Biocatalytic properties of a recombinant Fusarium proliferatum lactonase with significantly enhanced production by optimal expression in Escherichia coli.
The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC3.1.1.25) 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-beta-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.
Chen
Bing
B
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Fan
Li-Qiang
LQ
Xu
Jian-He
JH
Zhao
Jian
J
Zhang
Xian
X
Ouyang
Li-Ming
LM
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D011994:Recombinant Proteins; C005412:pantolactone; D002265:Carboxylic Ester Hydrolases; D015107:4-Butyrolactone