Utilization of one novel deep-sea microbial protease sin3406-1 in the preparation of ethyl (S)-3-hydroxybutyrate through kinetic resolution

Abstract

One novel protease sin3406-1 was identified from Streptomyces niveus SCSIO 3406, which was isolated from the deep sea of the South China Sea, and heterologously expressed in E. coli BL21(DE3). Protease sin3406-1 was further used as a green biocatalyst in the kinetic resolution of racemic ethyl-3-hydroxybutyrate. After careful process optimization, chiral product ethyl (S)-3-hydroxybutyrate was generated with an enantiomeric excess of over 99% and a conversion rate of up to 50% through direct hydrolysis of inexpensive racemic ethyl-3-hydroxybutyrate catalyzed by sin3406-1. Interestingly, protease sin3406-1 exhibited the same enantio-preference as that of esterase PHE21 during the asymmetric hydrolysis of the ester bonds of racemic ethyl-3-hydroxybutyrate. Through mutation studies and molecular docking, we also demonstrated that the four residues close to the catalytic center, S85, A86, Q87 and Y254, played key roles in both the hydrolytic activity and the enantioselectivity of protease sin3406-1, possibly through forming hydrogen bonds between the enzyme and the substrates. Deep-sea microbial proteases represented by sin3406-1 are new contributions to the biocatalyst library for the preparation of valuable chiral drug intermediates and chemicals through enzymatic kinetic resolution.

Actinobacteria as Microbial Cell Factories and Biocatalysts in The Synthesis of Chiral Intermediates and Bioactive Molecules; Insights and Applications

“…[7a] Recently, Huang et al isolated a novel protease (protease sin3406-1) from S. niveus SCSIO 3406. [73] The crude enzyme powder was obtained, after being expressed in E. coli BL21(DE3) cells and was directly used as a biocatalyst for the kinetic resolution of racemic ethyl-3-hydroxybutyrate. The latter was converted to the chiral ethyl S-3-hydroxybutyrate at a rate of 50 % with an excellent enantiomeric excess exceeding 99 % (Scheme S).…”

“…Markedly, ethyl S-3-hydroxybutyrate is a building block for various drugs, such as carbapenem antibiotics. [73][74] Another study presented the kinetic resolution of a putative epoxide hydrolase mined from S. griseus NBRC 13350. [7b] The putative epoxide hydrolase gene (sgeh) was cloned and overexpressed in E. coli BL21 (DE3).…”

“…Recently, Huang et al . isolated a novel protease (protease sin3406‐1) from S. niveus SCSIO 3406 [73] . The crude enzyme powder was obtained, after being expressed in E. coli BL21(DE3) cells and was directly used as a biocatalyst for the kinetic resolution of racemic ethyl‐3‐hydroxybutyrate.…”

“…The latter was converted to the chiral ethyl S ‐3‐hydroxybutyrate at a rate of 50 % with an excellent enantiomeric excess exceeding 99 % (Scheme S ). Markedly, ethyl S ‐3‐hydroxybutyrate is a building block for various drugs, such as carbapenem antibiotics [73–74] …”