嗜冷德沃斯氏菌来源GH1家族β-葡萄糖苷酶Bgl59的原核表达及酶学性质分析

Prokaryotic expression and characterization of the GH1 β-glucosidase Bgl59 from Devosia psychrophila

摘要:

β-葡萄糖苷酶在食品、医学以及生物能源等多个领域有着广泛的应用，因此挖掘新型高效的β-葡萄糖苷酶是十分必要的。【目的】 对嗜冷德沃斯氏菌(Devosia psychrophile)来源的GH1家族的葡萄糖苷酶原核表达并测定其酶学性质。【方法】 通过人工合成技术得到D. psychrophila来源的β-葡萄糖苷酶的编码基因，命名为bgl59。将该基因转化到大肠杆菌(Escherichia coli) BL21(DE3)中，诱导基因表达，对得到的蛋白进行纯化并测定其酶学性质。【结果】 Bgl59的分子量为48.8 kDa，最适温度为55 ℃，最适pH为6.0；Bgl59在pH 5.0−8.5范围内处理1 h后仍保持80%以上酶活；在8种供试底物中，Bgl59对4-硝基苯基-β-d-葡萄糖苷(4-nitrophenyl-β-d-glucopyranoside, pNPG)有着最高的水解能力，其K_m为3.090 mmol/L，V_max为194 μmol/(min·mg)，k_cat为159 s^–1；1 mmol/L的Ca²⁺、Co²⁺对Bgl59具有明显的激活作用，0.1%的SDS会使酶活全部丧失；0.10 mol/L葡萄糖和0.30 mol/L木糖具有促酶活作用，可分别使Bgl59酶活提高74%和91%；在1.25 mol/L葡萄糖或2.00 mol/L木糖存在的条件下，仍可保持50%以上酶活。【结论】 Bgl59的酶学性质优异，具有良好的pH稳定性，对金属离子或化学试剂都具有一定耐受能力，是少见的葡萄糖激活型β-葡萄糖苷酶，具有优良的糖促活性和耐受性，在未来的工业生产以及应用中具有潜在研究价值。

关键词:β-葡萄糖苷酶;酶学性质;GH1家族;葡萄糖耐受

Abstract:

β-glucosidases have been widely used in food, medicine, bioenergy and other fields, and thus it is necessary to explore new and efficient β-glucosidases. [Objective] To realize the prokaryotic expression of a GH1 glucosidase derived from Devosia psychrophila and characterize the enzymatic properties of the expressed protein. [Methods] The gene encoding the β-glucosidase derived from D. psychrophila was synthesized, named bgl59, and then transformed into Escherichia coli BL21(DE3). After the gene expression was induced, and the obtained protein was purified and characterized for the enzymatic properties. [Results] Bgl59 had a molecular weight of 48.8 kDa, with the highest activity at 55 ℃ and pH 6.0. After treatment for 1 h within the range of pH 5.0–8.5, Bgl59 maintained the relative activity over 80%. Bgl59 had the highest hydrolysis ability for 4-nitrophenyl-β-d-glucopyranoside (pNPG) among the eight substrates tested, with the K_m of 3.090 mmol/L, V_max of 194 μmol/(min·mg), and k_cat of 159 s⁻¹. The presence of 1 mmol/L of Ca²⁺ and Co²⁺ had a significant activating effect on Bgl59, while the presence of 0.1% SDS resulted in a complete loss of enzyme activity. The presence of 0.10 mol/L glucose and 0.30 mol/L xylose increased the activity of Bgl59 by 74% and 91%, respectively. Moreover, the enzyme remained the relative activity above 50% even when being cultured with 1.25 mol/L glucose or 2.00 mol/L xylose. [Conclusion] Bgl59 exhibits outstanding enzymatic properties, robust pH stability, and tolerance to metal ions and chemical reagents. It is a rare glucose-activated β-glucosidase with exceptional tolerance to glucose, holding significant potential for future industrial production and application.

Key words:β-glucosidase;enzymatic properties;GH1 family;glucose tolerance