Facile immobilization of his-tagged Microbacterial esterase on Ni-SBA-15 with enhanced stability for efficient synthesis of key chiral intermediate of d-biotin.

A series of nickel-incorporated SBA-15 mesoporous molecular sieves (Ni-SBA-15) were prepared as support for the immobilization of his-tagged recombinant Microbacterium esterase. The Ni-SBA-15 could strongly and specific absorb the his-tagged esterase from cell disrupted supernatant. It was found that the nickel amount in Ni-SBA-15 has dramatic influence on the activity and thermo-stability of immobilized enzyme, while the kinds of nickel precursor had little effect on enzyme stability. The morphology, chemical composition and structure of the best support NiCl2-SBA-15 (Ni-SBA-15 prepared from NiCl2 precursor) were characterized by various spectroscopy techniques. The immobilized esterase retained full activity of free esterase and showed high immobilized yield (> 90%) with higher thermo-stability, pH stability and organic solvent resistance compared with free enzyme. The optimum reaction temperature increased from 35 to 40 °C and the optimal reaction pH moved from 10.0 to 8.0 after enzyme immobilization. The immobilized esterase exhibited excellent storage stability and keeping 92% of the initial activity after 30 days' storage at 25 °C. In addition, the immobilized esterase had excellent reusability for the synthesis of key chiral intermediate of d-biotin and the substrate conversion could still keep 100% after 13 cycles continuously. Finally, optical pure (4S, 5R)-hemiester was obtained in 80.8% isolated yield and 99% purity in the gram preparative scale.