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(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 Es LeuDH-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 (Bm GDH) for NADH regeneration, Es LeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C. • A new chemo-enzymatic catalytic route for (S)− 2-chlorophenylglycine was developed. • Es LeuDH-F362L exhibited enhanced catalytic activity but decreased thermal stability. • Es LeuDH-F362L completely converted up to 0.5 M (2-chlorophenyl)glyoxylic acid in 8 h. [ABSTRACT FROM AUTHOR]

• A lipase-copper hybrid catalyst was developed via in-situ assembly strategy. • Copper nucleotide composite ensured uniformed distribution of copper nanoparticles. • Hybrid catalyst featured high enzymatic and copper catalytic activity. • Hybrid catalyst enabled highly efficient production of p -AP in cascade reaction. The integration of chemical catalyst and biocatalyst is of great interest due to the accompanying high designability of chemo-enzymatic cascade reactions for sustainable catalytic processes in chemical engineering. Here, a facile strategy is proposed by preparation of a copper ions coordinated cytidine monophosphate (CMP) encapsulated lipase composite via in-situ assembly in aqueous solution which followed by reduction to obtain an enzyme-copper hybrid catalyst. The as-synthesized enzyme-copper nucleotide composite exhibited both high enzymatic activity (6.05 U/mg) and copper-catalytic activity (with reaction rate constant of 8.85 min−1) in the chemo-enzymatic cascade reaction for hydrolysis of p -nitrophenyl butyrate (p -NPB) and generation of p -aminophenol (p -AP), which is a pharmaceutical intermediate for production of analgesics and antipyretics. This study thus sheds light on fabrication of hybrid catalyst under facile conditions, exhibiting potential in development of biosensors, industrial catalysis and biomanufacturing. [ABSTRACT FROM AUTHOR]