5. Production of enzyme in biotechnolgy.pptx

To give knowledge about enzymes.

1. Production of enzymes Prepared by: Ms. Harshada R. Bafna. M. Pharm (Quality Assurances)

2. Introduction Enzymes used in industry are isolated from microorganism, plants and animals . Enzymes are commercially produced by semisolid culture method and submerged culture. In semisolid culture method, enzyme producing culture is grown on the surface of suitable semisolid substrate supplemented with specific nutrients. Now a days, submerged culture methods are widely used in the production of enzymes. The fermentation equipment is used in manufacturing of antibiotics .

3. Microbial enzymes produced by fermentation includes : Amylases, proteases, catalase, penicillinase etc. The techniques used for microbial production of enzymes are described as; Isolation and selection of microbial strains. Formulation of medium Fermentation Extraction and purification of enzymes.

4. A. Isolation and selection of microbial strains Important criteria for selecting microbes are: To produce high amount of enzymes Less fermentation time Simple isolation and separation Non-pathogenic cell and Utilization of low cost culture medium Strain improvement techniques are used for optimization of enzyme production by the mutagens or UV rays method .

5. B. Formulation of medium The culture medium should contain all the nutrients to support adequate growth of microorganism that will result in maximum enzyme production. All ingredients should be easily available at low cost and with high nutritional value. The production media must contain sources of carbon, nitrogen, energy, minerals, macronutrients, micronutrients, growth factors, etc. Commonly used substrate for the media are soyabean meal, casein, yeast extract etc. The pH of medium should be adjusted to show optimal microbial growth and enzymes production.

6. C. Fermentation In Biotechnology, Fermentation means any process by which microorganisms are grown in large quantities to produce any type of useful materials . Submerged culture technique is commonly used for industrial production of enzymes. Submerged culture technique is most widely used because of less chances of infection and it give more yield. Surface culture technique is traditional method but still in use for production of fungal enzyme such as amylase, proteases, cellulose and pectinases .

7. Mechanically stirred capacities 20,000 to 1,00,000 liters are used for production of enzymes by submerged techniques. Duration is 2 to 7 days. The growth condition such as pH, temp and oxygen are maintained in optimum level. A small amount of oil is added to control the foam in fermentation. Most of enzymes are extracellular and produced at the end of exponential phase. Solid substrate culture method is mainly used for isolation of enzymes from fungi.

8. D. Extraction and purification of enzymes The desired enzyme produced may be extracted into the culture medium which are based on intracellular or extracellular nature of isolate. The physical, chemical and enzymatic methods are used to break the cells and release the intracellular enzyme. Recovery of extracellular enzyme is more simple than intracellular enzyme. Fungal broth is directly filtered or centrifuged after pH adjustment. Bacterial broth is first treat with calcium salts to precipitate calcium phosphate which help in separation of bacterial cells and colloids.

9. Isolated enzymes can be precipitated by using salts and organic solvents. Chromatographic techniques such as ion-exchange, size exclusion, affinity, hydrophobic interaction and dye ligand are commonly used for separation and purification of enzymes.

10. 1. Amylases It refer to a class of enzymes that specifically split or hydrolyze starch . In general, the amylases found in the animal kingdom are designated as α-amylases ; and the one encountered in plant kingdom are represented as β-amylases. It has been duly observed that a plethora of bacteria and fungi give rise to α-amylases , although the enzymes from these two sources are not identical. The bacterial amylases are relatively heat-resistant with an optimum temperature of ~ 55°C, which is a definite advantage over the corresponding fungal amylases when starch hydrolysis is to be performed at a higher temperature.

11. Amylase is an enzyme that break down the starch into sugar. It is present in human saliva, where it being the process of chemical digestion. Amylase was the first enzyme to be discovered and isolated. They are specially used in the industrial starch conversion process. The Alpha amylase is obtained from plants (barley and rice), Animals, or microbes. Cassava mash wastewater is also a source of Alpha amylase. This enzyme shows its activity in a wide range pH and temperature. The Beta-amylase is α-1, 4- glycanmaltohydrolases belong to plant origin there are certain specific microorganisms known to produce this enzyme, namely : Bacillus polymyxa ; Bacillus cereus ; Bacillus megaterium, Streptomyces sp. ; Pseudomonas sp. ; and Rhizopus japanicus.

12. Production of Amylase: Alpha amylase can be commercially produced by submerged fermentation and solid state fermentation techniques . The first traditional method of enzyme production from microbes, while the latest is a new method. A large no. of bacterial species are used for production of α -amylases such as Bacillus subtilis, B. cereus, B. licheniformis, b. amyloliquefaciens, Lactobacillus, Pseudomonas saccharophila etc.. Bacterial α -amylase is produced by submerged culture method. A careful balanced of carbohydrate and nitrogen ingredients of the medium is most important.

13. It necessary to maintain the pH near neutrality and incubation temp 30 to 40°C of the fermentation medium for the 3 to 5 days . After fermentation, culture is filtered or centrifuged to separate the cells . Amylases can be precipitated from aqueous solution by the addition of cold acetone, ethanol, isopropanol or ammonium sulfate. It purified by dialysis and chromatographic techniques. Fungal α -amylase was originally and is still produced in significant amounts in solid substrate culture. The β - amylase , α -1,4-glycanmalthodrolases hydrolyze starch and other amyloses by splitting off maltose molecules until the action is blocked by the occurrence of either 1,3 linkages or branch points.

14. Importance of amylase: It is used for the production of sweeteners in food industries , eg : glucose syrup, high mannose sugar, high fructose syrup. Dextrin's are produced by hydrolysis of starch with amylase followed by maltose and ultimately glucose It has a good heat stability. It dose not undergo browning reaction. It is used for converting glucose into fructose.

15. 2. Catalase Catalase is common enzyme found in nearly all living organism exposed to oxygen. It present in different sources such as microorganisms, plants and animals. It is a tetramer of four polypeptide chains , each over 500 amino acids long . Catalases are antioxidant enzymes present in all aerobic organism and it catalyze the conversion of hydrogen peroxide to water and oxygen molecule. 2H 2 O 2 Catalase 2H 2 O + O 2

16. This prevent conversion to hydroxyl radical and other non toxic reactive oxygen species (ROS). Hydrogen peroxidase is produced as a byproduct of aerobic respiration. Catalase can be classified into three categories on the basis of structure and sequence such as mono-functional or typical catalase , catalase-peroxide and pseudo-catalase or Mn -catalase. Different strains used for production of catalase enzyme such as A . niger, Penicillium variable, Thermoascus aurantiacus , Bacillius subtilis, Staphylococcus etc…this enzyme is located in the cytoplasm or in peroxisomes . The extracellular liberation of an enzyme is more advantageous for extraction and isolation.

17. This enzyme is used in several industrial applications such as food or textile processing to remove hydrogen peroxide which used for sterilization or bleaching. This enzyme is used to remove hydrogen peroxided from milk prior to cheese production. It is used in contact lens hygiene to decomposes the hydrogen peroxide which is used as disinfectant. Catalase enzyme also used in paper, and pharmaceutical industry.

18. 3. Peroxidase Peroxidase are also an oxidoreductase class of enzyme , which catalyze oxido reduction reaction . Many peroxidases contain an iron-porphyrin derivatives (heme) in their active site and they can catalyze oxidation of a wide variety of organic compounds using hydrogen peroxide. AH 2 + H 2 O 2 Peroxidase A + 2H 2 O Peroxide include a group of specific enzymes such as NAD peroxidase, fatty acid peroxidase and glutathione peroxidase. It isolated from different source of plants, animals and microbes. Peroxidase are primarily intracellular enzyme but many cells produce extracellular enzymes , which involved in degradation of complex organic compound.

19. Peroxidase are useful in a no. of industrial and analytical bioprocesses, due to high reduction potential. It used for reduction of water pollution by bioremediation of phenol and chlorinated phenolic compounds in waste water. Also used in degradation of wood compounds in paper industry by lignin and cellulose hydrolysis. It is also used in construction of biosensors for determination of hydrogen peroxide and phenolic compounds.

20. 4. Protease Complex mixture of true proteinases and peptidases are usually called proteases. Microbial protease divided into three groups based upon the pH range in which their activity is higher i.e. Acid, neutral or alkaline proteases. Proteolytic enzymes are produced by various bacteria such as Bacillus, pseudomonas, clostridium, proteus and serratia species and fungi such as aspergillus niger, Aspergillus oryzae, Aspergillus flavus, Penicillium roquefortii and Mucor pusillus. The enzyme associated with these microorganisms are actually mixtures of proteinases and peptidases.

21. Proteases are also employed as meat tenderizer Eg: Papain. Proteases also used in brewing industry, film industry, waste disposal management and manufacture of protein hydrolyzates. Industrial production of microbial proteases is carried out by cultivation of the microorganisms is submerged fermentation for the bacteria. Proteinases are excreted to the fermentation medium during growth while the peptidases are liberated only on autolysis of the cells. Protease are used on large scale in detergent, food and leather industries. Protease help in the liberation of silk fibers in silk industry.

22. 5. Penicillinase Penicillinase is bacterial extracellular enzyme produced from Bacillus species, Staphylococcus species and member of coliform group of bacteria. The enzyme penicillinase inactivates penicillin by the process of hydrolysis and converts to penicilloic acid. The enzyme penicillin amidase is normally produced by fungal species such as Penicilliun, Aspergillus and Mucor.

23. Penicillinase divided into two major classes based on their activity such as penicillin amidase or penicillin acylase and β -lactamase or penicillinase. The enzyme penicillin amidase is specific on attacking the acyl group attached to basic nucleus i.e. 6-amino-penicillanic acid. The enzyme β -lactamase acts on the basic nucleus itself. It breaks the β -lactam bond and produce penicilloic acid. This enzyme is more specific with penicillin G and penicillin X.

24. 6. Lipase Lipase are glycerol ester hydrolases that split or share fats into di-glycerides or mono-glycerides and fatty acids. A tri-glyceride is hydrolyzed by lipase to form glycerol and fatty acid.

25. Lipase are used to catalyze the trans-esterification reaction using alcohol together with fats, oil and free fatty acids to produce alkyl esters. Lipases are produced by animals, plants and microorganisms. The most common animal lipases produced from pancreatic gland. Papaya latex, oat seed and caster seed are the source of plant lipase. The lipases produced for commercial use they mainly isolated from fungi and bacteria. Screening of lipase produced on agar plate is performed by using tributyrin as a substrate and clear zones around the colonies indicate the cell produce lipase enzyme. Microbial lipases are generally extracellular nature and it produced by submerged fermentation or solid state fermentation.

26. Submerged fermentation requires large space, complex media different equipment's and control system. Solid state fermenter is alternative method for enzyme production due to use of residues and by products of agro-industries as nutrient sources. These components are high value and low cost substrates which are mainly responsible to reduce the cost of enzyme production. Lipase production is influenced by the type and concentration of carbon and nitrogen source, growth, temp, pH and dissolved oxygen concentration. Lipases are mainly used in the processing of fats and oil, detergents, food processing, synthesis of fine chemicals and pharmaceuticals, paper industry and production of cosmetics.

27. Lipases mainly modify the properties of lipids by altering the location of fatty acid chains in glycerol and replace some fatty acids with new ones. Lipases are also used to remove fat from meat and fish products to produce lean meat. Lipase are commonly used as biocatalyst in cosmetic industry for production of personal care products. It play a main role in production of specially lipids and digestive aids. Lipases modify monoglycerides for use as emulsifiers in pharmaceuticals.