Structure-based semi-rational engineering approach was applied to alter the binding pocket and substrate channel for enhancing the activity of CALB towards moxifloxacin chiral intermediate.
Candida antarctica lipase B (CALB) is a robust
biocatalyst for production of chemicals. In this study, an engineered
lipase B (CALB-I189K) was covalently immobilized onto a glutaraldehyde
activated amino resin (ESR-3) and used for the preparation of chiral
moxifloxacin precursor though enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate (cis-(±)-1). The immobilization conditions
were optimized, and the immobilized CALB-I189K exhibited improved
thermal stability, with a half-life of 247.5 h at 30 °C. It enables
the resolution of 100 g L–1
cis-(±)-1 for 50 cycles in a stirred tank reactor,
with an average productivity of 49.6 g L–1 h–1. The resolution reaction was also performed in a
recirculating packed bed reactor (RPBR) in a semicontinuous mode,
and the average productivity reached 59.4 g L–1 h–1 under the optimal operating condition. Moreover,
the RPBR bioconversion system has high operational stability and conserved
over 85.7% of the initial conversion capacity after 50 cycles. This
efficient bioconversion process demonstrated the potential of employing
immobilized CALB-I189K for industrial production of chiral moxifloxacin
precursor.
The Candida antarctica lipase B (CALB) was embedded in the metal-organic framework, zeolitic imidazolate framework-8 (ZIF-8), and applied in the enzymatic synthesis of L-ascorbic acid palmitate (ASP) for the first time. The obtained CALB@ZIF-8 achieved the enzyme loading of 80 mg g À1 with 11.3 U g À1 (dry weight) unit activity, 59.8% activity recovery, and 92.7% immobilization yield.Under the optimal condition, ASP was synthesized with over 75.9% conversion of L-ascorbic acid in a 10-batch reaction. Continuous synthesis of ASP was subsequently performed in a packed bed bioreactor with an outstanding average spacetime yield of 58.1 g L À1 h À1 , which was higher than ever reported continuous ASP biosynthesis reactions.
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