绿盲蝽水溶性海藻糖酶ALTre-1基因原核表达、 纯化与酶学特性

doi:10.3864/j.issn.0578-1752.2013.17.007

摘要/Abstract

摘要： 【目的】在前期克隆了绿盲蝽水溶性海藻糖酶（ALTre-1）基因的基础上，以获得具有酶活性的重组蛋白，明确酶促反应的最佳pH值及最适温度，为绿盲蝽海藻糖酶分子调控机制及其抑制剂的应用研究奠定基础。【方法】将含有绿盲蝽ALTre-1基因的T载体经Nde I和Not I双酶切，构建ALTre-1基因原核表达载体（pET28a-ALTre-1），表达载体经诱导表达和蛋白纯化，获得ALTre-1功能区纯化蛋白。在蛋白浓度为0.3 mg•mL-1的条件下，以海藻糖为底物，对纯化得到的重组Tre-1蛋白进行活性检测，确定其酶促反应的最佳pH值和最佳温度。【结果】ALTre-1基因在大肠杆菌中BL21中能够高效表达，纯化获得的重组蛋白在试验条件下有较高的海藻糖酶水解活性（（184.83±13.39）nmol•μg-1•min-1）。重组ALTre-1蛋白活性最适pH值为7.0（酶活性为（202.04±13.76）nmol•μg-1•min-1），表明重组ALTre-1蛋白是1个在中性环境中具有最佳活性的海藻糖酶，同时重组ALTre-1蛋白酶活性最适温度为55℃（酶活性为（228.59±4.62）nmol•μg-1•min-1）。【结论】本研究克隆得到了ALTre-1全长基因，获得了具有海藻糖酶活的重组蛋白，该重组蛋白在pH 7.0、温度55℃条件下酶活性最高。

关键词: 绿盲蝽 , 水溶性海藻糖酶 , 原核表达 , 酶学特性

Abstract: 【Objective】 The aims of this study are to obtain recombinant protein which has a trehalase enzyme activity and identify the best pH and the optimum temperature on the basis of cloned the soluble trehalase from Apolygus lucorum previous. This research expected to reveal the molecular mechanism of trehalase synthesis and offer the basic research of the development of trehalase inhibitor pesticide. 【Method】 The vector containing ALTre-1 gene was double enzyme restricted by Nde I and Not I, then the cDNA identified by sequencing was constructed to pET28a vector and transformed into BL21 of E. coli. The target recombinant protein was over expressed and been purified by using Ni-NTA agarose, and then its activity assay and enzymatic characteristics were studied. 【Result】 The ALTre-1 gene could over express in E. coli and the target recombinant protein which purified through Ni-NTA agarose had higher trehalase activity ((184.83±13.39)nmol•μg-1•min-1) by using trehalose as substrate, the suitable reaction temperature was 55℃ and the best pH was 7.0. 【Conclusion】 Recombinant protein with trehalase activity was obtained, and the suitable pH and temperature of the highest enzyme activity were 7.0 and 55℃, respectively.

Key words: Apolygus lucorum , soluble trehalase , prokaryotic expression , enzymatic characteristic

谭永安, 肖留斌, 孙洋, 柏立新. 绿盲蝽水溶性海藻糖酶ALTre-1基因原核表达、纯化与酶学特性[J]. 中国农业科学, 2013, 46(17): 3587-3593.

TAN Yong-An, XIAO Liu-Bin, SUN Yang, BAI Li-Xin. Prokaryotic Expression, Purification and Functional Activity Assay in vitro of Soluble Trehalse from Apolygus lucorum[J]. Scientia Agricultura Sinica, 2013, 46(17): 3587-3593.

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