A novel GH3-β-glucosidase from soda lake metagenomic libraries with desirable properties for biomass degradation

Abstract

Beta-glucosidases catalyze the hydrolysis of the glycosidic bonds of cellobiose, producing glucose, which is a rate-limiting step in cellulose biomass degradation. In industrial processes, β-glucosidases that are tolerant to glucose and stable under harsh industrial reaction conditions are required for efficient cellulose hydrolysis. In this study, we report the molecular cloning, Escherichia coli expression, and functional characterization of a β-glucosidase from the gene, CelGH3_f17, identified from metagenomics libraries of an Ethiopian soda lake. The CelGH3_f17 gene sequence contains a glycoside hydrolase family 3 catalytic domain (GH3). The heterologous expressed and purified enzyme exhibited optimal activity at 50 °C and pH 8.5. In addition, supplementation of 1 M salt and 300 mM glucose enhanced the β-glucosidase activity. Most of the metal ions and organic solvents tested did not affect the β-glucosidase activity. However, Cu²⁺ and Mn²⁺ ions, Mercaptoethanol and Triton X-100 reduce the activity of the enzyme. The studied β-glucosidase enzyme has multiple industrially desirable properties including thermostability, and alkaline, salt, and glucose tolerance.

Keywords: Beta-glucosidase; Enzyme characterisation; Glycoside hydrolase family 3 (GH3); Soda lakes.

Figure 1

Sequence analysis of CelGH3_f17 (A) The predicted modular architecture of CelGH3_f17. (B) The phylogenetic tree was constructed using the Neighbor-Joining method, based on the blast analysis of CelGH3_f17 with 31 amino acid sequences of proteins in the protein data bank. Branch reliability is indicated by the percentage of 500 bootstrap replicates supporting each cluster of taxa next to the branches. The evolutionary study was performed using MEGA11 software.

Figure 2

The predicted three-dimensional (3D) structural model of the CelGH3_f17 protein (A). The predicted active sites of the CelGH3_f17 protein (B). The predicted substrate recognition-pocket site of the CelGH3_f17 protein (C). The pairwise alignment of the CelGH3_f17 with c5g1mA, Crystal structure of NagZ from P. aeruginosa (D).

Figure 3

(A) Agar plate β-glucosidase assay of CelGH3_f17. (B) SDS-PAGE analysis of the purified, recombinant CelGH3_f17 protein. M, marker proteins; lane 1, IPTG-uninduced crude extract of BL21 (DE3) carrying pET-CelGH3_f17; lane 2, IPTG-induced crude extract of BL21 (DE3) carrying pET-CelGH3_f17; lane 3, Ni–NTA chromatography purified extract of BL21 (DE3) carrying pET-CelGH3_f17.

Figure 4

The CelGH3_f17’s β-glucosidase activity and stability analysis. (A) The relative activity of the enzyme across a range of temperatures, demonstrating optimal activity. (B) Residual activity of the enzyme over a 16-h period at various temperatures, demonstrating the stability (C) The relative activity of the enzyme across a range of pHs, demonstrating optimal activity. (D) Residual activity of the enzyme over a 16-h period at various pHs demonstrating the stability of CelGH3_f17 over time.

Figure 5

Effect of NaCl and glucose concentrations on the CelGH3_f17’s β–Glucosidase activity. (A) Relative enzyme activity at varying molar concentrations of NaCl. (B) Relative enzyme activity at different molar concentrations of glucose.