Biosynthesis Of α-keto-β-methylvaleric Acid From L-isoleucine By Whole-cell Biocatalyst Expressing L-amino Acid Deaminase

α-Keto-β-methylvaleric acid（KMV） is an important intermediate in several synthesis area. It is widely used and researched in the fields of medicine and chemistry. Compared with the chemical synthesis, biotransformaton is a potential alternative method based on the high selectivity and high efficiency of enzyme, and it is also environmentally friendly. Thus, biotransformaton can be beneficial to the industrial-scale production of α-keto acid. Wholecell biocatalyst is not only conducive to the membrane enzyme stability, but also to the isolation and purification of products. We expressed L-amino aicd deaminase gene laad, pma, pm1 and aad in Escherichia coli（DE3） respectively, and compared their KMV production by whole-cell biocatalyst. Then we choosed the Escherichia coli whole-cell biocatalyst which had the highest catalytic activity on L-Ile, and optimized the preparation conditions of the whole-cell biocatalyst and the reaction conditions of whole-cell bioconversion. After that, we made molecular modification on the L-amino acid deaminase gene. At last, the KMV production was improved. The main contents of my reseach are as below:（1） We successfully constructed four kinds of recombinant Escherichia coli named E. coli BL21（pET-20b-laad）, E. coli BL21（pET-20b-pma）, E. coli BL21（pET-20b-pm1） and E. coli BL21（pET-20b-aad）. They contained the L-amino acid deaminase gene laad, pma, pm1 and aad respectively. Among them, laad comes from Proteus myxofaciens, pma and pm1 come from Proteus mirabilis, and aad comes from Proteus vulgaris. Then we compared the KMV production of the four kinds of recombinant Escherichia coli whole-cell biocatalyst. At last, we found the Escherichia coli whole-cell biocatalyst expressing pma has the highest catalytic activity on L-Ile. So we made study on it.（2） We optimized the preparation conditions of the E. coli BL21（pET-20b-pma） wholecell biocatalyst in shake flasks. The optimization results were as following: The optimal seed broth age was 10 h. The most proper final concentration of IPTG was about 0.2 mmol×L^-1. The most proper induction temperature was 37℃. The optimum expression cell age was at OD600 1.0 or so. And the most proper induction time was about 4.5 h. Under the culture conditions described above, the whole-cell biocatalyst reaction rate could get 9.41 g×L^-1×h-1.（3） We optimized the reaction conditions of E. coli BL21（pET-20b-pma） whole-cell biocatalyst. The optimization results were as following: The optimal pH was 8.5. The most proper reaction temperature was 30℃. The optimal biocatalyst concentration（dry cells weight, DCW） was 1 g×L^-1. The optimal substrate concentration was 800 mmol×L^-1. And the optimal conversion time was about 22 h²4 h. Under the reaction conditions described above, the KMV production could get 94 g×L^-1, and the substrate conversion rate was 90%.（4） We used error-prone PCR method to make random mutation on the L-amino acid deaminase gene pma. Then we constructed mutant librarys and made high throughput screening on them. At last, two mutant strains named 5/13-26 and 7/23-6 were got. Under 900 mmol×L^-1 L-Ile, the KMV production of the wild strain whole-cell biocatalyst was only 90 g×L^-1, and its substrate conversion rate was 77%. By contrast, the KMV production of mutant 5/13-26 and 7/23-6 could get 97 g×L^-1 and 102 g×L^-1 respectively, 8% and 13% higher than the wild strain respectively. And their substrate conversion rate were 83% and 87% respectively.（5） According to the L-amino acid deaminase mutation sites of mutant strains 5/13-26 and 7/23-6, we made saturation mutagenesis at 209 th glutamic of the mutant 7/23-6 L-amino acid deaminase, and screened for good strains. At last, we got the mutant E209 Q. Under 900 mmol×L^-1 L-Ile, the KMV production of mutant E209 Q whole-cell biocatalyst was 104 g×L^-1, with 89% substrate conversion rate.