Screening,Identification Of Lipase-producing Strains And Enzymatic Study Of Lipase

Lipases(triacylglycerol hydrolases,E.C.3.1.1.3)are a class of hydrolase that catalyze the hydrolysis of ester bonds.As one of the earliest studied enzyme,lipase has strong substrate specificity and functional diversity.Lipases are commonly used in the processing of fats and oils,food processing,leather,textile,detergents and degreasing formulations,paper manufacture,synthesis of fine chemicals,production of pharmaceuticals,cosmetics.The filamentous fungus,from South Africa,were used to screen lipase-producing strains with substrate specificity.The lipase genes of Mucor circinelloides were cloned and expressed in Pichia pastoris.Also,their characteristics were analyzed.The main results of this project were as follows.(1)56 alkalophilic filamentous fungus and 22 thermophilic filamentous fungus that could produce lipase were selected from 106 alkalophilic strains and 53 thermophilic strains,with Rhodamine B flat method and titrimetric method.After the molecular identification of nuclear ribosomal internal transcribed spacer(ITS),the strains belonged to 19 genera and 32 species.(2)After analysising the activity of hydrolyzing different carbon chain length substrates,4 strains had diverse activity and Mucor circinelloides F4-95 had higher hydrolyzing activity to pNPC12 than others,prominently.Besides,lipase gene of the strains that had similar evolutionary relationships hadn't been heterologoug expressed.So,it was used for further study.(3)lip A and lip B of Mucor circinelloides F4-95,amplified by PCR,were used to recombine pPIC9K-lip A and pPIC9K-lip B.Follow it up,overexpressed recombinant Pichia pastoris strains GS-Lip A and GS-Lip B were constructe,and the lipase activity of Lip A anf Lip.B to pNPC12 could reach 14.34 U/mL and 21.66 U/mL,induced by 0.5%(v/v)methanol for 120 hour in shaking flask fermentation.(4)The biochemical properties of the recombinant lipases were examined.The optimal temperature and pH of Lip A and Lip B were 40?,pH 8.0 and 30?,pH 7.5,respectively.They had similar influence,took by metal ion.Lip A was stimulated by 10 mmol/L N2+ and K+,while Co2+and Cu2+had strong inhibition.Lip B was stimulated by 10 mmol/L K+,while Cu had strong inhibition.The hydrolysis activity of two recombinases to pNPC12 were 2-5 times as others.The activity of Lip B was higher than Lip A,so Lip B had more powerful effect on substrates specificity,compared with Lip A.