Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the l-amino acid deaminase (PmiLAAD)

Licheng Wu; Xiaolei Guo; Gaobing Wu; Pengfu Liu; Ziduo Liu

doi:10.1016/j.enzmictec.2019.109393

Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the l-amino acid deaminase (PmiLAAD)

Enzyme Microb Technol. 2020 Jan:132:109393. doi: 10.1016/j.enzmictec.2019.109393. Epub 2019 Aug 8.

Authors

Licheng Wu¹, Xiaolei Guo², Gaobing Wu³, Pengfu Liu⁴, Ziduo Liu⁵

Affiliations

¹ Department of R&D of Zhejiang zhengshuo Biological Co., Ltd, Huzhou 313000, Zhejiang, China; College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
² College of life science, Fujian normal university, Fujian 350000, China.
³ College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
⁴ Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China.
⁵ College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: ziduoliu_hzauedu@sina.cn.

PMID: 31731950
DOI: 10.1016/j.enzmictec.2019.109393

Abstract

In our previous study, we produced α-keto acids by using an L-amino acid deaminase PmiLAAD (wide-type) from Proteus mirabilis, however, the catalytic efficiency was low due to its low substrate affinity. In this study, protein engineering of PmiLAAD was performed to improve the α-keto acid production. PmiLAAD was engineered by iterative CASTing to improve its catalytic performance. The four mutant PmiLAAD-SAVS (PmiLAAD-Phe93Ser-Pro186Ala- Met394Val-Phe184Ser) with 6.6 -fold higher specific activity compared with that of wild-type PmiLAAD has been obtained by high-throughput screening. Comparative kinetics analysis showed that the four mutant PmiLAAD-SAVS had a higher substrate-binding affinity and catalytic efficiency than that of PmiLAAD wild-type. The K_m, k_cat, and k_cat/K_m values of the PmiLAAD(SAVS) variant was better (-42.7%, 75.11%, and 85.79%, respectively) than the corresponding values of PmiLAAD wild type. Finally, the whole cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS) has been applied to α-keto acids production. The conversion rate of L-phenylalanine reached 99% by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS). The conversion of (D/L)-4-phenylalanine was reached 49.5% after 7 h by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS), while the conversion of E. coli-pETDuet-1-PmiLAAD (wild type) was only 18% after an extension of the reaction time (24 h). This study has developed a robust whole-cell E. coli biocatalyst for α-keto acids production by protein engineering, and this strategy may be useful for the construction of other biotransformation biocatalysts.

Keywords: Biocatalysis; Protein engineering; Site-saturation mutagenesis; l-Amino acid deaminase.

MeSH terms

Amino Acids / metabolism*
Aminohydrolases / metabolism*
Biocatalysis
Biotransformation
Keto Acids / metabolism*
Protein Engineering / methods*
Proteus mirabilis / enzymology

Substances

Amino Acids
Keto Acids
Aminohydrolases