嗜热古菌Thermofilum adornatum来源的高温热激活β-葡萄糖苷酶TaBgl3的原核表达及酶学性质研究

Expression and characterization of thermo-activated β-glucosidase TaBgl3 from Thermofilum adornatum

摘要:

【目的】β-葡萄糖苷酶，又称β-D-葡萄糖苷水解酶，属于纤维素酶类，是一种降解纤维素的关键限速酶。来源于嗜热古菌的β-葡萄糖苷酶已被广泛验证具有酸性高温等特性，已成为高温酶的研究热点之一。本文对尚未报道的来源于嗜热古菌中一种热丝菌(Thermofilum adornatum)的GH3家族的葡萄糖苷酶，进行了原核表达和酶学性质测定，以期找到更优的β-葡萄糖苷酶。【方法】从NCBI数据库中获得了嗜热古菌(T.adornatum)来源的GH3氨基酸序列，构建重组质粒pET-30a (+)-TaBgl3，并在大肠杆菌(Escherichiacoli) BL21(DE3)感受态细胞中诱导表达重组蛋白；采用磁珠纯化，研究其酶学性质。【结果】重组蛋白TaBgl3的分子量为77.0 kDa；酶学性质结果表明，其最适反应条件为80℃和pH 5.0，在70℃保温处理1–4 h，对TaBgl3的酶活力有促进作用，在最适温度80℃处理2 h后，其激活作用更加明显，能提高40%以上的酶活；其在pH 5.0–8.0下37℃保温1 h，仍具有60%以上的活性；底物为对硝基苯-β-D-吡喃葡萄糖苷(pNPG)时酶的比活力为144.23 U/mg，米氏常数K_m值和最大反应速率V_max分别为1.81 mmol/L和268.10 μmol/(mg·min)，催化效率为115.47/s；终浓度为5 mmol/L的Cu²⁺、Li⁺和EDTA对TaBgl3的酶活有较大的促进作用，可提升39%以上的酶活，而5 mmol/L Fe³⁺和5%β-巯基乙醇对酶活有抑制作用，SDS、乙醇和葡萄糖对酶具有很强的抑制作用。【结论】本研究中，TaBgl3是酸性高温酶，且具有较好的热稳定性和热激活特性，这些特征在以后的理论研究及在工业生产中具有一定的科学价值。

Abstract:

[Objective] β-glucosidase, also known as β-D-glucosidase hydrolase, is a key rate-limiting enzyme for cellulose degradation, which is a cellulase. The β-glucosidases from thermophilic archaea have been verified to have tolerance to acid and high temperature, which have become one of the research hotspots of thermostable enzymes. We studied the prokaryotic expression and enzymatic properties of a GH3 family glucosidase from Thermofilum adornatum, which has not been reported, aiming to mine superior β-glucosidase. [Methods] We obtained the GH3 amino acid sequence of T.adornatum from NCBI database and constructed the recombinant plasmid pET-30a(+)-TaBgl3. The recombinant protein TaBgl3 was expressed in Escherichia coli BL21(DE3) competent cells under induction. The properties of the enzyme were studied after purification with magnetic beads. [Results] TaBgl3 had a molecular weight of 77.0 kDa and the best performance at pH 5.0 and 80℃. The treatment at 70℃ for 1-4 h improved the enzyme activity, and that at the optimum temperature of 80℃ for 2 h showed the most significant effect, which increased the enzyme activity by more than 40%. The enzyme still had the relative activity of above 60% after being treated 37℃ and pH 5.0-8.0 for 1 h. When the substrate was 4-p-nitrophenyl-β-D-glucopyranoside (pNPG), the enzyme had the specific activity of 144.23 U/mg, the K_m value of 1.81 mmol/L, the maximum reaction rate of 268.10 μmol/(mg·min), and the catalytic efficiency of 115.47/s. Cu²⁺, Li⁺ and EDTA at the final concentration of 5 mmol/L all increased the enzyme activity, which had the maximum increase of 39%. Fe³⁺ (5 mmol/L) and 5% β-mercaptoethanol had inhibitory effect on the enzyme activity. In addition, SDS, ethanol and glucose greatly inhibited the enzyme activity. [Conclusion] TaBgl3 is an acidic thermostable enzyme with good thermal stability and thermal activation characteristics, which can shed light on the future theoretical research and industrial production.