来自构巢曲霉FGSCA4的β-葡糖苷酶基因被克隆并在构巢曲霉A773中过表达。所得纯化的β-葡萄糖苷酶,名为AnGH3,是一种分子量约为80kDa的单体酶,如通过SDS-PAGE确认的。圆二色性进一步验证了其独特的典型桶形折叠(β/α),在AnGH3的3D同源模型中也观察到这一特征。这种重组酶最引人注目的方面是它的坚固性,因为它在45和50ºC和pH6.0孵育24小时后保留了100%的活性。即使在55°C,在相同的pH下孵育6小时后,它保持了72%的酶活性。动力学参数Vmax,KM,还测定了ρ-硝基苯基-β-D-吡喃葡萄糖苷(ρNPG)和纤维二糖的Kcat/KM。使用ρNPG,当在pH6.0和65°C下孵育时,该酶的Vmax为212Umg-1,KM为0.0607mmolL-1,Kcat/KM为4521mmolL-1s-1。KM,Vmax,使用纤维二糖的Kcat/KM分别为2.7mmolL-1、57Umg-1和27mmol-1s-1。木糖和乙醇在高达1.5molL-1和25%的浓度下显着增强了AnGH3活性,分别。即使在具有挑战性的条件下,在65°C和pH6.0下,该酶保持其活性,在200mmolL-1糠醛和5-羟甲基糠醛(HMF)存在下保留其初始活性的100%和70%,分别。该酶的潜力进一步证明了其在牧草的糖化中的应用,在24小时后,它导致葡萄糖释放增加48%。这些独特的特征,包括高催化性能,在水解温度下具有良好的热稳定性,以及对高浓度乙醇的耐受性,D-木糖,糠醛,和HMF,将这种重组酶定位为木质纤维素生物质水解的有前途的工具,作为有效的多酶混合物的一部分,从而在生物技术和酶学领域开辟了新的途径。
The β-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A. nidulans A773. The resulting purified β-glucosidase, named AnGH3, is a monomeric enzyme with a molecular weight of approximately 80 kDa, as confirmed by SDS-PAGE. Circular dichroism further validated its unique canonical barrel fold (β/α), a feature also observed in the 3D homology model of AnGH3. The most striking aspect of this recombinant enzyme is its robustness, as it retained 100% activity after 24 h of incubation at 45 and 50 ºC and pH 6.0. Even at 55 °C, it maintained 72% of its enzymatic activity after 6 h of incubation at the same pH. The kinetic parameters Vmax, KM, and Kcat/KM for ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) and cellobiose were also determined. Using ρNPG, the enzyme demonstrated a Vmax of 212 U mg - 1, KM of 0.0607 mmol L - 1, and Kcat/KM of 4521 mmol L - 1 s - 1 when incubated at pH 6.0 and 65 °C. The KM, Vmax, and Kcat/KM using cellobiose were 2.7 mmol L - 1, 57 U mg - 1, and 27 mmol -1 s - 1, respectively. AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L - 1 and 25%, respectively. Even in challenging conditions, at 65 °C and pH 6.0, the enzyme maintained its activity, retaining 100% and 70% of its initial activity in the presence of 200 mmol L - 1 furfural and 5-hydroxymethylfurfural (HMF), respectively. The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum, where it led to a 48% increase in glucose release after 24 h. These unique characteristics, including high catalytic performance, good thermal stability in hydrolysis temperature, and tolerance to elevated concentrations of ethanol, D-xylose, furfural, and HMF, position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail, thereby opening new avenues in the field of biotechnology and enzymology.