Development of a new chemo-enzymatic catalytic route for synthesis of (S)- 2-chlorophenylglycine.

(S)-2-chlorophenylglycine ((S)-CPG) is a key chiral intermediate for the synthesis of clopidogrel. Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure (S)-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to (S)-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant EsLeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability. Coupled with a glucose dehydrogenase from Bacillus megaterium (BmGDH) for NADH regeneration, EsLeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C.

Comparative proteome analysis of Actinoplanes utahensis grown on various saccharides based on 2D-DIGE and MALDI-TOF/TOF-MS.

Comparative proteomes of Actinoplanes utahensis ZJB-03852 grown on various saccharides (glucose, maltotriose, maltose, glucose + maltose) were analyzed using 2D-DIGE and MALDI-TOF/TOF-MS. Acarbose was detected in all groups except in the glucose only culture. The abundance of acarbose synthesis proteins AcbV, AcbK, AcbL and AcbN was highest in the medium containing mixed glucose and maltose. The accumulation of Zwf and Xpk1 in acarbose-producing media indicated that the cyclitol moiety of acarbose was derived from pentose phosphate pathway. The elevation of GlnA supported that glutamine was a good nitrogen source of the nitrogen-atom in acarbose synthesis. SIGNIFICANCE: Non-insulin-dependent diabetes mellitus, also known as Type II diabetes, constitutes >90% of the diabetes mellitus worldwide. Acarbose is clinically utilized to treat Type II diabetes, but the fermentation process of acarbose-producing Actinoplanes is usually accompanied with structural analogues of acarbose. In this study, we compared the proteomics of Actinoplanes utahensis ZJB-03852 grown on various saccharides by 2D-DIGE and MALDI-TOF/TOF-MS. Our findings highlighted the importance of key proteins in the formation of acarbose and its analogues when A. utahensis was cultivated in various saccharides. These results revealed fundamental data to elucidate the complexity of formation of acarbose analogues.