Production of enzymes

bacterial enzymes,procwss enzyme production

1. Bacterial Enzymes, Industrial Enzymes And Production Of Enzymes

2. BACTERIAL ENZYMES, INDUSTRIAL ENZYMES AND PRODUCTION OF ENZYMES Assignment of PHARMACEUTICAL BIOTECHNOLOGY PHS C C 1203 Session 20 23-2024 Department of Pharmaceutical Sciences Dr. Harisingh Gour Vishwavidyalaya, Sagar, (M.P.) (A Central University) Supervisors: PROF. UMESH K. PATIL DR. UDITA AGRAWAL DR.PRIYANKA JAIN MR.SATYAM SHYAMVISHWAKARMA Submitted by: ADARSH SHARMA Y 23254001

3. ACKNOWLEGEMENT I sincerely appreciate the assistance and support I received from my guide and other faculty members during my assignment PROF. UMESH K. PATIL , DR. UDITA AGRAWAL ,DR. PRIYANKA JAIN and MR. SATYAM SHYAMVISHWAKARMA.

4. CONTENT: 1. INTRODUCTION 2. STEPS INVOLVED IN PRODUCTION OF ENZYMES 3. BACTERIAL ENZYMES 4. INDUSTRIAL ENZYMES 5. REFERENCES

5. ENZYMES: Enzymes are colloidal, organic, polymer, proteinaceous substance that acts as biocatalyst and alters the speed of any reaction. Enzymes have an active site . This active site is the place where the substrate binds with the enzymes and holds the substrate. Active site has specific shape due to tertiary structure of proteins. 1. INTRODUCTION

6. STEPS: 2. PRODUCTION OF ENZYMES (6.) Recovery and purification of enzymes

7. (2.1) Selection of microorganism Microorganism should not be pathogenic. Raw material should be cheap. Fermentation time taken should be less. Organism should be able to produce maximum quantities of enzymes in short time.

8. (2.2) Isolation o f microorganism We always get microorganism in mixture of different strains. So, isolation becomes essential from the mixture to obtain PURE CULTURE . Importance of pure culture: Once purified, isolated species can be cultivated with the knowledge that only desired microorganism is being grown. Pure culture are correctly identified. Experiments with pure culture ensures same result regardless of how many times experiment is performed. METHODS OF ISOLATION STREAK PLATE METHOD POUR PLATE METHOD SPREAD PLATE METHOD SERIAL DILUTION METHOD

9. STREAK PLATE METHOD Widely used technique used to isolate a pure strain from single species especially bacteria. Streak  literally means “ a long, thin line ”: and the streak plate method is a microbiological culture technique where a sample is spread in a petri dish in the form of a long, thin line over the surface of solid media. The sample is picked by using different tools, mostly using a sterile inoculating loop or swab. The sample is placed over a surface of sterile solid media at one edge of the petri dish and a smear is prepared. Using the tool, the smear is successively streaked over the agar medium on different patterns..   As the streaking proceeds, the inoculum is gradually diluted to the point where bacterial cells are separated as individual cells or as a colony-forming unit (CFU) at a gap of a few millimeters. When these inoculated plates are incubated, the isolated bacterium or a CFU will give rise to a well-isolated colony. This will allow us to get a pure culture as well as describe the colony morphology of the organism

10. POUR PLATE METHOD The sample is either added to the Petri plate and then the molten agar medium is poured over it, or the sample is mixed with the molten agar medium prior to pouring. After pouring in the Petri plate, the plate must be swirled quickly to properly mix the sample with the medium. The mixed medium is allowed to solidify and is incubated under the suitable condition to grow the microorganisms present in the sample. Following the incubation, the numbers of isolated colonies are counted.

11. SPREAD PLATE METHOD Spread Plate Method  is one of the widely used culture techniques in microbiology laboratories due to its ease and simplicity . The spread plate method is a microbiological laboratory technique for isolating and counting the viable microorganisms present in a liquid sample by spreading a certain volume of the sample over an appropriate solidified culture media. The sample in the spread plate method must be liquid or in suspension. Before plating, the samples are serially diluted. 

12. SERIAL DILUTION METHOD This method is implied for pure culture which generally doesn’t grow on solid media and grow only in liquid media. A microorganism that pre-dominates in mixed culture can be isolated in pure form by series of dilutions. The inoculum is subjected to serial dilutions in sterile liquid medium and a large number of tubes of sterile liquid medium are inoculated with aliquots of serial dilutions.

13. (2.3) Strain improvement: Once the microorganism is selected and isolated, strain improvement for optimizing enzyme production can be done. It is done to provide desired qualities to microorganism. To increase production of enzymes. It is performed by: Physical Methods- X-Rays UV Methods Chemical Methods

14. (2.4) Formulation of Medium: Culture media should contain all nutrients to support adequate growth of microorganisms that results in adequate quantity production of enzymes. Ingredients of media: Readily available Low cost Nutritionally safe Growth of microorganism should be proper Composition of media: Macronutrients- Magnesium (Mg), Sulphur(S),Potassium(K) etc. Micronutrients- Iron(Fe), Zinc(Zn), Manganese(Mn) etc. Carbohydrates- Sugars, Starch, Cellulose Vitamins- Thiamine, Adenine Amino acids Hormones Antibiotics * Gelling Agent added if solid culture media is made.

15. Preparation of 1L of Culture Media:

16. (2.5) Production Process: It is carried out by: Submerged Culture Solid Substrate Culture In submerged solid media, the yield is more, and chances of infection is less. The solid substrate culture is historically important and used for fungal enzymes such as amylase, cellulase. The fermentation is started by inoculating the medium. The growth conditions (pH, temperature, O 2 supply) are maintained at optimal levels. The bioreactor system must be maintained sterile throughout the fermentation process. Duration of fermentation is 2-7 days. Besides desired enzymes, several other metabolites are produced. So, enzymes have to be recovered and purified.

17. (2.6) Recovery and Purification of Enzymes: The desired enzyme produced may be excreted into culture or may be present within the cells. Depending upon requirement, the enzyme is purified. For release of intracellular enzymes: Sonication High pressure Osmotic shock Done for microbial cell disruption Removal of Cell Debris: Filtration or Centrifugation can be used Removal of Nucleic acids: They are precipitated and removed by adding Poly-Cation such as Polyamines. Enzyme Precipitation: Using ammonium sulphate salts and organic solvents (isopropanol, ethanol, acetone). More enzyme Purification by: Ion-exchange chromatography Size-exclusion chromatography Affinity chromatography Enzymes is dried and stored using freeze dryers.

18. OUTLINE OF PRODUCTION OF ENZYMES:

19. BACTERIAL ENZYMES: LIST OF ENZYMES OBTAINED FROM BACTERIA: AMYLASE PROTEASES LIPASE ESTERASE CELLULASE GLUCANASE XYLANASE GLUCOSE ISOMERASE ᵦ - LACTAM AMYLASE

20. BACTERIAL ENZYMES:

21. PROPERTIES BACTERIAL ENZYMES: AMYLASE Starch hydrolysing activity Thermotolerant Thermostable Alkali resistant Cyclodextrin producing enzyme PROTEASES Proteolytic activity Acidic Thermophilic Active in presence of inhibitory compounds LIPASE Lipolytic enzyme Fat splitting Stereoselectivity Racemic-resolution activity CELLULASE Cellulolytic complex enzyme Saccharification of crystalline and amorphous cellulose Thermostable

22. INDUSTRIAL ENZYMES: 1. TEXTILE INDUSTRY- AMYLASE For desizing of fabric like cotton Hydrolyses starch into soluble dextrin and oligo saccharide CELLULASE Biopolishing of cellulosic fabrics under acidic conditions Partially digests excess yarns, loosening them from fabric PECTINASE Bio-scouring of cellulosic fabrics under alkaline conditions Hydrolyses pectin and associated hemicellulose matter from fabrics thus assisting eco-friendly removal of waxes from fabrics CATALASE Breaks hydrogen peroxide into nascent oxygen and water Used for bleach cleanup.

23. 2. DETERGENT INDUSTRY- ALKALINE PROTEASE Decomposes protein-based stains like blood, mucus etc. ALKALINE AMYLASE Automatic dish-washing liquid detergent formulations to decompose starch-based stains like potato, food, carbohydrates etc ALKALINE LIPASE Decomposes fatty based stains like fats, butter, salad etc. ALKALINE CELLULASE Degradation of cellulose and Modifying structure of cellulose fibre to increase colour brightness 3. PULP AND PAPER INDUSTRY- CELLULASE Pulp cleanliness Improves drainage LIPASE Allows secondary fibre to loosen up, releasing embedded ink with reduced usage of detergents LIGNINASE Removes lignin to soften paper LACCASE Bleach to improve brightness

24. 4. LEATHER INDUSTRY- ALKALINE AND ACID PROTEASE Removes unwanted proteins, materials like elastin, albumin, mucoids , globulins without damaging collagen. ALKALINE AND ACID LIPASE Hydrolyses insoluble fat and oil matter into soluble fatty acids and glycerol giving high degreasing performance. 5. STARCH AND SUGAR INDUSTRY - AMYLASE Hydrolyses alpha-1,4-glucosidic bonds to reduce viscosity of gelatinized starch, producing soluble dextrin. GLUCOAMYLASE To saccharify liquified starch from various sources such as corn, wheat . Resultant are glucose rich syrups. DEXTRANASE FOR SUGAR INDUSTRY Dextran are undesirable compounds in sugar production which reduces viscosity and reduces industrial recovery. GLUCOSE ISOMERASE Catalyses isomerization of glucose to fructose.

25. 6. DAIRY INDUSTRY- CHYMOSIN, LYSOZYME Cheese manufacturing LIPASE Enhances ripening of blue Mold cheese 7. BAKING INDUSTRY - AMYLASE For starch modification XYLANASE To break down Xylan. 8. ANIMAL FEED INDUSTRY - PHYTASE For breakdown of phytic acid Increases digestibility of feeds BETA-GLUCANASE To break down beta-glucans present in Animal Feed

26. REFERENCES: 1. Vyas S.P, Dixit V.P, “Pharmaceutical biotechnology”, CBS publishers and distributors, 1 st edition, 1998, Pg no.288-296 2. Gad S.C, “Handbook of pharmaceutical biotechnology”, Wiley publications, 2007, Pg.no.691-698 3. Smith J.E, “Biotechnology”, Cambridge publications, 5 th edition, 1995, Pg.no 73-88 4. Sharma A.K, Beniwal V, “Industrial Enzymes”, Nova publications, 2014, Pg.no 15-49 5. Quax w, “Bacterial enzymes”, Prokaryotes(2006), 1;777-796