INDUSTRIALY PRODUCED ENZYMES BY MICROORGANISMS .PPTXpptx

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1. Industrialy produced enzymes by microorganisms M.S.SWETHA(BP231508)

2. Enzymes: Enzymes are bio molecules that catalyse chemical reaction, Nearly all enzymes re-proteins however certain RNA molecules can be effective catalyst too. These are RNA molecule is known as Coenzyme . In Enzymatic reaction, the molecule at the Beginning of the process are called Substrates, Enzymes converts then to different molecules called Products. Industrial enzymes are these which are used for industrial production and also these are produced in industry Eg : Amylase, protease and pectinase

3. Amylase: Amylase is an enzyme that catalyses the hydrolysis of Starch to Sugars. Amylases are the large group of enzymes. They are characterized by their ability to hydrolyze 1,4 – glucosidic linkage in Polysaccharides (E.g. Starch and Glycogen). Amylases play the most important part in Food technology (Bread making, Beer making, etc.). Amylases are classified into two sub-groups. They are: ( i ) α – Amylases (ii) β - Amylases

4. MICROORGANISMS USED FOR AMYLAZE PRODUCTION (Bacteria and fungi) Bacterial strains : Most are Bacillus species ( B. subtilis, B.mesentericus , B. vulgaris, B. megaterium, B. cereus ).Amylases from Rhodothermus marinus, Corynebacterium gigantea, Chromohalobacter sp., Lactobacillus fermentum have also been isolated. Halophilic strains that produce amylases include Haloarcula hispanica , Halobacillus sp., Chromohalobacter sp., Bacillus dipsosauri , and Halomonas meridiana . Fungal strains : Efficient amylase-producing species include those of genus Aspergillus ( A. oryzae, A. niger , A. fumigatus and A. flavus ), as well as Penicillium species ( P. brunneum , P. expansum , P. chrysogenum and P. olsonii ), Streptomyces rimosus , Thermomyces lanuginosus , Cryptococcus flavus, Thermomonospora curvata , and Mucor sp.

5. Bacterial amylase Bacterial Amylase – Submerged fermentation. Fermentation Medium Soya bean meal – 1.85 % Brewer’s Yeast Fraction – 1.50 % Distiller’s dried solubles – 0.76 % N – Z Amine – 0.65 % Lactose – 4.75 % MgSO4.7H2O – 0.04 % Antifoam - 0.05 % Water - 90.40 L

6. Fungal amylaze Fungal Amylase – Submerged fermentation, Wheat bran was used as a substrate. Aeration and Agitation is problem due to high viscosity. Amylase synthesis is inhibited when medium contains Glucose . Fermentation Medium Corn Starch – 24 g/L Corn steep liquor – 36 g/L KCl – 0.2 g/L Na2HPO4 – 47 g/L CaCl2 – 1 g/L MgCl2.6H2O – 0.2 g/L

7. Fermentation conditions: Temperature – 30 °C to 40 °C . pH – 7.0 ; Neutral or close to Neutral; pH below 6 results in denaturation of Amylase. Calcium carbonate is used to maintain Neutral pH. The Amylase production begins when the bacterial count reaches 10 9 to 10 10 cells per ml after 10 – 20 hrs and continues for 100 – 150 hours. Duration of Fermentation – 4 to 6 Days .

8. Recovery of amylase: Separation of fungal mycelium is accomplished by Filtration of the refrigerated broth. For Bacterial amylase, there is no need of Filtration . The suspended particles present in the broth are removed with Flocculating agents like Calcium phosphate . The enzyme is precipitated , in order to get high degree of purity, by using acetone or alcohol or even inorganic salts like ammonium sulphate or sodium sulphate . Sometimes Fractional precipitation of the enzyme is done to obtain it in purest form .

9. Applications of amylase Alpha and beta amylases are important in brewing beer and liquor made from sugars derived from starch. Amylases are used in bread making and to break down complex sugars, such as starch (found in flour), into simple sugars. Amylase also has medical applications in the use of Pancreatic Enzyme Replacement Therapy . Bacilliary amylase is also used in clothing and dishwasher detergents to dissolve starches from fabrics and dishes.

10. Pectinase: Pectinase is the collective term for row of enzyme that are able to breakdown or transform pectins . Pectinase is a general form for enzymes such as pectolyase , pectozyme , polygalacturonase etc. commonly referred as Pectic enzymes . Pectinase breakdown Pectin , a polysaccharide that is found in the cell wall of plants . Pectinase is involved in the metabolism of the cell wall as well as in the growth of the cell, senescence (biological ageing), ripening of fruits, pathogenesis and abscission process (shedding of various parts). The basic structure of Pectin is 1,4 – linked galuctronic acid with up to 90 % of its Carboxyl group, esterified with Methanol. According to the Enzyme Commission and the International Union of Biochemistry, Pectinase enzymes are classified under Hydrolase group . The majority of commercial enzymes are obtained by employing fungal cultures . Pectinases have been commercially prepared from the microorganisms especially from the fungi since the 1970s .

11. Microbes used for pectinase production : Pectinase Produced by various bacteria and fungi although commercial microbial pectinase production utilizes species of Penicillium and Aspergillus. Microbial strains : Aspergillus niger , Aspergillus japonicum , Bacillus subtilis and Penicillium italicum are common pectinase producers.

12. Fermentation conditions: Solid or submerged fermentation process are used. Substrates - Wheat bran, Rice bran, Potato starch, Rice husk, 0.1% NH4NO3, NH4H2PO4 and MgSO4. Enhancer - 2% sucrose and 2% pectin Inoculum - Spore of Aspergillus niger (107 spores per gram of dry substrate) Aeration is necessary. Temperature – 37 °C . pH – 3 to 4 Duration of Fermentation – 60 to 80 hours .

13. Recovery and purification : Pectinase can be extracted from the cell and the medium so can be recovered from both sources. In the process of harvesting, the mycelium is filtered or centrifuged, dried, grind and pectinase extracted with water. Recover aqueous portion is concentrated by evaporation to yield pectinase concentrate. As an alternate recovery procedure, pectinase can be precipitated from aqueous solution by the addition of cold acetone, ethanol, isopropanol or ammonium sulphate.

14. Applications of pectinase: Pectinases are widely used in the food industry - To improve the cloud stability of fruit and vegetable nectars and also For production and clarification of fruit juices For haze removal from wines. Pectinases are also involved in Clarification of wine,Oil extraction,Removal of citric fruit peels,Degumming fibres Pectinase usage accelerates Tea fermentation and also destroys the Foam forming property of Instant tea powders by destroying pectins . Pectinase used in Coffee fermentation to remove mucilaginous coat from coffee beans. Pectinase used in the extraction of pigments , food coloring from plant based raw materials. Pectinase used in the Industrial manufacture of Feeds . Pectinase used in Retting (separation of Fiber from the plant parts).

15. Proteases: Proteases represent a broad group of enzymes which break down or hydrolyze proteins or peptides . Complex mixtures of Proteinases and Peptidases are called Proteases. Proteases act on the peptide bonds joining the adjacent amino acid residues in a protein molecule and cleave them leading to formation of shorter peptides and amino acids .Proteases occupy a central position in commerce, accounting for nearly 65% of the global enzyme market. Proteases are hydrolytic enzymes are ubiquitous in nature and have been found in eukaryotes (plants, animals, fungi) and prokaryotic domains of bacteria and archaea . Even several Viruses are also known to encode their own proteases. Cysteine proteases , identified in Adenovirus, Alphavirus; Serine protease reported in Hepatitis C Virus; Aspartic protease found in HIV.

16. Types of proteases: Proteases are classified into three sub-groups . They are: ( i ) Alkaline protease (pH range 9 to 11) – Serine protease, Cysteine protease and Subtilisin Carlsberg (most widely used Detergent protease) (ii) Acid protease (pH range 3.8 to 5.6) – Aspartic protease, Pepsin and Retropepsin . (iii) Neutral protease (pH range 5 to 8) – Neutrase ( Thermolysin )

17. Alkaline protease Bacteria - Bacillus sp .,Bacillus megaterium Enterococcus hirae , Pseudomonas aeruginosa,Streptomyces griseus and Streptomyces rectus. (Proteases are mainly produced from Bacillus licheniformis ) Fungi – Aspergillus niger , Aspergillus oryzae, Aspergillus flavus, Aspergillus sojae & Chetomium globosum Acid protease Acid proteases are found in animal cells, yeast and molds but never in bacteria. Microbial renin-like enzymes are derived from Mucor sp ..Aspergillus sp. And Rhizopus spp. Neutral protease Bacteria - Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Pseudomonas aeruginosa and Streptomyces griseus. Fungi – Aspergillus oryzae and Aspergillus sojae

18. Alkaline protease(fermentation medium) Subtilisin Carlsberg from Bacillus licheniformis is the most widely used Detergent protease obtained by Submerged culture method . In 1971, Japanese Scientist Koki Horiloshi first reported the production of Alkali Protease from Bacteria. Fermentation Medium Starch hydrolysate – 50 g/L Soya bean meal – 20 g/L Casein – 20 g/L Disodium phosphate – 3.3 g/L

19. Fermentation conditions Temperature – 30 °C to 40 °C . pH – 7.0 Calcium carbonate is used to maintain Neutral pH. The Acid Protease production begins after 10 – 20 hrs of microbial cultivation. Duration of Fermentation – 2 to 3 Days . Yield – 10 % of the initial Protein content of the medium. Enzyme stability – Stable in Liquid preparation and the Liquid preparation contains 2 % of enzyme

20. Acid protease(fermentation medium) Mostly produced by Fungi through Semi-solid culture or Submerged culture . Normally, the Fermentation medium contains 60 % of Wheat bran with water. Fermentation Medium Starch – 4 g/L Soya bean meal – 3 g/L Ground Barley – 10 g/L Calcium carbonate – 0.5 g/L

21. Fermentation conditions Temperature – 30 °C . pH – 7.0 Duration of Fermentation – 3 Days . Yield – 3200 Soxhlet units per gram of Wheat bran in Mucor pusillus fermentation and 10000 to 150000 Soxhlet units per ml of Fermentation medium in Mucor miehei fermentation

22. Recovery and purification (alkaline and acid protease) Different methods can be applied for purification of enzyme like Ultrafiltration . Chromatographic technique (ion exchange) purification by treatment with activated charcoal and H2O2.

23. Applications of protease: Alkaline protease – Detergent industry and Leather industry. Acidic proteases - Soy sauce, Protein hydrolysate, Digestive aids and in production of Seasoning material, Clearing beer and fruit juice, Improving texture of flour paste and Tendering the fibril muscle. Neutral protease - Food industry and Brewing industry.

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