Isolation And Identification Of Lipase Producing Bacteria And Optimization Of Enzyme Production Conditions And Lip7 Gene Cloning

Lipase is a special ester hydrolase,which is widely found in animals,plants and microorganisms.In recent years,lipases are widely used in food processing,bio-diesel preparation,paper making,detergent,medicine and other fields,has shown significant potential application prospects.Currently used for industrial production of enzymes are mostly imported,due to lipase expensive,severely limited the promotion of its usage.Therefore,application of lipase become a hot research.This experiment isolated lipase production strains from the oil and grease,and strains were isolated from species identification,meanwhile its fermentation conditions were optimized for enzyme production,the preliminary study of enzymatic properties is also part of the test.The results showed that a strain which has high lipase activity,named HDY-76 is obtained.A fter identification of morphological and molecular biology,the strain HDY-76 is Yarrowia lipolytica.This test determined the best conditions for the enzyme production of HDY-76 were,that is:peptone 20.0 g/L,sucrose 5.0 g/L,NH4Cl 0.5 g/L.MgSO4 0.3 g/L,NaCl 1.0 g/L,Tween-80 0.4 ml/L,speed 200�5 r/min,pH 3.0,incubation temperature 28?,1.0%inoculation,the best loading capacity of 60 mL/250 mL,shaking 50 h.lipase production increased 62.53%,reach to(252.89�4.12)U/mL which is(155.6�3.87)U/mL before optimization.Optimum temperature of the lipase produced by HDY-76 strain is 45?,optimum pH is 5.5.Mg2+ is activator for lipase production,and Na+?Fe2+?EDTA?Cu2+?Mn2+ has significant inhibition for lipase production.When the temperature is 30-50 and in pH 4.0-6.0 lipase has better stability.At the same time,by using PCR technology,the lipase gene(lip 7)was successfully cloned and expressed in Pichia pastoris GS115,which made the enzyme activity increased 30.27%,reach to(329.43�6.52)U/mL,compared to(252.89 � 4.12)U/mL before optimized.