Lab scale production of bacterial enzyme.pptx

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1. Lab scale production of bacterial enzyme: M.Phool Badshah

2. Enzymes: Enzyme are macromolecular biological catalysts. Enzymes accelerate or catalyze chemical reaction. The molecules at the beginning of the process are called substrates and enzymes converts these into different molecules called products. Microbial enzymes are biological catalysts for the biochemical reactions leading to microbial growth and respiration, as well as to the formation of fermentation products.

3. Methods of enzyme production : There are two methods of enzyme production which are generally used at industry level : Solid-state Fermentation.. Submerged Fermentation .

4. a Solid-State Fermentation There are many biotechnological processes that involve the growth of organisms on solid substrates in the absence or near absence of free water. Solid state method is another method used for the production of enzymes, which involves the cultivation of microorganisms on solid substrate, such as grain , rice and wheat. Solid state fermentation employs natural raw materials as carbon source such as cassava , barley, wheat bran etc.

5. a Solid State Fermentation (SSF) is a fermentation method used by several industries like the pharmaceuticals, food, textile etc., to produce metabolites of microorganisms using solid support in place of the liquid medium. Solid state fermentation (SSF) deals with substrates that are solid and contain low moisture levels. The most regularly used solid substrates are cereal grains (rice, wheat, barley, and corn), legume seeds, wheat bran, ligno -cellulose materials such as straws, sawdust or wood shavings, and a wide range of plant and animal materials.

6. a Selection of Microorganisms: This is one of key factor to improve yields of the product. Bacteria, Yeast and Filamentous fungi can be used. Filamentous fungi has shown better results growing in the solid substrate fermentation . Substrate: Substrate also plays important role in determining the growth of microorganisms, there by increasing the product yield. Substrate is chosen in such a way that it should provide as well as nutrients to the growing culture.

7. a Applications Solid-state fermentation has emerged as a potential technology for the production of microbial products such as feed, fuel, food, industrial chemicals, and pharmaceutical products. It is widely applied to producing several enzymes, organic acids, flavoring compounds etc., which must be extracted and purified and then used in different products. Its application in bioprocesses such as bioleaching, bio-beneficiation, bioremediation, bio-pulping, etc. has offered several advantages.

8. a Submerged Fermentation Submerged liquid fermentations are traditionally used for the production of microbial derived enzymes. In the submerged process, the substrate used for fermentation is always in liquid state which contains the nutrients needed for growth. The fermentor which contains substrate is operated continuously and the product biomass is continuously harvested from the fermenter by using different techniques then the product is filtered or centrifuged and then dried. Submerged fermentation is the method of manufacturing bimolecular in which enzymes and other reactive compounds are submerged in a liquid such as alcohol, oil and nutrient broth.

9. a The process is used for a variety of purposes, mostly in industrial manufacturing. The process can be used to make products such as citric acid, glycerol or lactic acid. Submerged culture fermentation has been widely used for the production of enzyme because in submerged fermentation unwanted metabolites are not produced and purification of enzymes takes place in an easy way. Parameters like temperature, pH, oxygen consumption and carbon dioxide formation are measured and controlled to optimize the fermentation process. Fermentation takes place in large vessels ( fermenter ) with volumes of up to 1,000 cubic meters. The fermentation media sterilizes nutrients based on renewable raw materials like maize, sugars, and soya.

10. a Applications Submerged Fermentation/Liquid Fermentation utilizes free-flowing liquid substrates, such as molasses and broths. The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients. This fermentation technique is best suited for microorganisms such as bacteria that require high moisture. An additional advantage of this technique is that the purification of products is easier. It is primarily used in the extraction of secondary metabolites that need to be used in liquid form

11. a Solid-state fermentation Organism require less water for growth and preferred such as filamentous fungi. Less chances of contamination because of less availability of water. Less consumption of energy for aeration and gas transfer. Liquid waste is not produced. Downstream processing is easy, cheaper and less time consuming. Limiting factor for growth is diffusion for nutrients. Submerged Fermentation Media concentration is very much lower as compared to water content. Higher water activity becomes the major cause of contamination in SLF. Higher air pressure consumes more power and there is poor transfer of gas in SLF. High quantity of liquid waste is produced, causing difficulties in dumping. Water makes downstream process difficult and very expensive. Vigorous mixing makes diffusion easy.

12. Protease Enzyme Protease (mixture of peptidases and proteinases ) are enzymes that perform the hydrolysis of peptide bonds. Peptide bonds links the amino acids to give the final structure of a protein. Proteinases are extracellular and peptidases are intracellular. Second most important enzyme produced on a large scale after amylase.

13. a Classification based upon the residues in the catalytic sites: Serine protease. Threonine protease. Aspartate protease. Cysteine protease. Glutamic acid protease. Metalloprotease . Classification based upon the PH in which the proteases are active: Alkaline serine proteases Acid proteases. Neutral proteases.

14. a Alkaline serine Proteases PH of the production medium is kept at 7.0 for satisfactory results. Optimal temperature maintained is 30’ to 40’C. Important producers are B, lechinoformis , B.amyloliqueifaciens , B.firmus , A.flavus . Enzymes used in detergents are chiefly proteases from bacillus strains ( Bacillopeptidases ). Best Known proteases are Subtilisin Carlsberg from B,licheniformis and Subtilisin BPN and Subtilisin Novo from B. Amyloliquefaciens .

15. a Substrate for Protease : Molasses. Nutrient broth. SCREENING: Because enzyme should be stable in alkaline conditions, screening for better producers is done by using highly alkaline media. It was found that B.Lichenoformis and B.subtilis showed growth in the range of PH 6,7 by new strains were found to grow even in PH 10-11. Genetic manipulation can also be carried out.

16. a Fermentation Process Culture was stored in the lyophilized state or under liquid nitrogen. Initial culture are carried out in shaken flasks and small fermenters (40-100m3) at 30-37’C. Fed-Batch culture is generally used to keep down the concentration of ammonium ions and amino acids as they may repress protease production. High oxygen partial pressure is generally necessary for optimal protease titers, time span for fermentation is 48-72 hours depending upon the organism. Protease must be converted in a particulate form before they are added to detergents. To produce a suitable encapsulated product, a wet paste of enzyme is melted at 50’ to 70’C with a hydrophobic substance such as poly- ethy lene -glycol and then converted into tiny particles.

17. a Neutral protease: They are relatively unstable and calcium, sodium and chloride must be added for maximum stability. Not stable at higher temperature. Producing organisms are B.subtilis and B.megaterium etc. They are quickly inactivated by alkaline protease. Acid protease Similar to mammalian pepsin. It consist of Rennin like proteases from fungi which are chiefly used in cheese production. They are used in medicine, in the digestion of soy protein for soy sauce production and to break down wheat gluten in the baking industry

18. a Application Textile industry to remove proteinaceous sizing. Silk industry to liberate silk fibers from naturally occuring proteinaceous material in which they are embedded. Tanderizing of meat. Used in detergent and food industries.