嗜酸古菌Cuniculiplasma divulgatum来源的GH1家族中温β-葡萄糖苷酶CdBglA的原核表达及酶学性质分析

Expression and characterization of mesophilic GH1 β-glucosidase CdBglA from acidophilic Cuniculiplasma divulgatum

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的热点。本文对尚未报道的来源于嗜酸古菌(Cuniculiplasma divulgatum) GH1家族的葡萄糖苷酶进行了克隆表达和酶学性质测定，以期找到更优的β-葡萄糖苷酶。从NCBI数据库中获取了C. divulgatum来源的GH1葡萄糖苷酶氨基酸序列，然后制备重组质粒pET-30a(+)-CdBglA，并在大肠杆菌BL21(DE3)中诱导表达重组蛋白，对纯化后的CdBglA进行酶学性质研究。结果发现，重组酶CdBglA的分子量为56.0 kDa，最适pH为5.5，最适温度为55 ℃。该酶具有良好的pH稳定性，在pH 5.5-11.0范围内处理1 h，仍维持92.33%以上的酶活力。以pNPG为底物时的酶促反应动力学参数K_m、V_max和K_cat/K_m分别为0.81 mmol、291.99 μmol/(mg·min)和387.50 s^-1 mmol^-1。其在终浓度5 mmol/L重金属离子的影响下均能保持90.33%以上的酶活力；在15%的乙醇溶液中酶活力被提高了28.67%，在20%的乙醇溶液中酶活力保持不变，在30%的乙醇溶液中仍保持43.68%的酶活力；该酶在0-1.5 mol/L的NaCl溶液中具有明显的激活作用并且可以耐受0.8 mol/L葡萄糖。本研究表明，CdBglA是酸性中温酶，具有宽泛的pH稳定性，且对大部分金属离子、有机溶剂、NaCl和葡萄糖都具有很强的耐受性。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:

β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters K_m, V_max and K_cat/K_m were 0.81 mmol, 291.99μmol/(mg·min), and 387.50 s^-1 mmol^-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.