Candida Antarctic Lipase Displaying on Pichia Pastoris Catalytic Reaction System and Application

Author:Zhang Zuo

Supervisor:lin ying


Degree Year:2018





Yeast cell surface displaying technology also known as ‘Arming yeast’,it combines the expression,purification and immobilization of heterologous protein or polypeptide,making the recombinant strain directly used as whole cell biocatalyst,therefore saving cost.Using yeast cell-surface displayed enzymes to produce chemicals is a promising green chemistry procedure.Pichia pastoris cell surface display Candida antarctica lipase B(Pp-CALB)and Candida antarctica lipase A as whole cell biocatalyst catalyze reactions in non-aqueous phase were studied in this article base on cell surface display system,further modification the whole cell biocatalyst were researched to expand its application range.In order to provide theoretical and technical basis for the yeast cell surface displaying as whole cell biocatalyst application in the field of green biological production.(1)Pp-CALB catalyzes complex mixtures as substrate esterification system and enzymatic process enhances the flavour profile of citrous essential oils13 kinds of citrus essential oil(CEO)were selected as substrate and solvent to studied Pp-CALB catalyze modification of CEO.By GC-MS,the change of citrus essential oil composition was determined.The proportion of esters in citrus essential oils increase after enzyme modification,from 10% to 1170%,the system can provide citrus essential oil with novel flavour profile.The bacteriostatic properties were also slightly enhanced.The carboxylic acid in citrus essential oils is an acyl donor for the addition of ethyl ester in essential oils.(2)Pp-CALB catalyze transesterification system and synthesis of terpene esterPp-CALB catalyze transesterification system could avoid the byproduct water of the esterification system,thus solving the problem of the adsorption and coagulation of the Pp-CALB,and improving the reutilization rate of the enzyme.Pp-CALB was applied to catalyze transesterification synthesis of benzyl acetate in solvent free system and optimize the reaction condition.The molar conversion of benzyl alcohols reached 97.2% and Pp-CALB remained the original vitality of 82.3% after repeated reaction 5 times.The ability of Pp-CALB catalyze resolution of structural isomer geraniol and nerol was also studied.(3)The enantioselectivity characteristics of Pp-CALB catalyze transesterification kinetic resolution of sec-alcohosBase on the Pp-CALB catalyze transesterification system,Pp-CALB was applied to catalyze kinetic resolution of sec-alcohols in solvent system.To study the influence of CALB displaying on pichia pastoris cell surface on CALB enantioselectivity characteristics.Pp-CALB catalyzed kinetic resolution of all test sec-alcohols with enantioselectivity value more than 600 and had preferences to short straight-chain secondary alcohol.By comparison with the commodity enzyme Novozyme 435,CALB was found that displaying on the surface of pichia pastoris did not change its stereoselective properties.The kinetic resolution of Pp-CALB catalyzed secondary alcohol was evaluated in solvent-free and high concentration substrate system,and the solvent-free system had higher time and space efficiency than the organic solvent system.(4)Linkers as spacer to increase the catalytic performance of Pp-CALBThe catalytic performance of Pp-CALB needs to be further improved.The catalytic activity was enhanced by insertion of a link peptide between the anchoring protein and the display protein CALB.The hydrolysis activity of recombinant with insreting of PAPAP linked peptide increased by 114.8%,and the non-water phase esterification activity and transesterification activity increased by 6.58 times and 1.34 times respectively.Flow cytometry and lipase active-site titration method were used to analysis the recombinant strains and the result shown that inserting linker can improve the amount of CALB displayed on cell surface.(5)Constriction and Non-aqueous catalytic characteristic of Pichia pastoris cell surface display Candida antarctica lipase ACALA different from CALB has a high activity toward steric hindrance secondary and tertiary alcohols.First,pichia pastoris cell surface display CALA was successfully constructed and its hydrolysis activity reached 6331.6 U/g,this is much higher than the reported 80.4 U/g.Pichia pastoris cell surface display CALA selectivity of the substrate alcohol in the non-aqueous phase system was discussed.Its lyophilized powder was applied to synthesis of neryl butyrate and kinetic resolution of DL-menthol.In addition,the stereoselectivity of CALA for DL-menthol was improved by the point mutation.The eep of CALAL367 I mutant protein increased from 63% to 72.9%.