COMMERCIAL PRODUCTION OF ENZYMES.pptx

Commercial pro

1. COMMERCIAL PRODUCTION OF ENZYMES

2. introduction Enzymes- Biocatalysts- Complex protein molecules that bring about chemical reactions- Nontoxic and biodegradable. Produced in large amounts by microorganisms- industrial applications Enzyme technology- broadly involves selection, production, isolation, purification and use of enzymes recombinant DNA technology and protein engineering- efficient use of enzymes

3. The first enzyme produced industrially was taka-diastase (a fungal amylase) in 1896, in United States. It was used as a pharmaceutical agent to cure digestive disorders. Europe- softening of hides with faeces of dogs and pigeons- before tanning- pancreases- German scientist- Otto Rohm Proteases in laundry industry- impurities caused allergic reactions- 80 % from microbial sources.

4. Uses of enzymes from different organisms Fungi – 60% Bacteria – 24% Yeast – 4% Streptomyces – 2% Higher animals – 6%

5. Enzymes from plant and animal sources Quantities are limited D ifficulties in isolating, purifying the enzymes, and the cost factor W ide variation in their distribution Mammalian cell cultures- cost constraints- Tissue plasminogen activator from cell cultures

6. Microbial sources Inexpensive media, short period, easy to manipulate, isolation and purification are easy Extracellular enzymes- safe, stable and active Stable over a range of pH and temperature Optimal fermentation conditions- cheap substrates, low cost production Co- production- amylase, lipase and protease- used in detergent industry rDNA- overproduce, substrate specific and stable Bacillus and Aspergillus- most used

7. Isolation and screening of microorganisms Sources: Plant bark, watery environment, skim milk , marine sediment , municipal solid wastes and from grapes Serial dilution and spread plate method- specific agar Thermophilic microbes- thermostable enzymes Enzyme inducers in media Laccase- CuSO4 and MgSO4 Proteases- casein, skim milk, gelatin Amylases, cellulases and lipases- starch, CMC, oil substances

8. Identification

9. screening Reaction Presence of enzyme Starch hydrolysis amylase Casein/ gelatin hydrolysis protease Tributyrin/Tween 80 hydrolysis or change in phenol red color to orange Lipases (breakdown of oil to FA) Gram’s iodine solution amylases 0.1% Congo red solution followed by 1 M sodium chloride solution cellulase Phenol red to pink colour L-asparaginase Sodium carbonate Alkalophilic organisms

10. identification Bergey’s manual of determinative bacteriology- cultural, morphological, microscopic and biochemical characteristics Fungi- 18S rRNA sequencing Bacteria- 16S rRNA or 16S rDNA sequencing

11. Production process

12. production Mostly used inocula - Acinetobacter, Pseudomonas, Staphylococcus, Streptomyces, Fusarium, Mucor, Penicillium , and Trichoderma species Fermentation process- depends on plant equipment, yield, convenience and application SmF - extracellular enzymes secreted in media, needs expensive synthetic media SSF- inexpensive media, downstream processing is simple

13. Examples: Invertase ( β D fructofuranosidase )- p roduced by Aspergillus sojae – SSF- orange peels moistened with molasses -production of alcoholic beverages L ignocellulosic biomass was used to produce cellulase -with Aspergillus awamori D etergent protease by the bacterium Alcaligenes sp . - using fed batch fermentation

14. Inducers in medium Surfactants- Triton X-100/ Tween Aromatic and phenolic compounds Cheap substrates Chicken feathers- Bacillus megaterium- keratinase Orange peel- Aspergillus sojae - invertase Wheat bran- Aspergillus sp.- keratinase, laccase and phytase

15. Optimization Optimization of various nutritional parameters (C, N, and P sources) physico -chemical aspects (inoculum age, incubation time and temperature) fermentation factors- inoculum level , agitation/ aeration

16. extraction of extra and intracellular enzymes

17. Strain improvement Cycles of random mutagenesis and screening Target for- 1. secretion efficiency- EC enzymes 2. overcomes organism’s regulatory mechanism- avoid catabolite repression mRNA half life increased Gene dosage increase- plasmid/ chromosomal amplification Use of GRAS listed organisms- Bacillus, Aspergillus and Saccharomyces

18. Protein engineering