期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 137, 期 -, 页码 -出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2020.109551
关键词
Threonine aldolase; beta-Hydroxy-alpha-amino acid; Random mutagenesis; Stereoselectivity; Enzymatic synthesis
资金
- National Natural Science Foundation of China [21602246]
- Tianjin Municipal Science and Technology Commission [15PTCYSY00020]
L-Threonine aldolases (L-TAs) catalyze the aldol condensation of aldehyde and glycine, offering direct enzymatic synthesis of beta-hydroxy-alpha-amino acids under mild conditions. However, this method suffers from moderate yield and low stereoselectivity at the beta-carbon. Given the importance of 4-(methylsulfonyl)phenylserine for the synthesis of florfenicol and thiamphenicol, mutations of a L-threonine aldolase from Pseudomonas sp. (L-PsTA) were performed in this study by error-prone PCR and combinatorial mutation. Some beneficial mutants were obtained by screening the mutant library using a stepwise visual method. 4-(Methylsulfonyl)phenylserine was synthesized in up to 71 % diastereomeric excess (de), which are much higher than the previously reported 2 % de value, by using the newly identified mutants. The mutants V200I and C187S/V200I were found to improve the product yield and stereoselectivity for the aldol condensation of various benzaldehydes with glycine. These results show that the amino acid residues outside of the substrate-binding cavity of L-PsTA play an important role in determining its C-beta-stereoseletivity.
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