嗜热淀粉芽孢杆菌来源β-葡萄糖苷酶的重组表达与酶学性质

Recombinant expression and characterization of β-glucosidase from Bacillus thermoamylovorans

【背景】β-葡萄糖苷酶(EC 3.2.1.21,β-glucosidase),是纤维素分解酶系中的重要组成部分,目前工业上应用的β-葡萄糖苷酶多数来源于植物和真菌,来源于细菌的较少,且应用中还存在酶活力偏低、热稳定性差、反应条件适用范围窄、酶活力易受产物反馈抑制等问题,增加了经济成本。嗜热微生物具有特殊的遗传信息资源,极有可能从中挖掘到酶学性质优良的新型β-葡萄糖苷酶,从而解决工业难题。【目的】从嗜热淀粉芽孢杆菌(Bacillus thermoamylovorans)基因组中挖掘新型β-葡萄糖苷酶基因,通过基因重组、异源表达和蛋白纯化技术制备新型β-葡萄糖苷酶,并探究其酶学性质,为新型β-葡萄糖苷酶在纤维素水解等领域的应用奠定基础。【方法】人工合成新型β-葡萄糖苷酶基因bgl52,构建重组表达质粒pET22b-bgl52,并用电脉冲法转化到大肠杆菌BL21(DE3)中实现可溶性表达,利用Ni-NTA亲和层析纯化得到高纯度的β-葡萄糖苷酶Bgl52。【结果】实现重组表达质粒pET22b-bgl52在大肠杆菌BL21(DE3)中的可溶性表达,并获得β-葡萄糖苷酶Bgl52纯蛋白,蛋白分子量为52 kD,在70°C和pH 6.5条件下表现出最佳活性;以p-nitrophenyl-β-D-glucopyranoside(p NPG)为底物时的比酶活为223.7±5.3 U/mg;Km为9.3±1.2 mmol/L,Vmax为270.3±4.3μmol/(min·mg);Bgl52偏好性水解β-1,4糖苷键的底物;Fe2+和Mg2+对酶的激活作用明显,Co2+、Cu2+和SDS可抑制其活性;Bgl52是少有的几种葡萄糖和木糖激活型β-葡萄糖苷酶之一,当反应体系中外源添加0.2 mol/L葡萄糖时可提升活力至2.84倍,外源添加0.4 mol/L木糖时可提升活力至3.24倍,同时Bgl52在生理条件下基本不受产物的反馈抑制。【结论】利用嗜热微生物基因组中蕴藏的遗传信息资源,通过现代生物技术方法,可以从中挖掘到酶学性质优良的β-葡萄糖苷酶,为其在纤维素降解等工业领域的应用奠定基础。

[Background]β-glucosidase(EC 3.2.1.21)is an important component of cellulase system.At present,mostβ-glucosidases used in industry come from plants and fungi,but few come from bacteria,and there are some problems such as low enzyme activity,poor thermal stability,narrow reaction conditions,products inhibition,which increase the economic cost.Thermophilic microorganisms have special genetic information resources,so it is possible to excavate novelβ-glucosidases with good enzymatic characterization from the genome to solve the industrial problems.[Objective]A novelβ-glucosidase gene was extracted from the genome of Bacillus thermoamylovorans,and purified protein was obtained by gene recombination,heterologous expression and protein purification.The enzymatic characterization was studied systematically.It can lay the foundation for the application ofβ-glucosidase in the fields of cellulose hydrolysis.[Methods]The recombinant plasmid pET22 b-bgl52 was constructed and transformed into Escherichia coli BL21(DE3)by electric pulse method.The recombinant protein was expressed in soluble form and purified by Ni-NTA affinity chromatography.[Results]The recombinant plasmid pET22 b-bgl52 was expressed in E.coli BL21(DE3)and purifiedβ-glucosidase Bgl52 protein was obtained.The molecular weight of the Bgl52 was 52 kD and it showed the best activity at 70°C and pH 6.5.When p-nitrophenyl-β-D-glucopyranoside(p NPG)as substrate,the specific enzyme activity was 223.7±5.3 U/mg,Km was 9.3±1.2 mmol/L,Vmax was 270.3±4.3μmol/(min·mg).Bgl52 preferred substrate for hydrolysis ofβ-1,4 glycosidic bond.Fe2+and Mg2+activated the enzyme activity obviously,Co2+,Cu2+and SDS inhibited the activity of enzyme.Bgl52 is one of the few glucose and xylose-activated glucosidases.A maximal 2.84-fold stimulation by glucose was observed at 0.2 mol/L,and a maximal 3.24-fold stimulation by xylose was found at 0.4 mol/L.At the same time,under physiological conditions,Bgl52 was not substantially inhibited by the feedback of the product glucose.[Conclusion]Using the genetic information resources contained in the genomes of thermophilic microorganisms,and through modern biotechnological methods such as gene synthesis,we can excavate theβ-glucosidase with excellent enzymatic characterization,it lays a foundation for its application in cellulose degradation and other industrial fields.