE

6. INDUSTRIAL PRODUCTION Commercially produced microbial proteases contribute to approximately 2/3 of all enzyme sales. Bacteria- Bacillus, Pseudomonas, Clostridium, Proteus, and Serratia , Fungi- Aspergillus niger , Aspergillus oryzae , Aspergillus flavus, and Penicillium roquefortii . Bacillus species are mostly used in the commercial production of proteases. The fungal proteases present a wider pH activity range- wider range of uses. There are two types of proteases: (a) alkaline serine proteases and (b) acid proteases. Alkaline serine proteases- Bacillus licheniformis by submerged culture method. Acid proteases- fungi by either semisolid culture or submerged culture method.

7. MICROBIAL PROTEASES Microbial proteases are one of the important groups of industrially and commercially produced enzymes. Emphasis is on the microorganisms producing proteases with desired characters. Demand for novel proteases .  Microorganisms represent an excellent source- broad biochemical diversity, susceptibility to genetic manipulation. Optimization of culture media is important to yield an economically viable amount of proteases. SOURCES FOR PROTEASE PRODUCTION

8. FUNGAL PROTEASE Commercial production of fungal protease- Aspergillus flavus, Aspergillus wentii , Aspergillus oryzae , Mucor delemar , Mucor miehei and Amylomyces rouxii . The fungus is usually grown on wheat bran , although other media are sometimes employed, under fermentation conditions similar to those for amylase production. At sporulation, the various fungal proteolytic enzymes are present in the medium, and the proteases are recovered by procedures similar to those for mold amylases. The optimum temperature of the fermentation is 30°C & requires 3 days for completion. Mucor miehei - Acid proteases by submerged culture method (The optimum temperature of the fermentation is 30°C but requires 7 days for completion).

9. BACTERIAL PROTEASE Bacterial protease production- strains of Bacillus subtilis , and the fermentation conditions are similar to those for amylase production by this organism. However, the Bacillus subtilis strains are specially selected for high protease activity and not for amylase activity. As stated previously, a high carbohydrate content medium is utilized to stimulate protease activity and depress amylase production, although the final product does contain some amylase activity. The fermentation is incubated 3 to 5 days at 37°C in pans containing a shallow layer of fermentation medium, and the harvest procedure is similar to that for bacterial amylase. Bacillus licheniformis - alkaline serine proteases by submerged culture method.

10. PLANT PROTEASES EXAMPLES: Papain, bromelain and ficin represent some of the well-known proteases of plant origin. Papain- latex of Carica papaya fruits- active between pH 5-9 and is stable up to 90°C. Bromelain- stem and juice of pineapples- also called as cysteine protease. A neutral protease- purified from Raphanus sativus leaves. An aspartic protease- potato leaves. Thiol Protease- pineapple Crown Leaf. Serine protease- artificially senescing parsley leaves. Endo proteases- alfalfa, oat and barley senesced leaves.

11. ANIMAL PROTEASES EXAMPLES: The most common proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin, and rennins . Trypsin- intestinal digestive enzyme- type of serine protease- hydrolyzes peptide bonds. Chymotrypsin- animal pancreatic extract- expensive enzyme used only for diagnostic and analytical applications- used extensively in the de- allergenizing of milk protein hydrolysates. Pepsin- acidic protease- present in the stomachs of vertebrates. Rennet- pepsin-like protease (rennin, chymosin)- used exclusively in the dairy industry to produce good flavored curd.

12. BIOREACTOR A  bioreactor  may refer to any manufactured or engineered device, (large fermentation chamber), for growing organisms such as bacteria or yeast under controlled conditions that which supports a biologically active environment or a  chemical process  is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be  aerobic or anaerobic . These bioreactors are commonly cylindrical, ranging in size from liters to cubic meters, and are often made of stainless steel. Bioreactors  are  used  in the biotechnological production of substances such as pharmaceuticals, antibodies, or vaccines, or for the bioconversion of organic waste.

13. TYPES OF BIOREACTORS Types of Bioreactor Continuous Stirred Tank Bioreactor Airlift  Bioreactor Fluidized Bed  Bioreactor Packed Bed  Bioreactor Photobioreactor Membrane Bioreactor

14. CSTR Air lift bioreactor

15. Packed Bed  Bioreactor Photobioreactor

16. Fluidized bed bioreactor Membrane Bioreactor

17. FERMENTATION Fermentation  is a metabolic process that produces chemical changes in organic substrates (sugars) through the action of microbes and enzymes. It is also defined as the extraction of energy from carbohydrates in the absence of oxygen or refer to any process in which the activity of microorganisms brings about a desirable change to a foodstuff or beverage .  The science of fermentation is known as  zymology . It is carried out in a fermenter, which is type of bioreactor . Humans have used fermentation to produce foodstuffs and beverages since the Neolithic age .

18. SOLID STATE FERMENTATION Solid state fermentation  (SSF) is an industrial process, in which mostly metabolites generated by microorganisms grown on a solid support selected for this purpose. This technology for the culture of microorganisms is an alternative to liquid or submerged fermentation, used predominantly for industrial purposes (bread, maturing of cheese). SUBMERGED STATE FERMENTATION Submerged fermentation  is an industrial process in which enzymes and other reactive compounds are  submerged  in a liquid such as alcohol, oil or a nutrient broth. The process is used for a variety of purposes, mostly in industrial manufacturing (enzymes, antibiotics..).

19. SOLID STATE FERMENTOR

20. SUBMERGED FERMENTOR

21. INDUSTRIAL APPLICATION Detergent industry Leather industry Food industry Dairy industry Baking and brewing industry Soy sauce production Meat tenderization Synthesis of aspartame Pharmaceutical industry Therapeutics Photography industry Management of industrial wastes Degumming of silk

22. RECENT ADVANCES AND FUTURE PROSPECTS Extension of biotechnological approach in terms of both quality and quantity. Qualitative improvements : low-temperature screening methods, protein engineering, r-DNA technology, novel cold-active proteases. Quantitative enhancement needs : Strain improvement, especially through site-directed mutagenesis, and standardizing the nutrient media for the overproduction. Strain development- achieved by changes at the gene level . Extensive attempts to engineer cold-adapted proteases from subtilisin BPN have previously been made.

23. Genetic and protein engineering can bring about a great yield of the enzymes by the microbes. Offer a new strategy for site specific drug targeting and tumor imaging (cross linking). Proteases from extremophiles have a great market value due to their desirable unique features, eg. , microbes in hot springs.

24. CONCLUSION Microbes are a reliable source for protease production in order to attain a standard output. Plant and animal sources are not reliable for industrial production as they are limited. Advancement in biotechnology leads to a constructive position in protease production. The need for the protease enzyme is constantly increasing till now and there will be a great need for industries producing proteases. Future of enzyme technology also depends on the research and development sector in modifying a microbial strain to tolerate various physical conditions and produce a good yield.

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