Fermentation Optimization And Cell Immobization Of Recombinant Esterase From Microbacterium Sp.SIT101

In the previous work,the microbiological strain SIT101 was screened,which can produce the esterase,and then asymmetrically catalyze the production of biotin intermediate dimethyl ester(4S.5R)-monomethyl ester with >95% ee values and >99% yield.It is an important intermediate in the total synthesis of D-biotin,which has great industrial application value.At present,our team has completed the construction of the genetically engineered strain of Escherichia coli and completed the fermentation optimization(shake flask level)of the recombinant strain induced by the inducer IPTG.This project has continue to carry out more systematically and deeply on the fermentation process of this bacterium,and explored a low-cost and suitable route for esterase production from recombinant esterase E.coli BL21(DE3)-p ET-21a-estsit01.At the same time,the immobilization of E.coli recombinant esterase whole cells was also studied.In the first part of the dissertation,batch fermentation optimization experiment of the 5L fermentor was performed on the recombinant E.coli.The influence of the four factors was studied in detail including the concentration of the inducer,the initial induction point,the induction temperature and the p H value.The optimum enzyme activity of 3886.0 U/L was obtained at 12 h.At the same time,the specific enzyme activity was 383.9 U/g and the cell OD600 was 26.29.In the second part of the thesis,a single-factor method was used to optimize the self-inducible expression conditions of the recombinant esterase,including the three aspects of the culture medium components,culture conditions and induction conditions.After fermentation optimization,the cell concentration(3.7 g/L)and enzyme activity(1654.7 U/L)from the induction medium were both increased by about 1.5 times comparing to the initial medium with IPTG induction(the cell concentration was 3.7 g/L and enzyme activity was1072.7 U/L).The cost of the AR-grade reagent used to produce 1KU of recombinant esterase is about 3.8 yuan.Compared with the previous group using the inducer IPTG culture,the cost of using AR grade reagents has been reduced 35%.In the third part of the thesis,the immobilization technology of E.coli whole cells was studied.Several common immobilization methods were carried out.The cross-linking agent glutaraldehyde was selected to immobilize the cells,which can retain 61% of the initial enzyme activity.The optimal preparation conditions of the immobilized cells were obtained.The optimal preparation conditions for the final immobilized cells are 5% final concentration of glutaraldehyde,90 minutes of the cross-linking time,and 7 of p H value of the cross-linking PB buffer solution.The results about the enzymatic properties of immobilized cells and free cells were found that the immobilized cells showed superior stability compared to free cell at p H 6-10 and in 30 ?-60 ?.The immobilized cells still had a 100% conversion rate after repeated use for 16 times,and still had 40% residual enzyme activity after 30 days' storage at4 ?.