白蚁肠道元基因组来源高温β-葡萄糖苷酶Bgl17的酶学性质

Characterization of a β-glucosidase Bgl17 from a metagenomics library of termite gut

摘要:

【目的】对短角球白蚁(Globitermes brachycerastes)肠道元基因组文库中筛选得到的一个新型β-葡萄糖苷酶编码基因bgl17进行酶学性质研究。【方法】通过克隆与异源表达得到纯的Bgl17酶蛋白，根据Bgl17对底物的水解活性测定其稳定性及动力学参数，利用薄层层析确定其水解产物。【结果】该酶属于糖基水解酶第一家族(GHF1)，对其特异性底物4-硝基苯基-β-D-吡喃葡萄糖苷(pNPGlc)的最适反应温度为70 °C，最适pH为5.0。在最适反应条件下，该酶以pNPGlc为底物比活力为115.69 U/mg，以水杨苷为底物比活力为297.39 U/mg。以pNPGlc为底物时，其动力学参数Km值和Vmax分别为0.81 mmol/L和227.27 μmol/(mL·min)。在稳定性方面，该酶在50 °C处理1 h仍可保持50%的活性，在pH 5.0和6.0条件下，该酶的半衰期为1 h。【结论】该酶在较高的温度下具有较高的活性，且对水杨苷水解活性高，这点不同于已知的β-葡萄糖苷酶，推测其更有利于木质纤维素复杂结构的降解；该酶的最适温度远高于白蚁生存环境温度，可为研究白蚁降解纤维素的机理提供参考。

Abstract:

[Objective] A novel β-glucosidase encoding gene bgl17, which was functional screened from a metagenomic library constructed from the gut of Globitermes brachycerastes, was expressed in Escherichia coli BL21 and its characteristic were studied. [Methods] The recombinant enzyme Bgl17 was purified, and the stability and kinetic were identified. And the hydrolysates were analyzed by thin layer chromatography. [Results] Bgl17 was belong to glycoside hydrolase family 1 (GH1), and the optimal temperature and pH of Bgl17 with p-nitrophenyl-β-D-glucopyranoside (pNPGlc) were 70 °C and 5.0, respectively. The specific activity of purified Bgl17 was 115.69 U/mg and 297.39 U/mg with pNPGlc and salicin as substrate, respectively. The Km and Vmax of Bgl17 were 0.81 mmol/L and 227.27 μmol/(mL·min) with pNPGlc, respectively. Bgl17 can remain 50% activity at 50 °C for 1 hour and 50% residual activity was detected after 1 h at pH 5.0 and 6.0. [Conclusion] The β-glucosidase Bgl17 showed the high activity with salicin, which might be beneficial to the degradation of lignocellulose. It showed the potential for industrial applications because of high thermostability. The optimal temperature of Bgl17 was much higher than that of termites’ living environment, which would contribute to the study of cellulose degradation mechanism of termites.