Abstract:

The aim of this study is to conduct the bio-enzymatic synthesis of β-hydroxy-α-amino acids,the important pharmaceutical intermediates,and to explore and identify new L-threonine aldolases. Using pET-28a（+）as an expression vector,protein expression purification,thin layer chromatography（TLC）,and high-performance liquid chromatography（HPLC）were performed to analyze the properties of L-threonine aldolases and their catalytic products. A new activity detection method of L-threonine aldolases was developed based on 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole（Purpald）color reagent. The results demonstrated that the aldolases from Streptomyces coelicolor SCO1844 and Streptomyces xinghaiensis SFR7A were both L-threonine aldolases and could be used to synthesize β-hydroxy-α-amino acids. The aldol condensation reaction of benzaldehyde and glycine was achieved using these two L-threonine aldolases,producing L-threo/erythron-phenylserine. The developed visual activity detection method allows rapid identification and high-throughput screening of aldolases to be feasible. The identification of two new L-threonine aldolases and the development of activity detection methods not only enrich the biosynthetic enzyme library for β-hydroxy-α-amino acids,but also provide the research basis for molecular modification to improve its catalytic activity and selectivity in the future.

Key words: L-threonine aldolase, β-hydroxy-α-amino acid, biosynthesis, activity detection, streptomyces