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

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 Km, kcat, and kcat/Km 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.

journal_name

Enzyme Microb Technol

journal_title

Enzyme and microbial technology

authors

Wu L,Guo X,Wu G,Liu P,Liu Z

doi

10.1016/j.enzmictec.2019.109393

subject

Has Abstract

pub_date

2020-01-01 00:00:00

pages

109393

eissn

0141-0229

issn

1879-0909

pii

S0141-0229(19)30131-0

journal_volume

132

pub_type

杂志文章

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