RRC ID 34494
著者 Hossain GS, Li J, Shin HD, Du G, Wang M, Liu L, Chen J.
タイトル One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.
ジャーナル PLoS One
Abstract α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.
巻・号 9(12)
ページ e114291
公開日 2014-1-1
DOI 10.1371/journal.pone.0114291
PII PONE-D-14-29015
PMID 25531756
PMC PMC4273966
MeSH Biocatalysis* Biotransformation Cloning, Molecular Deamination Directed Molecular Evolution Enzymes, Immobilized / chemistry Enzymes, Immobilized / genetics Enzymes, Immobilized / metabolism Escherichia coli / cytology* Escherichia coli / genetics Escherichia coli / metabolism Gene Expression Lyases / chemistry Lyases / genetics* Lyases / metabolism Methionine / analogs & derivatives* Methionine / metabolism* Mutagenesis, Site-Directed Protein Engineering* Protein Transport Proteus vulgaris / enzymology* Proteus vulgaris / genetics Substrate Specificity
IF 2.74
引用数 22
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
一般微生物 JCM 20339