Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.

Lichun Qian,Xinxin Xu,Ying Cui,Xiuan Zhan,Pengjun Shi,Yingguo Bai,Bin Yao,Huiying Luo,Wei Xia,Wei Zhang

doi:10.1038/srep27062

Abstract

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50–60 °C and pH 5.5–6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

Highlights

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose
Complete hydrolysis of cellulose needs the synergistic action of a whole cellulolytic enzyme system, which includes endo-β-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21)1,5,6. β-Glucosidase can accelerate the decomposition of cellulose and improve the glucose yield by catalyzing the rate-limiting step of cellobiose hydrolysis[7,8]
Several conserved substrate bind sites were verified in single protein by crystallisation of inhibitor complex or experimental determination, such as Arg[156] and Tyr[511] of GH3 β-glucosidase AaBGL1 from Aspergillus aculeatus (PDB: 4IIB)[15], and Trp[49] of β-glucosidase from Aspergillus niger[18]

Summary

Introduction

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. We analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50–60 °C and pH 5.5–6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Cost-effective process of enzymatic hydrolysis requires low production cost and highly active enzymes with great inhibitor tolerance and synergistic actions. The enzymes were all most active under neutral and mesophilic conditions, but showed distinguished substrate specificity, catalytic efficiency and glucose tolerance. Further site-directed mutagenesis revealed the vital role of three residues in the substrate specificity of GH3 β-glucosidases

Methods

Results

Discussion

Conclusion