Lipases & Industrial Production of Lipases

Introduction to Lipases
Industrial Production of Lipases by Fermentation
Industrial Significance of Lipases

1. ‘LIPASES’ PREPARED BY: Nahal jehangir (18006231-011 ) BS. BIOTECHNOLOGY COURSE TITLE: Food Biotechnology

2. CONTENTS Introduction to Lipases Sources Classification Production by Fermentation Technology Applications Industrial & Medical Aspects Significance in Food Biotechnology

3. Introduction A lipase is any enzyme (extracellular) that catalyzes the hydrolysis of fats . Splits fats into fatty acids and glycerol. (Glycerol Ester Hydrolases) A subclass of Esterases . Lipases perform essential roles in digestion , transport and processing of dietary lipids in most organisms. Genes  encoding lipases are even present in certain  viruses .

4. What are Lipases?

5. Enzymatic Activity The mode of action of lipase is:

6. Sources Many microbial lipases have been commercially available in free or immobilized form. Produced in pancreas , seeds, Euphorbiaceae ( Ricinus communis ). Numerous species of; Bacteria (Bacillus, Pseudomonas, and Burkholderia ) Yeasts (Candida rugosa , Yarrowia lipolytica and Candida antarctica ) Molds ( Aspergillus , Trichoderma viride ) Microbes are potent lipase producer .

7. Pancreatic Lipase Also known as pancreatic triacylglycerol lipase , is secreted from the pancreas The primary lipase that hydrolyzes dietary fat molecules in the human digestive system. Requires a high pH for its activation. That is why fats are the hardest of all foods to digest. Mechanism: Bile salts secreted from the liver and stored in gallbladder are released into the duodenum where they coat and emulsify large fat droplets into smaller droplets, thus increasing the overall surface area of the fat, which allows the lipase to break apart the fat more effectively.

8. Classification

9. Industrial Production The industrial production of lipases involves the following steps; Sterilization Isolation of pure culture Characterization & Screening Optimization Substrate addition Inoculation Automation of bioreactors Harvesting / recovery

10. Production Steps: 1. Isolation of Lipase P roducing Bacteria: Sludge and oil contaminated samples are collected from effluents . STRAINS : Candida lipolytica Asperigillus niger (Fungus used mostly) Pseudomonas , Bacillus and Lactobacillus sp. Samples are subjected to serial dilution. Incubation at 37°C for 24-48 hours Nutrient medium Agar A clear zone around the colonies indicates the production of lipase.

11. 2. Screening Screening is done on Tributyrin agar rich media. (Primary Screening) P-NPP Assay for estimating lipolytic activity (Para-nitro phenyl phosphate method). 3. Characterization Effect of pH and Temperature Effect of organic solvent and detergents Analytical  characterization methods are necessary to ensure the identity, purity, structural and conformational integrity, and activity of an enzyme product.

12. 4. Substrate Addition Solid State Fermentation (SSF) is used for the commercial production of lipases due to; Higher yields and productivities Extended stability of products Lower production cost Different types of agricultural wastes and oily substrates. Rice bran, Wheat bran, Gingelly oil cake Almond meal, Mustard oil cake, Neem oil cake, Groundnut oil cake, Gingerly seed and groundnut kernel & Olive oil.

13. 5. Fermentation Process Optimal medium composition: glucose 0.1, olive oil 3.0, NH 4 Cl 0.5, yeast extract 0.36, K₂HPO₄0.1, MgCl 2  0.01, and CaCl 2 0.4 mM. Carbon source: Wheat bran, rice husk, lentil husk, banana waste, watermelon waste. Olive oil as inducer and yeast extract as nitrogen source . To maintain sufficient oxygen concentration for optimum production, fermentation has been carried out at 600 rpm and at different aeration rates. Gas flow rate of 50.34 cm 3 /s yielded optimum production of lipase. 6. Optimization Lipase production was optimized at different pH (7 - 10), temperature (30 - 60°C) incubation period (1-5 days) with constant shaking at 120 rpm . Bacteria were cultured in nutrient broth with 1% olive oil . During the cultivation, lipase activity will be measured every 12 h to determine the maximum lipase producing period.

14. 7. Purification / Harvesting (DOWNSTREAMING) Calcium Chloride precipitation Ammonium Sulphate precipitation Colum Chromatography Supernatant containing extracellular lipase obtained from fermented broth is treated with 0.4M CaCl 2 to precipitate fatty acids followed by centrifugation at 4°C and 12000rpm for 30 min. The supernatant was collected in a glass beaker and to it chilled acetone was added slowly to allow protein precipitation . The precipitates were then harvested by centrifugation at 4°C and 12000rpm for 30 min. Supernatant then subjected to ultrafiltration and dialyzed overnight.

15. Experimental Design

16. APPLICATIONS Processing of fats and oils Detergents industry Food Processing (Improvement of flavor) Synthesis of fine chemicals and pharmaceuticals Paper manufacture (Control of pitch) Production of cosmetics & beverage Waste water treatment (By removing oil) Biocatalysts in industrial applications Diagnostic and medical use

17. Industrial Aspects: Lipases serve important roles in human practices as ancient as yogurt and cheese fermentation. A  biotechnology company has brought recombinant lipase enzymes to market for use in applications such as baking, laundry detergents. As   biocatalysts in  alternative energy strategies to convert vegetable oil into fuel .   ( Biodiesel ) Can be more environmentally friendly and safe. Detergent formulations (Minimize toxic phosphate u sage) Food, pharmaceutical, leather, textile, cosmetic, and paper industries .

18. Medical & Diagnostic Aspects Blood tests for lipase may be used to help investigate and diagnose  acute pancreatitis and other disorders of the pancreas. Cystic fibrosis, Alzheimer's disease, atherosclerosis Measured serum lipase values may vary depending on the method of analysis. Lipase can also assist in the breakdown of fats into lipids in those undergoing  pancreatic enzyme rep lacement therapy (PERT). Lipase is used for  indigestion, heartburn, allergy to gluten in wheat products  (celiac disease), Crohn's disease, and cystic fibrosis.

19. Lipases in Food Biotechnology: Blood tests for lipase may be used to help investigate and diagnose acute pancreatitis and other disorders of the pancreas. Cystic fibrosis, Alzheimer's disease, atherosclerosis Measured serum lipase values may vary depending on the method of analysis. Lipase can also assist in the breakdown of fats into lipids in those undergoing pancreatic enzyme replacement therapy (PERT). Lipase is used for indigestion, heartburn, allergy to gluten in wheat products (celiac disease), Crohn's disease, and cystic fibrosis.

20. REFRENCES https ://labpedia.net/lipase-serum / https :// www.mdpi.com/2077-0472/11/6/540/htm https :// www.researchgate.net/figure/Application-of-lipases-in-different-industries_fig2_340974234 https :// www.informit.com/articles/article.aspx?p=2783636&seqNum=4 https :// www.slideshare.net/LIKHITHK1/lipase-production-and-urification-likhith-k?qid=e9f4792d-cab5-41be- 994b-0788ddf6a047&v =&b=& from_search=8 https :// www.slideshare.net/ponkalaivanis/production-of-lipases-and-cellulase https :// www.slideshare.net/MDCrules/enzymes-their-production https ://microbenotes.com/lipase-test-objectives-principle-procedures-and-results / https :// www.researchgate.net/profile/Ali-Nawaz-3/publication/288933945/figure/fig4/AS:669283327696898@1536580988204/Effect-of-lipid-substrate-for-lipase-production-by-Aspergillus-niger-in-solid-state.ppm

21. Thank you for the attention! Any questions please?