Construction Of Recombinant Strains Capable Of Producing Lipase And Application Of Recombinant Enzyme

Candida antarctica lipase B(CALB)is a widely used lipase,which can catalyze hydrolysis,esterification,transesterification reactions.It has great catalytic activity and broad substrate spectrum.It is popular both in theoretical research and industrial application.In particular,with the development of non-aqueous enzymology,CALB opens up more applications in various fields,including the synthesis of vitamin A ester derivatives.Vitamin A ester derivatives are often used as additives of food,cosmetics and others.The synthesis of vitamin A acetate is very mature in industrial production.But vitamin A acetate is unstable while the long-chain esters of vitamin A have good stability.So the synthesis of vitamin A palmitate using vitamin A acetate as the substrate becomes a trend.In the present dissertation,CALB gene was cloned from C.antarctica ZJB09193 using RT-PCR technology.The enzyme was successfully high-level expressed in P.pastoris X33 by the vector pPICZa A.The enzyme production and growth curves were measured in shake flask and fermentor.The results showed that the enzyme activity and biomass were 0.8 U·mL-1,19.1g·L-1 and 5.16 U·mL-1,80.2 g·L-1,respectively.Microfiltration,ultrafiltration and nickel ion affinity chromatography technology were used to purify the recombinant lipase.The enzyme was purified to 7.6 fold with a yield of 52.9%.The highest specific activity of the purified enzyme was 7.7 U·mg-1.The biochemical properties of recombinant lipase were determined using the p-nitrophenyl actate(pNPA)as substrate.The results showed that recombinant lipase was highly active between pH 5.0-10.0 with an optimum around pH 8.0.The optimal temperature range was found to be from 50-55?,and showed optimal activity at 52 ?.This lipase was a temperature-sensitive enzyme.The enzyme was activated by all metal ions used except Co2+ at low concentration(1 mmol·L-1).However,when the concentration of metal ions increased to 10 mmol·L-1,the enzyme was inhibited by several metal ions,such as Ni2+,Mn2+,Ba2+,Co2+.The enzyme activities were enhanced in the presence of Tween 20 and Sorbitol,whereas,SDS and Tween 80 had a little inhibition on enzyme activities.The values of Km and Vmax for pNPA were calculated to be 0.34 mmol·L-1and 7.36 ?mol·min-1·mg-1,respectively.Immobilized enzyme can overcome the disadvantage of free enzyme reaction in organic phase,such as insoluble,product separation.Using commercial fabric as a carrier,the immobilized enzyme was prepared.The best immobilization conditions were following:commercial cotton 2 seted as the carrier,the proportion of textile fabrics and co-immobilization solution was 1:7,one gram activated cotton was added 584 U free enzymes,and the immobilization time was 10 h.The synthesis of vitamin A palmitate was achieved using immobilized enzyme.The best reaction conditions were following:25 ?,180 rpm,hexane seted as a solvent,vitamin A acetate and palmitic acid concentrations were 100 mmol·L-1 and 300 mmol·L-1 respectively,immobilized enzyme dosage was 130 g·L-1,catalyzed transesterification for 12 h.The conversion rate was 77%at last.The substrate specificity of immobilized enzyme was also studied;the results showed that it can catalyze most of the transesterification reaction between long-chain fatty acids and vitamin A acetate.Finally,the kinetics of synthesis of vitamin A palmitate was discussed.The results indicated that the reaction was correspond with ping-pong Bi Bi mechanism,the Vmax,KmA,KmB were 19.2?mol·min-1·g-1,0.086 mol·L-1,0.072 mol·L-1,respectively.