Optimization Of Enzymatic Preparation Of D-pantolactone And The Cloning And Expression Of Enzyme Gene

In this paper,based on the limited ability of microbial enzymatic resolution of DL-pantolactone in industry,we hope to solve the problem of spontaneous hydrolysis of keto pantolactone substrate and the problem of enzyme production and catalysis of D-pantolactone hydrolase by biological means.Based on Saccharomyces cerevisiae and Fusarium moniliforme preserved by the research group,the substrate was prepared by optimizing the organic synthesis of raw material keto pantolactone,and the catalytic efficiency of ketopantoate lactone reductase of Saccharomyces cerevisiae was further improved by biphasic catalytic technology.Later,through the optimization of enzyme production conditions and immobilization process of Fusarium moniliforme,the optimal fermentation process and immobilization process parameters were determined.Finally,two recombinant expression plasmids were constructed by extracting the c DNA structure sequence of D-pantothenic acid lactone hydrolase,and transformed into E.coli and Pichia pastoris.It was verified by HPLC that the two recombinant host strains produced active enzyme protein after induction.The research content and main conclusions of this paper are as follows:1.After the improved organic synthesis method,the purity of the homemade substrate ketopantoic acid lactone is significantly improved,with an increase rate of10%.After rectification by distillation column,it can be used as a subsequent substrate for two-phase catalysis.Through the study of the types and proportion of biphasic substances in the biphasic catalytic technology,the proportion of 70%dibutyl phthalate+30%PBS buffer had the best catalytic effect.Compared with the normal PBS buffer,the yield of keto pantothenic acid lactone was increased by 45%,which effectively inhibited the hydrolysis of substrate and improved the enzymatic hydrolysis efficiency of reductase.2.The optimal fermentation conditions and immobilization parameters were as follows:initial p H5.5,inoculum amount 8%,temperature 28?,culture time 48h,biomass 16.05g/L,enzyme activity 426.78IU/L.sodium alginate concentration 12g/L,embedding rate 100g/L,Ca Cl₂concentration 40g/L,immobilization time 2h,and the relative enzyme activity and immobilization yield were the best.3.The structural gene encoding D-pantolactone hydrolase was amplified by gene extraction and RT-PCR.The structural gene encoding D-pantolactone hydrolase was inserted into E.coli expression vector p ET22b(+)and Pichia pastoris expression vector p PIC9K respectively,and the recombinant plasmids p ET22b(+)-fsp and p PIC9K-fsp were constructed.The recombinant vector p ET22b(+)-fsp was transformed into E.coli BL21(DE3),and a number of active enzyme protein was expressed under IPTG induction.The enzyme activity per wet cell was 1007IU/g.The recombinant vector p PIC9K-fsp was transformed into Pichia pastoris GS115,and a number of active enzyme protein was expressed under the induction of methanol supplement.The enzyme activity per wet cell reached 90.5IU/g and the enzyme activity of fermentation broth reached 3180IU/L after 120h cultivation by HPLC.