Gene Cloning, Prokaryotic Expression, and Biochemical Characterization of a Soluble Trehalase in Helicoverpa armigera Hubner (Lepidoptera: Noctuidae)

文献类型： 外文期刊

第一作者： Cheng, Shenhang

作者： Cheng, Shenhang;Yu, Caihong;Chang, Hetan;Yang, Ting;Wang, Guirong

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关键词： castanospermine; Helicoverpa armigera; inhibitor; prokaryotic expression; soluble trehalase

期刊名称：JOURNAL OF INSECT SCIENCE （ 影响因子：1.857； 五年影响因子：1.904 ）

ISSN： 1536-2442

年卷期： 2018 年 18 卷 3 期

页码：

收录情况： SCI

摘要： Trehalase is an indispensable component of insect hemolymph that plays important role in energy metabolism and stress resistance. In this study, we cloned and expressed the gene encoding soluble trehalase (HaTreh-1) of Helicoverpa armigera (cotton bollworm) and characterized the enzyme. HaTreh-1 had a full-length open reading frame encoding a protein of 571 amino acids. Sequence comparison indicated that HaTreh-1 was similar to some known insect trehalases. Two essential active sites (D321 and E519) and three essential residues (R168, R221, and R286) were conserved in HaTreh-1. The recombinant trehalase was expressed in Escherichia coli and purified by nickel exchange chromatography. Molecular weight of the recombinant protein was about 71 kDa, and the optimum HaTreh-1 enzyme activity is at 55 degrees C with pH 6.0. Enzymatic assays showed a K-m value of 72.8 mmol/liter and a V-max value of 0.608 mmol/(liter.min). Inhibition assays in vitro indicated that castanospermine, a polyhydroxylated alkaloid, was an effective competitive inhibitor of trehalase with a K-i value of 6.7 mu mol/liter. The inhibitor action of castanospermine was linked to its modification effect on trehalase structure. The circular dichroism spectrum showed that the percentage of a-helix increased under the presence of castanospermine. Results of our study will aid in developing effective trehalase inhibitors for controlling H. armigera in the future.

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