Micro-organism Lipase Gene Cloning And Expression

Lipase is a kind of ester bond hydrolase.Microbial lipase is an important part of enzyme application,is widely used in food,medicine,detergent,leather,paper and other industrial fields.In this study,two lipase gene from Serratia marcescens BOB was cloned,and analysed its heterologous expression.Stain BOB degraded olive oil was screened from Arctic tundra and identified as Serratia marcescens by morphology and 16S rRNA methods,then named as Serratia marcescens BOB.After ultrafiltration and ammonium sulfate precipitation,wild stain BOB lipase has optimum temperature at 30? and optimum pH at 7.0,with enzyme activity of 25.93 U/mL.Serratia marcescens BOB was found two lipase gene,a novel gene lip6 consists of 801 bp,encoding 266 amino acids and 1 stop codon,was cloned.Recombinant stain E.Coli BL21-pET-28a(+)-lip6 was constructed,after optimization of inducing conditions,the recombinant stain was induced under IPTG(final concentration 0.5 mmol/L),30? 20 h,then disrupted cell by ultrasonic,has mass soluble protein LIPA with enzyme activity of 520 U/L.After purified by affinity chromatography column,SDS-PAGE detected a single band at 30 kD,with specific enzyme activity of 22.29 U/mg.Another lip18 gene consists of 1 842 bp,encoding 614 amino acids,was cloned.Recombinant stain E.Coli BL21-pET-28a(+)-lip18 was constructed,with soluble enzyme LIPC activity of 5.29 U/mL.And enzyme activity increased 1.7 times after optimization of inducing conditions.After purified by affinity chromatography,SDS-PAGE detected a single band at 65 kD.Enzymatic property of the recombinant enzyme showed that:LIPC was cold-active and neutral lipase,with optimum pH and temperature of 7.0 and 30 ?,respectively.And LIPC was thermal instability,has high catalytic activity at low temperature(Km was 0.634 mmol/L,Vm was 1.131 mmol/L/min).The recombinant enzyme has better tolerance of the organic solvent(methanol,ethanol,propanol,glycerin,etc),among them,50%(v:v)propanetriol tolerance of LIPC was up to 130%.Moreover,the recombinant enzyme has highly selectivity in medium and long chain fatty acid(C10-C16).The lip18 gene was amplified and integrated into the genome of P.pastoris GS115 via the pPIC9k vector,with LIPE lipase activity of 19.75 U/mL.After optimization of inducing conditions and ammonium sulfate precipitation,enzyme activity increased 3.89 times.Enzymatic property of the recombinant enzyme showed that:the optimum pH and temperature for recombinant enzyme LIPE were at 7.5 and 35? has preferable pH stability at 6.0-9.0 and thermal stability at temperature lower than 40?.Furthermore,the organic solvent tolerance and substrate specificity of LIPE lipase was similar with LIPC lipase.