| The extracellular lipase from Candida sp.99-125(ClLip), an important industrial biocatalyst, has been widely used in esterification and transesterification reactions, chiral resolution of various chiral compounds and production of biodiesel because of its high reactive activity. To clarify the characterization of gene and protein of ClLip, some researches have been done as follows:1. Purification and characterization of ClLipC1Lip was purified by acetone precipitation, ion exchange chromatography on Q-sepharose Fast Flow and hydrophobic interaction chromatography on Butyl-sepharose Fast Flow. After three-step purification, the specific activity of ClLip was5035U/mg. The purification factor and yield was6.27and27%, respectively. SDS-PAGE showed a nearly homogenous, pure ClLip (purity>92%) with the molecular weight of38kDa. Analysis of native PAGE, activity-stained isoelectric focusing (IEF) and two-dimensional electrophoresis indicated that ClLip contained four isoenzymes which have the same molecular weight (38kDa) and close isoelectric point (pI5.0-6.5). N-terminal amino acid sequencing and MALDI-TOF mass spectral analysis showed that the four isoforms matched with extracellular lipase Lip2from Yarrowia lipolytica. The four isoforms were encoded by the same gene (LIP2) and differed in N-glycosylation patterns. Enzymatic deglycosylation showed that ClLip was a glycosylated protein which contained about12%sugars. The optimum temperature and pH of ClLip was37℃and8.0, respectively. The activity was stable up to35℃and began to decline at40℃. Lipase was stable at pH between5and8. Lipase activity was stimulated by Ca2+and Mg2+and inhibited by Zn2+, Ni2+and Cu2+, whereas EDTA, DTT and β-mercaptoethanol had no effect on its activity. Triton X-100, Tween80and Span60increased slightly the lipase activity and SDS inhibited it. CILip showed high activity towards long chain and unsaturated fatty acid triglycerides.2. Cloning and expression of CILip in Pichia pastorisCILip DNA encoding mature peptides of lipase was cloned into the expression vector pPICZaAby PCR using primers designed by the extracellular lipase gene Lip2from Y. lipolytica. CILip gene consists of903bp encoding301amino acids. A conserved lipase pentapeptide GHSLG was found in CILip amino acid sequence. Multiple alignments showed that CILip showed a high amino acid identity to the lipases from fungi. The three-dimensional structure of ClLip was modeled by SWISS-MODEL. CILip DNA and CILip DNA with a six-histidine tag was cloned into vector pPICZa A to construct expression vectors pPICZaA-ClLipå'ŒpPICZaA-ClLip-His. Two resulting plasmids were linearized and transformed into P. Pastoris GS115and selected on high Zeocin concentration plates (500μg/ml). Two clones (CILip2-8and CILip3-5) which secreted high lipase activity (550U/ml) were obtained. Southern blot analysis showed that three CILip gene copies were integrated into the genomic DNA of clones of CILip2-8and3-5. The lipases with histidine-tag showed low activity (50U/ml) possibly because the histidine-tag disturbed the correct folding of the lipase.3. Fermentation, purification and characterization of recombinant CILipFed-batch cultivation was performed in a51reactor containing31basal salt medium. When glycerol was used up, clone of0-8(single lipase gene copy) was induced to express the lipase by feeding methanol controlled by the dissolved oxygen (DO). The OD600and lipase activity increased continuously and reached a final value of346and4460U/ml, respectively, after192h of cultivation. The OD600and lipase activity was350and4280U/ml, respectively, when constant methanol concentration (5g/1) feeding strategy was used. The OD600and lipase activity was350and11000U/ml, Lipase activity was stimulated by Ca2+and Mg2+and inhibited by Zn2+, Ni2+and Cu2+, whereas EDTA, DTT and β-mercaptoethanol had no effect on its activity. Triton X-100, Tween80and Span60increased slightly the lipase activity and SDS inhibited it. ClLip showed high activity towards long chain and unsaturated fatty acid triglycerides.2. Cloning and expression of CILip in Pichia pastorisCILip DNA encoding mature peptides of lipase was cloned into the expression vector pPICZaAby PCR using primers designed by the extracellular lipase gene Lip2from Y. lipolytica. CILip gene consists of903bp encoding301amino acids. A conserved lipase pentapeptide GHSLG was found in CILip amino acid sequence. Multiple alignments showed that ClLip showed a high amino acid identity to the lipases from fungi. The three-dimensional structure of CILip was modeled by SWISS-MODEL. ClLip DNA and CILip DNA with a six-histidine tag was cloned into vector pPICZa A to construct expression vectors pPICZaA-ClLipå'ŒpPICZaA-ClLip-His. Two resulting plasmids were linearized and transformed into P. Pastoris GS115and selected on high Zeocin concentration plates (500μg/ml). Two clones (ClLip2-8and CILip3-5) which secreted high lipase activity (550U/ml) were obtained. Southern blot analysis showed that three ClLip gene copies were integrated into the genomic DNA of clones of ClLip2-8and3-5. The lipases with histidine-tag showed low activity (50U/ml) possibly because the histidine-tag disturbed the correct folding of the lipase.3. Fermentation, purification and characterization of recombinant CILipFed-batch cultivation was performed in a51reactor containing31basal salt medium. When glycerol was used up, clone of0-8(single lipase gene copy) was induced to express the lipase by feeding methanol controlled by the dissolved oxygen (DO). The OD600and lipase activity increased continuously and reached a final value of346and4460U/ml, respectively, after192h of cultivation. The OD600and lipase activity was350and4280U/ml, respectively, when constant methanol concentration (5g/1) feeding strategy was used. The OD600and lipase activity was350and11000U/ml, respectively, using clone of2-8(three lipase genes integrated) by constant methanol feeding (5g/1). The culture supernatant contained2.3g/1of pure rClLip and the lipase productivity was67900U/L/h. After fermentation, the culture supernatant was purified by ultrafiltration and ion exchange chromatography. The specific activity of purified rClLip (purity>90%) was4860U/mg. The purification factor and yield was2.29and85%, respectively. The molecular weight of rClLip was38kDa. The optimum temperature and pH of rClLip was37℃and8.0, respectively. Lipase rClLip showed high activity towards long chain fatty acid methyl esters (C12-C18). Enzymatic deglycosylation showed that rClLip contained about12%sugars. These results showed that CILip and rClLip had the same characterization.4. Effect of N-glycosylation on secretion and characterization of recombinant CILipWe used the site-directed mutagenesis to remove the two potential glycosylation sites one by one, by mutating the Asn from glycosylation consensus sequence (Asn-X-Ser/Thr) into Gln, in order to determine the role of the glycan moieties on the secretion, activity and stability of YlLip2. The results showed that two glycosylation sites were occupied by2-3kDa sugar for each site when CILip was expressed in P. Pastoris. The secretion level of lipases from N113Q and N134Q mutants decreased about37%and36%, respectively, while the secretion level of lipases from N113Q/N134Q mutant decreased77%as compared with wild-type rClLip2lipase. The T50value of lipases from N113Q, N134Q and N113Q/N134Q mutant decreased3.5℃,2.6℃and4.2℃, respectively. Kinetic parameters analysis indicated that Vmax value of N113Q and N113Q/N134Q mutants was44%and54%lower than that of wild-type lipase, respectively, and Kmax value of N134Q mutant was15%higher. These results indicated that N-glycosylation at Asn113and Asn134played an important role on synthesis and functions of rClLip2lipase. |
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