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Cloning, Expression And Characteriation Of Lipase

Posted on:2015-01-28Degree:MasterType:Thesis
Country:ChinaCandidate:J F ZhangFull Text:PDF
GTID:2180330464471006Subject:Microbiology
Abstract/Summary:PDF Full Text Request
In this study, a lipase producing bacterium was isolated from soil samples in WuMing.The strain was identified as Staphylococcus by homology alignment and phylogenetic analysis 16S rDNA,named STL-01.We cloned two lipase genes from STL-01 and GX-T6 that had been accomplished genome sequence, named lip1 and lip3, respectively.Two genes connected with expression vector to construct recombinant plasmids, which were transformed into E. coli to expression. Nickel affinity chromatography was used to purify the recombinant lipase Lip1 and Lip3, the research of enzymatic properties revealed:The optimum temperature of Lip1 was 25℃,and it exhibited 32.5%、70.2% relative activity at 0℃ and 10℃.The optimum pH of Lip1 was 8.0,and it kept stable between pH6.0 and 9.0.Lip1 could hydrolyze substrate from C2 to C16,and the maximal activity was on Cg (pNPH).Five mM metal ions, including Ba2+、Zn2+、Ni2+、Ca2+、Fe3+, inhibited its activity obviously.Lipl had little activity in 1%(w/v) ionic surfactant SDS and CTAB. It could tolerate 15%(v/v) methanol and 10%(v/v) ethanol,1, 2-propylene glycol, acetone,n-hexane and DMF. The optimum temperature and pH of Lip3 were 40℃ and 7.0, respectively.Lip3 remained stable in the range of pH5.0 to 9.0. The optimum substrate of Lip3 was C2(pNPA), in addition Lip3 also exhibited rather high activity as substrate was C6(pNPO).Five mM Zn2+ almost completely inhibited its hydrolytic activity.Lip3 could tolerate 55%(v/v) methanol and 10%(v/v) ethanol,1,2-propylene glycol, acetone, n-hexane and DMSO.
Keywords/Search Tags:Lipase, Cloning and expression, Enzymatic property
PDF Full Text Request
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