Molecular Modification And Application Of Candida Antarctica Lipase B

Candida antarctica lipase B(CALB)showing a high catalytic activity both in aqueous and non-aqueous media,is a highly versatile biocatalyst.It can be used to catalyze hydrolysis,esterification,polymerization and transesterification and so on.Because its advantages in high activity,generally high regio-and enantioselectivity,environment friendly,mild reaction conditions and broad substrate spectrum,CALB has drawn more and more attentions.It is reported that the CALB has been used successfully both in resolution of numerous key intermediates of drugs,the synthesis of esters and biodiesel oil,and oil modification.Studies on CALB have been widely carried out in terms of their structure,cloning and expression,immobilization,and molecular modification.Recently,directed evolution of CALB and exploring their new applications are becoming hot issues.In this study,CALB was modified by rational design.Due to the three-dimensional structure of CALB,we selected five sites(D134?1189?V190?V221?A281)which play the significant roles in the structure.Then five pairs of primers were designed for mutations.The recombinant vector pPICZ?A-CALB was used as the template and the plasmid amplification method was using for single-point or complex point saturation mutagenesis.Approximately,about 4500 bp mutant fragment of the restructuring plasmid library was accessed and expressed in P.pastoris to rebuild the recombinant CALB mutants(P.pastoris X-33/pPICZaA-CALBmut).After screening by p-NPA,a number of mutants with increased activities were obtained.The purification and biochemical properties of D-4(A281G/V190L)were determined using the p-nitrophenyl acetate(p-NPA)as substrate,which showed the highest hydrolysis activity.The results showed that the optimum pH and temperature were 7.0 and 35?,respectively.D-4 was highly active between pH 5.0-10.0,but it was sensitive to heat.The enzyme was activated slightly by part of metal ions and completely inhibited by Fe2+.The enzyme activities were inhibited in the presence of SDS,Tween-20,Tween-80 and Sorbitol,whereas,TritonX-100 had a slightly increasing effect on enzyme activity.Four mutants(V190L,281-1-1,281-11-2,D-4)and the original strain(WT)were selected for the study of their performance in different reactions.Substrate specificity of mutants and WT were determined using p-nitrophenol esters(p-NPs)of varying chain length.Compared to WT,D-4 had short and mid chain specificity,whereas 281-1-1 was long chain specific.Further D-4 had approximately 41-and 60-fold higher activities as compared to WT on p-NPA and p-NPB,respectively.In the esterification of vitamin A palmitate,only V190L showed 145%activity.There is no difference of esterification activity between D-4 and WT.The resolution of racemic 3-cyano-5-methylhexanoic acid esters was carried out by mutants and WT as well.The results indicated that activities of all mutants have improved.The enantioselectivity of all substrates except 3-cyano-5-methylhexanoic acid methyl ester have been swifted.WT has no selectivity towards to 3-cyano-5-methylhexanoic acid n-amyl ester,wheras D-4 obtained selectivity and E value reached 12.8.