Biocatalytic Properties of a Recombinant Fusarium proliferatum Lactonase with Significantly Enhanced Production by Optimal Expression in Escherichia coli
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The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC220.127.116.11) was cloned and expressed in Escherichia coli JM109 (DE3) for biocatalytic resolution of industrially important chiral lactones, including DL-pantoyl lactone which was a key precursor to calcium d-pantothenate. By increasing the biomass concentration and lowering the inducer (isopropyl-β-d-thiogalactoside) concentration and induction temperature, the lactonase production was significantly enhanced up to 20 kU/L, which was 20 times higher than that of wild-type strain F. proliferatum ECU2002. The recombinant Fusarium lactonase was purified using immobilized metal affinity chromatography, and its SDS-PAGE revealed a molecular mass of 50 kDa for the recombinant protein, suggesting that the enzyme was a simplex protein. Furthermore, biocatalytic properties of the recombinant lactonase were investigated, including kinetic parameters, additive’s effect, and substrate specificity. The results reported in this paper provide a feasible method to make the whole cells of E. coli JM109 (DE3) expressing lactonase gene to be a highly efficient and easy-to-make biocatalyst for asymmetric synthesis of chiral compounds.
KeywordsLevo-lactonase DL-pantoyl lactone Recombinant protein Immobilized metal affinity chromatography Biocatalytic property Substrate specificity
This research was financially supported by National Natural Science Foundation of China (grant No. 20506037 & 20773038), Ministry of Science and Technology (grant No. 2007AA02Z225 & 2009CB724706), and China National Special Fund for State Key Laboratory of Bioreactor Engineering (grant No. 2060204).