Enzyme immobilization in MOFs offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. In this work, Pseudomonas cepacia lipase (PCL) was successfully immobilized into the Zeolitic imidazolate framework-8 (ZIF-8) by physical adsorption. The amounts of PCL in the immobilized enzyme (PCL@ZIF-8) was determined to be 16.7 % (200.5 mg PCL/g ZIF-8). Furthermore, the immobilized enzyme was applied as efficient bio-catalyst for enantioselective hydrolysis of 2-phenylpropionic acid (2-PPA) ester enantiomers and enantioselective transesterification of 1-phenylethanol enantiomers. The enzymatic activity of the immobilized enzyme was three times more than that of free PCL in enantioselective hydrolysis system, and the enantiomeric excess was maintained above 99 %. There was no significant difference in enzyme activity between immobilized PCL and free PCL in transesterification system. In addition, the immobilized enzyme showed good reusability in both hydrolysis (30.57 % of initial activity, 4 cycle) and transesterification reaction systems (64.38 % of initial activity, 6 cycle).
A novel carboxylesterase Ac Est1 was identified from Acinetobacter sp. JNU9335 with high efficiency in the biosynthesis of chiral precursor of Edoxaban through kinetic resolution of methyl 3-cyclohexene-1-carboxylate (CHCM). Sequence analysis revealed Ac Est1 belongs to family IV of esterolytic enzymes and exhibits <40% identities with known carboxylesterases. The optimum pH and temperature of recombinant Ac Est1 are 8.0 and 40 °C. Substrate spectrum analysis indicated that Ac Est1 prefers substrates with short acyl and alcohol groups. Ac Est1 was highly active in the hydrolysis of CHCM with k cat of 1153 s −1 and displayed high substrate tolerance. As much as 2.0 M (280 g·L −1 ) CHCM could be enantioselectively hydrolyzed into ( S )-CHCM by merely 0.08 g·L −1 Ac Est1 with ee s of >99% ( S ) and substrate to catalyst ratio (S/C) of 3500 g·g −1 . These results indicate that the novel Ac Est1 is a promising biocatalyst in the synthesis of chiral carboxylic acids.
An efficient chemoenzymatic route has been developed for the synthesis of optically active pregabalin (PGB) from isobutylsuccinonitrile (IBSN). (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), a critical chiral intermediate of PGB, was synthesized using regio- and enantioselective hydrolysis of IBSN by immobilized Escherichia coli cells harboring nitrilase BrNIT from Brassica rapa. The catalytic performances of immobilized cells were investigated, and high enantioselectivity (E > 150) and substrate conversion (>41.1%) were obtained at a substrate loading of 100 g/L by immobilized cells after 12 batches of reaction. The unreacted (R)-IBSN was recycled by racemization with a high yield of 94.5%, and the resultant (S)-CMHA was hydrogenated directly to the desired PGB with a high purity of 99.6% and optical purity of 99.4%. The input of raw materials and E factor of this chemoenzymatic route were demonstrated to be much lower than those of the first- and second-generation routes for PGB synthesis.
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