| Pyruvate,an important intermediate in pharmaceutical synthesis,is widely used in the pharmaceutical and chemical industries.At present,the pyruvic acid synthesis in the industry mainly includes direct microbial fermentation and chemical synthesis.The chemical synthesis method has serious environmental pollution,and the cost is relatively high.However,in the direct microbial fermentation method,the conversion rate of the substrate to pyruvic acid is not high,and the product components are complex and difficult to separate.The synthesis of pyruvic acid by biocatalysis can overcome the deficiencies and shortcomings of the above two methods.Therefore,it has broad prospects in industrial applications.In this study,pyruvate was catalyzed by a recombinant strain expressing L-amino acid deaminase（pm1）derived from Proteus mirabilis.Then,several aspects such as expression of cytochrome b562（cybC）,modification of pm1 and the optimization of conditions in shake flasks and fermentor were carried out to improve the efficiency of pyruvate production by whole-cell biocatalyst.The main content of the paper are as follows:（1）The pm1 derived from Proteus mirabilis was heterologously expressed in E.coli BL21（DE3）.The fermentation conditions and catalytic conditions were optimized.The induction temperature was 25°C,the induction time was 14 h,and the IPTG concentration was 0.04 m M.The cell concentration during the catalytic process was determined to be 1.5g·L-1,the substrate concentration was 80 g·L-11 D/L-alanine,the pH of the reaction solution was 7.0,and the yield of pyruvic acid was 17.47 g·L-1;（2）The recombinant E.coli strain expressed cybC and pm1 was constructed,and the fermentation conditions of the recombinant strain were optimized.The results showed that the expression of cybC did not affect the optimum induction temperature of pm1,but it could increase the rate of pyruvate catalyzed by the recombinant strain,and the reaction time decreased from 27 h to 21 h;（3）A homologous model of pma from Proteus mirabilis was obtained with a homology of 93.74%by modeling on the website of SWISS MODEL.By analyzing the crystal structure of the model,amino acids at key positions such as Gln100,Phe318,Arg316,and Trp439 were verified,and four amino acids such as Leu278,Glu418,Var438,and Glu341 were selected for mutating.After primary screening and rescreening,three mutants with improving catalytic capacity were obtained,which were L278I,E418A,and V438I,respectively.The yields of pyruvic acid were 20.18,22.57,and 23.79 g·L-1,respectively.In addition,the yield of pyruvic acid in the mutant E418A/V438I/L278I obtained by the combined mutation was 25.58 g·L-1,which increased by about 44.6%compared with the control strain;（4）The fermentation conditions and catalytic conditions were optimized in a 3 L NBS fermentor.The optimal conditions for fermentation process in the fermentor were as follows:IPTG was added for inducing during the end of logarithmic phase,the induction time was about 10 h,the stirring speed during the induction process was 250 rpm,and the ventilation volume was 1.5 L·min-1.Under this condition,the ability of per unit mass of cells to catalyze pyruvate was the same as that in shake flasks.The optimum conditions for the catalytic process in the fermentor were as follows:the substrate was added in three batches,and the yield was increased by about 59%compared to one batch,which was 14.74 g·L-1;the three-phase control of ventilation volume during the catalytic process was better,and the final yield was 16.25 g·L-1. |
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