The ene reductase can reduce the carbon-carbon double bond of α, β-unsaturated alkenes and convert carvone into dihydrocarvone.In this study, an ene reductase gene KlebER2 was cloned from the genome of Klebsiella sp.O852, and was expressed heterologously in Escherichia coli.Besides, the expression conditions of KlebER2 were optimized, and enzymatic properties were explored after purification by nickel column.Finally, the catalytic performance of KlebER2 on dihydrocarvone production was investigated.Results indicated that KlebER2 protein was composed of 369 amino acids, and was a hydrophilic and non-transmembrane protein.The optimal expression condition was induced by 0.8 mmol/L IPTG for 22 h at 20 ℃ and 100 r/min when the concentration of OD600 was 0.5.Enzymatic property studies showed that the optimum reaction temperature and optimal reaction pH value were 30 ℃ and pH 6.0 respectively, and KlebER2 remained good stability below 30 ℃.In addition, KlebER2 was strongly activated by Mn2+, Mg2+, Ba2+, and glycerol, and had a catalytic effect on various unsaturated alkenes.KlebER2 was capable of converting carvone into dihydrocarvone, and the conversion rate was over 50%.These results can provide guidance for the heterologous expression of ene reductase and further industrial applications.
WANG Wanying
,
LI Zhijian
,
LI Zehao
,
WANG Zixuan
,
ZHANG Jiayue
,
WANG Guoguo
,
WANG Jingyue
,
DUAN Zhirou
,
ZHANG Lulu
. Heterologous expression and characterization of Klebsiella sp.ene reductase gene[J]. Food and Fermentation Industries, 2025
, 51(14)
: 65
-73
.
DOI: 10.13995/j.cnki.11-1802/ts.040927
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