Enzyme Production.pptx

Industrial Production of enzymes using microbes

1. Production of Enzymes-Amylase, Protease & Lipase

2. Many microbes synthesize and excrete large quantities of enzymes into the surrounding medium. Using this feature of these tiny organi s ms, many e n zyme s have produced commercially. These been incl u de Amylase, Cellulase, Protease, Lipase, Pectinase, Streptokinase, and many others. Enzymes are extensively used in food processing and preservation, washing powders, leather industry, paper industry and in scientific research.

3. ENZYMES PRODUCER ORGANISM (Bacteria) Penicillinase Bacillus cereus Amylase Bacillus coagulans L-asparaginase Citrobacter sp. Penicillin acylase Bacillus megaterium Protease Xanthomonas ENZYMES PRODUCER ORGANISM (Fungi) Amylase Aspergillus niger Lipase Candida lipolytica Invertase Saccharomyces cerevisiae Glucose oxidase Penicillium notatum Cellulase Trichoderma reesei Dextranase Penicillium funiculosum Trysinase Neurospora crassa

4. In early days, animal and plant sources largely contributed to production of enzymes. Even now, they act as the major source for certain enzymes Animal organs and tissues are very good sources for enzymes such as lipases, esterases and proteases. Some plants are excellent sources for certain enzymes-papain (papaya), bromelain (pineapple)

5. The most important limitations are the difficulty in isolating, purifying the enzymes and the cost factor . For this very reason, microbial production of enzymes is preferred. Microbes are the most significant and convenient sources of commercial enzymes They can be made to produce abundant quantities of enzymes under suitable growth conditions. Microbes can be cultivated by using inexpensive media and production can take place in a short period.

6. Its easy to select microbes for the production of specific type of desired enzymes. Recovery, isolation and purification processes are easy with microbial enzymes than that with animal or plant sources. In fact, most enzymes of industrial applications have been successfully produced by microbes. Various fungi, yeast and bacteria are employed for this purpose.

7. Submerged fermentation (SMF) Solid state fermentation (SSF)

8. SUBMERGED FERMENTATION :- It employs free flowing liquid substrates, such as molasses and broth The products yielded in the fermentation are secreted into the fermentation broth. This method is suitable for those microbes such as bacteria that require high moisture content for their growth. The sterilization of the medium and purification process of the end products can be done easily. Also the control of process parameters like temperature, pH, aeration, oxygen transfer and moisture can be done conveniently.

9. SOLID STATE FERMENTATION :- Method used for microbes which require less moisture content for their growth The solid substrates commonly used in this method are bran, bagasse and paper pulp The main advantage is that, nutrient-rich waste materials can be easily recycled and used as substrates in this method. It requires simple instruments over SMF, higher concentration of products with less effluent generation

10. AMYLASE Amylases are important hydrolase enzymes. It usually degrades complex polysaccharide molecules such as starch into glucose. Amyla se Starch G l ucose 🞂 🞂 Present in saliva of humans (for digestion) This enzyme randomly cleave internal glycosidic linkages in starch molecules Hydrolysis of starch with amylase results in formation of dextrin and then disaccharide maltose (2 glucose molecules) and finally glucose. Types of amylases:-α-amylase, β-amylase, γ-amylase The substrate that α-amylase acts upon is starch. Starch is composed of 2 polymers- amylose (20-25% of the starch) and amylopectin (75-80% of starch). Amylose is broken down to give maltotriose and maltose molecule A m ylo p ec t i n i s b r o k e n dow n t o g iv e de x tri n an d g l u c ose molecule

11. Primary source of β-amylase are the seeds of higher plants and sweet potatoes. BACTERIAL SOURCE OF α-Amylase:- Bacillus subtilis, B. stearothermophilus, B. licheniformis, B. amyloliquefaciens Carbon source:- maltose, sucrose, glucose Nitrogen source;- Inorganic nitrogen sources such as ammonium sulphate, ammonium chloride, ammonium hydrogen phosphate, Organic sources such as soyabean meal, peptone, yeast extract

12. Bacillus licheniformis has been used industrially for the production of α-amylases. The fermentation of bacterial amylases is accomplished in submerged culture at neutral pH and at a temperature of 30-40ºC. Cereal meal and starch rich medium are used along with an organic source of nitrogen . After 10-20 hours, formation of α-amylase starts and continues for another 100 hours pH must be below 6 during fermentation to prevent the denaturation of α-amylase.

13. SELECTION OF MICROORGANISM :- Aspergillus niger, A. oryzae , A. kawachii T hey produce amylase and secrete acid hence easily degrades complex raw material. SELECTION OF RAW MATERIALS :-fungi cannot grow on natural media, so we provide synthetic or artificial media. CHEMICALS QUANTITY USED Corn starch 24g/L KCl 0.2g/L Na 2 HPO 4 4.7g/L CaCl 2 /CaCO 3 1g/L MgCl 2 .6H 2 O 0.2g/L STEPS INVOLVED IN PRODUCTION OF FUNGAL AMYALSE

14. FERMENTATION PROCESS Fermenter is filled with the raw material or fermentation medium Inoculate mycelium of A. niger Maintain temperature at 30ºC-45ºC After 72 hours recovery process is carried out.

15. RECOVERY PROCESS F i ltrat e c o ntains a m ylase , sy n thet i c m e d i a an d my c el i a of fungi By filtration, remove mycelia A m ylas e i s sepa r a t e d from synthe t i c media by addi n g ammonium hydroxide which forms precipitate with amylase B y filtra t io n c o l l ec t th e p rec i p i t at e ( amylase+a m monium hydroxide) Precipitate is subjected to crystallization at 4ºC Amylase in form of crystals are separated and collected

16. Fungal amylase differ from bacterial amylases by low pH 4-5. The production of fungal amylase is carried out in su b mer g e d cul t ure w it h s e lec t e d st r ains solid - su b s trat e cultu r e an d someti m e s in of Aspergillus . Fermentation medium used remains the same as in the case of bacterial α-Amylase but the concentration of glucose inhibits the formation of amylase, therefore the concentration of glucose is kept low. Fungal amylases- used in manufacture of baked products and production of maltose rich syrups

17. LIPASE It is an enzyme that catalyzes the breakdown of most triglycerides into fatty acids and glycerol (lipolysis). Lipase enzyme is usually found naturally in pancreatic juice and stomach . They also control the volume of fat in body that is synthesized and burned by reduction of adipose tissue. Lipase can be purified or extracted from plant, animal, yeast, bacteria and fungal sources.

18. Uses:-used in detergents (laundry) due to the wide use of washing machine, -fat and oil processing (inexpensive and less needed lipid can be converted into greater value fat), PUFAs (Poly Unsaturated Fatty Acids) used remarkably as pharmaceutical, nutraceutical etc. - obtained by using microbial lipases from plant, and animal lipids.

19. Selection of microorganism Formulation of medium Production process Recovery and purification of enzymes

20. SELECTION OF MICROORGANISM Bacterial source:- Pseudomonas aeruginosa, Staphylococcus caseolyticus, Bacillus coagulans, Bacillus subtilis, Bacillus stearothermophilus Fungal source:- Aspergillus niger, Candida rugosa, Candida utilis, Penicillium citrinum, Penicillium restrictum, Candida cylindracea Candida cylindracea was grown on yeast agar medium and maintained at 4ºC. A loop full of cells from freshly grown culture (agar slant) of Candida cylindracea was transferred to flask. Flask was then incubated at 30ºC on a rotary shaker at 200 rpm for 36 hrs.

21. FORMULATION OF MEDIUM:-medium contai n in g flask w a s s t erilized i n an autoclave at 121ºC for 20 min CHEMICALS REQUIRED QUANTITY (g/L) KH 2 PO 4 6 MgSO 4 .7H 2 O 1 Urea 4 FeCl 3 .6H 2 10 mg Inositol 0.4 mg Thiamine hydochloride 0.2 mg Biotin 0.8 mg Glucose 10 mg Distilled water 1L

22. PRODUCTION PROCESS:- The medium used in preparing the inoculum is the same as the production medium. The only difference is the use of glucose as the carbon source . Palm oil can be used as the main carbon source in production medium but not used in the inoculum preparation medium 50g of sterile palm oil was transferred aseptically to the sterilized medium in the fermentor 10% of the inoculum was added to the total volume. Samples of 50ml was withdrawn aseptically at regular time interval for analysis. Temperature= 2 8 -3 3 º C, pH = 6 - 7, st i rring speed=500rpm

23. RECOVERY AND PURIFICATION OF ENZYMES:- Removal of debris by filtration After f erm e ntation t h e ext r ac t w a s precipitated with ammonium sulphate Ultr a f i lt r ati o n w a s u sed t o con c e n tra t e t h e sample

24. PROTEASE These are a group of enzymes belonging to a class of hydrolases whose catalytic function is to hydrolyze peptide bonds of proteins They are also called proteolytic enzymes USES:-for cleaning contact lenses to remove large variety of stains of food, blood and body secretions -in the manufacture of cheese -meat tenderization (reduce toughness of meat) -to correct lytic enzyme deficiency syndrome -treatment of industrial waste:- keratinase used as depilatory to remove hair from drains

25. SELECTION OF MICROORGANISM FORMULATION OF MEDIUM PRODUCTION PROCESS RECOVERY AND PURIFICATION OF ENZYMES

26. SELECTION OF MICROORGANISM Ba c teri a l sourc e : - Bac ill u s l iche ni formis, B . amyloliquefaciens, B. stearothermophilus Hi g h car b ohydr a t e le v e l i n th e medium stimulates protease production

27. FORMULATION OF MEDIUM Medi a u sed contain s ground ba r l e y a s t h e carbon source Starch level is limited Protein hydrolysates or sodium glutamate is used as the nitrogen source

28. PRODUCTION PROCESS Th e me d iu m has a pH o f 6.5 - 7 . 5 an d inc u bation temperature of 37ºC Fermentation proceeds for 3-5 days Preserved inoculum Inoculum development Inoculation tank Fermenter Cell disruption Filtration (to remove debris) Remove nucleic acids Salt treatment Cool storage Final purification (chromatography etc.) F it ration Freeze drying *During fermentation, insoluble protein of the medium is partially hydrolysed by boiling in dilute acid/enzymatic treatment.

29. RECOVERY AND PURIFICATION OF ENZYMES Culture is filtered Aqueous portion is concentrated by evaporation at reduced pressure and at temperature not less than 40ºC Recovery can also be carried out by precipitation.

30. SELECTION OF MICROORGANISM 1. Various fungi produce protease such as Aspergillus oryzae, A. sojae , A. niger, A. wentii, Mucor pusillus, Mucor miehei, Mucor delemar, Amylomyces rouxii , Endothia parasitica

31. FORMULATION OF MEDIUM The fungus is grown on wheat bran under fermentation conditions similar to those for amylase production Trace elements used in Inoculation medium:- CHEMICALS REQUIRED QUANTITY Fe(NH 4 ) 2 (SO 4 ) 2 1 mg ZnSO 4 1 mg MnSO 4 0.5 mg CuSO 4 0.08 mg CoSO 4 0.1 mg H 3 BO 3 0.1 mg Distilled water 1 L

32. Medium for inoculum preparation (g/L):- Medium for production (g/L):- Chemicals required Quantity Casein hydrolysate 5 Soya protease digest 2 Yeast extract 2 Soluble starch 10 D-mannitol 5 Trace element 1 ml FeSO 4 .7H 2 O 15 mg Chemicals required Quantity Soyabean meal 30 Glucose 10 NaNO 3 3 Skim milk 10 KH 2 PO 4 0.5 MgSO4.7H 2 O 0.25

33. PRODUCTION PROCESS 1. End othi a p arasitic a i s employe d for the production of this enzyme in lab scale A 5% inoculum from 96 hrs flask is utilized for the production in a stirred, aerated vessel for 48 hrs at 28ºC. Optimum pH is 4-5 Proteases from Aspergillus oryzae and A. sojae are produced by solid substrate fermentation.

34. RECOVERY AND PURIFICATION OF ENZYMES After growth the harvest can be dried at 50ºC or less Protease can also be extracted by water followed by addition of alcohol for precipitation and dried at 55ºC

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