β-葡萄糖苷酶Bgl747的定向筛选、重组表达与酶学性质

Targeted screening, recombinant expression and enzymatic properties of β-glucosidase Bgl747

摘要:

[背景] β-葡萄糖苷酶（β-Glucosidase，EC3.2.1.21）是3种纤维素酶中的重要成分之一。目前工业用纤维素酶大都来源于木霉等真菌，较少来源于细菌，而且在应用中还存在反应条件（温度、pH等）适用范围窄、酶活力较低、获取成本偏高等问题，这大大限制了β-葡萄糖苷酶的应用。从秸秆还田土壤细菌中筛选β-葡萄糖苷酶有极大地可能性筛选出酶学性质较好的酶，从而解决现存的工业问题。[目的] 从土壤中筛选β-葡萄糖苷酶，通过基因重组、表达优化和蛋白纯化获得一株新型β-葡萄糖苷酶，探究其酶学性质，为其在工业上的应用奠定基础。[方法] 利用功能筛选法从土壤中筛选出β-葡萄糖苷酶，全长为747 bp，命名为Bgl747，构建重组表达质粒pET-28a-Bgl747，以Escherichia coli BL21（DE3）为宿主菌株，经IPTG诱导实现可溶性表达并优化表达条件，通过His标签蛋白纯化试剂盒纯化获得纯化酶，探究其酶学性质。[结果] β-葡萄糖苷酶Bgl747属于BglB超家族，分子量为27.23 kD，最适反应温度为45℃，最适pH 4.0；最佳诱导条件：当OD₆₀₀为1.0，加入终浓度为0.6 mmol/L的IPTG，于37℃、220 r/min诱导10 h后β-葡萄糖苷酶Bgl747蛋白获得最高表达量1.82 mg/mL；底物为对硝基苯-β-D-半乳糖苷（p-nitrophenyl-β-D-galactopyranoside，pNPG）时的比酶活225.07 U/mg，米氏常数K_m值和最大反应速率V_max分别为0.268 mmol/L、547.23 μmol/（L·min）；1 mmol/L K⁺、1 mmol/L和10 mmol/L Fe²⁺、30%甲醇、30%乙醇、1 mmol/L和10 mmol/L盐酸胍对酶活都有促进作用，30% TritonX-100及10 mmol/L SDS抑制其酶活效果较为明显；该酶受到产物葡萄糖的反馈抑制，葡萄糖浓度越高，抑制效果越明显，但当葡萄糖浓度为1 mol/L时，酶活仍保持50%以上。[结论] Bgl747反应温度范围较广且稳定，酶学性质优异，为其在纤维素降解等工业应用奠定基础。

Abstract:

[Background] β-glucosidase (EC3.2.1.21) is one of the important components of the three cellulase enzymes. At present, most of the industrial cellulase are derived from fungi such as Trichoderma, and less derived from bacteria, and there are still problems in application such as narrow application range of reaction conditions (such as temperature, pH), low enzyme activity, and high acquisition cost. These greatly limit the application of β-glucosidase. Screening β-glucosidase from soil bacteria has a great possibility to screen out enzymes with better enzymatic properties, thus solving existing industrial problems. [Objective] Use functional screening method to screen β-glucosidase from soil, obtain a new type of β-glucosidase through gene recombination, expression optimization and protein purification, explore its enzymatic properties, and its industrial application lay the foundation. [Methods] The β-glucosidase was screened from the soil by the functional screening method. Because its full length is 747 bp, it was named Bgl747, and the recombinant expression plasmid pET-28a-Bgl747 was constructed with Escherichia coli BL21(DE3). Induced by IPTG to achieve soluble expression and optimize expression conditions, the purified enzyme was purified by His-tag protein purification kit, and its enzymatic properties were explored. [Results] β-glucosidase Bgl747 as part of the BglB superfamily, its molecular weight is 27.23 kD, and an optimal pH of 4.0, an optimal reaction temperature of 45℃; The optimal induction conditions are:when OD₆₀₀ 1.0, add the final concentration IPTG 0.6 mmol/L, the highest expression level of β-glucosidase Bgl747 protein was 1.82 mg/mL after being induced at 37℃ and 220 r/min for 10 h. The specific enzyme activity when the substrate is p-nitrophenyl-β-D- galactopyranoside (pNPG) is 225.07 U/mg, the Michaelis constant K_m value and the maximum reaction rate are respectively 0.268 mmol/L, 547.23 μmol/(L·min); 1 mmol/L K⁺, 1 mmol/L and 10 mmol/L Fe²⁺, 30% methanol, 30% ethanol, 1 mmol/L and 10 mmol/L guanidine hydrochloride all have a promoting effect on enzyme activity, 30% TritonX-100 and 10 mmol/L SDS inhibits enzyme activity more obviously; The enzyme is feedback inhibition by the product glucose, the greater the glucose concentration, the more obvious the inhibitory effect, but when the glucose concentration is 1 mol/L, the enzyme activity remains above 50%. [Conclusion] Bgl747 has a wide and stable reaction temperature range and excellent enzymatic properties, and lays the foundation for its industrial applications such as cellulose degradation.