Expression and characterization of a bifunctional thermal β-glucosidase IuBgl3 from thermophilic archaeon <i>Infirmifilum uzonense</i>.

Xinhan Liu; Fengfei Shen; Pengjun Shi; Huiqin Liu

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Expression and characterization of a bifunctional thermal β-glucosidase IuBgl3 from thermophilic archaeon Infirmifilum uzonense.

Author: Xinhan LIU ¹ ; Fengfei SHEN ² ; Pengjun SHI ³ ; Huiqin LIU ¹
Author Information

1. College of Horticulture and Landscape, Tianjin Agricultural University, Tianjin 300392, China.
2. College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China.
3. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China.
Publication Type:Journal Article
Keywords: GH3 family; bifunctional enzymes; enzymatic properties; thermophilic archaea; β-glucosidase
MeSH: beta-Glucosidase/chemistry*; Archaea/metabolism*; Escherichia coli/metabolism*; Hydrogen-Ion Concentration; Temperature; Glucosides; Enzyme Stability; Substrate Specificity; Kinetics
From: Chinese Journal of Biotechnology 2022;38(12):4644-4657
CountryChina
Language:Chinese
Abstract: β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense. Sequence analysis showed that the full length of Iubgl3 was 2 106 bp, encoding 702 amino acids, with a theoretical molecular weight of 77.0 kDa. The gene was cloned and expressed in E. coli and the enzymatic properties of purified IuBgl3 were studied. The results showed that the optimal pH and temperature for pNPG hydrolysis were 5.0 and 85 ℃, respectively. The enzyme has good thermal stability, and more than 85% of enzyme activity can be retained after being treated at 80 ℃ for2 h. This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0-11.0 for 1 h. It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-d-glucoside (pNPG) and p-nitrophenyl β-d-xylopyranoside (pNPX). When pNPG was used as the substrate, the kinetic parameters K_m and V_max were 0.38 mmol and 248.55 μmol/(mg·min), respectively, and the catalytic efficiency k_cat/K_m was 6 149.20 s^-1mmol^-1. Most metal ions had no significant effect on the enzyme activity of IuBgl3. SDS completely inactivated the enzyme, while EDTA increased the enzyme activity by 30%. This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.