嗜热古菌Infirmifilum uzonense来源的双功能高温β-葡萄糖苷酶IuBgl3的原核表达及酶学性质分析

Expression and characterization of a bifunctional thermal β-glucosidase IuBgl3 from thermophilic archaeon Infirmifilum uzonense

摘要:

β-葡萄糖苷酶在食品、医药、生物质转化等领域具有重要的应用价值，因此发掘适应性强、性质优良的β-葡萄糖苷酶是国内外研究热点。本研究从嗜热古菌Infirmifilum uzonense中成功克隆出一个GH3家族的β-葡萄糖苷酶基因，命名为Iubgl3。基因序列分析显示Iubgl3全长为2109bp，编码702个氨基酸，理论分子量为77.0kDa。将该基因在大肠杆菌中进行克隆表达并对纯化后的IuBgl3进行酶学性质研究。结果显示，重组酶IuBgl3最适pH5.0，最适温度85℃。该酶具有良好的热稳定性，80℃处理2h后仍能保持85%以上的酶活力。其具有优良的pH稳定性，在pH4.0−11.0范围内处理1h，仍维持85%以上的酶活力。通过底物特异性测定发现，该酶对对硝基苯-β-d-吡喃葡萄糖苷(p-nitrophenylβ-d-glucoside,pNPG)和对硝基苯-β-d-吡喃木糖苷(p-nitrophenyl β-d-xylopyranoside,pNPX)均有很高的水解能力，是典型的双功能酶。以pNPG为底物时的动力学参数K_m和V_max分别为0.38mmol和248.55μmol/(mg·min)，催化效率k_cat/K_m=6149.20s⁻¹mmol⁻¹。大多数金属离子对IuBgl3的酶活力没有显著影响，SDS可导致酶完全失活，而EDTA却能提高30%的酶活力。本研究丰富了高温古菌GH3家族的β-葡萄糖苷酶基因，获得了一个稳定性优良的高温酸性双功能酶，具有良好的工业应用前景。

Abstract:

β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense. Sequence analysis showed that the full length of Iubgl3 was 2 106 bp, encoding 702 amino acids, with a theoretical molecular weight of 77.0 kDa. The gene was cloned and expressed in E.coli and the enzymatic properties of purified IuBgl3 were studied. The results showed that the optimal pH and temperature for pNPG hydrolysis were 5.0 and 85℃, respectively. The enzyme has good thermal stability, and more than 85% of enzyme activity can be retained after being treated at 80℃ for 2 h. This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0−11.0 for 1 h. It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-d-glucoside (pNPG) and p-nitrophenyl β-d-xylopyranoside (pNPX). When pNPG was used as the substrate, the kinetic parameters K_m and V_max were 0.38 mmol and 248.55 μmol/(mg·min), respectively, and the catalytic efficiency k_cat/K_m was 6 149.20 s⁻¹mmol⁻¹. Most metal ions had no significant effect on the enzyme activity of IuBgl3. SDS completely inactivated the enzyme, while EDTA increased the enzyme activity by 30%. This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.