Screening of multimeric β-xylosidases from the gut microbiome of a higher termite, Globitermes brachycerastes

Abstract

Termite gut microbiome is a rich reservoir for glycoside hydrolases, a suite of enzymes critical for the degradation of lignocellulosic biomass. To search for hemicellulases, we screened 12,000 clones from a fosmid gut library of a higher termite, Globitermes brachycerastes. As a common Southeastern Asian genus, Globitermes distributes predominantly in tropical rain forests and relies on the lignocellulases from themselves and bacterial symbionts to digest wood. In total, 22 positive clones with β-xylosidase activity were isolated, in which 11 representing different restriction fragment length polymorphism (RFLP) patterns were pooled and subjected to 454 pyrosequencing. As a result, eight putative β-xylosidases were cloned and heterologously expressed in Escherichia coli BL21 competent cells. After purification using Ni-NTA affinity chromatography, recombinant G. brachycerastes symbiotic β-xylosidases were characterized enzymatically, including their pH and temperature optimum. In addition to β-xylosidase activity, four of them also exhibited either β-glucosidase or α-arabinosidases activities, suggesting the existence of bifunctional hemicellulases in the gut microbiome of G. brachycerastes. In comparison to multimeric protein engineering, the involvement of naturally occurring multifunctional biocatalysts streamlines the genetic modification procedures and simplifies the overall production processes. Alternatively, these multimeric enzymes could serve as the substitutes for β-glucosidase, β-xylosidase and α-arabinosidase to facilitate a wide range of industrial applications, including food processing, animal feed, environment and waste management, and biomass conversion.

Keywords: Globitermes brachycerastes; fosmid library; glycoside hydrolases; gut microbiome; multimeric hemicellulase; β-xylosidase.

Figure 1

Phylogenetic analysis of the predicted hemicelluases from G. brachycerastes gut microbiome. These genes belong to three glycoside hydrolase families, GH1, 3, and 43, and their protein sequences were aligned using clustalX. This unrooted phylogenetic tree was established with MEGA 5.0 by the neighbor-joining method. Bootstrap values were derived from 1,000 replications.

Figure 2

Predicted functional domains of β-xylosidases from G. brachycerastes gut microbiome. Besides the signature motifs for GH1, GH3, and GH43 families, respectively, four of the recombinant G. brachycerastes symbiotic β-xylosidases, Xy12, Xy15, Xy16, and Xy17, possess a Fn3-like domain, which mostly exists at the C-terminus. BgIX, an E. coli gene encoding a β-D-glucosidase (EC 3.2.1.21), is a shared feature among Xy12, Xy15, and Xy17.

Figure 3

Optimal pH for recombinant β-xylosidases from G. brachycerastes gut microbiome. Experiments were conducted at their optimal temperature using 4 mM pNPX as the substrate. Enzyme activity was measured in 100 mM sodium acetate buffer (pH 3.0~6.0), 100 mM sodium phosphate buffer (pH 7.0~8.0), and 100 mM Tris-HCl buffer (pH 9.0), respectively. Error bar represents the standard deviation between three independent measurements.

Figure 4

Optimal temperature for recombinant β-xylosidases from G. brachycerastes gut microbiome. Experiments were conducted at their optimal pH using 4 mM pNPX as the substrate. Enzyme activity was tested under a temperature gradient ranging from 20 to 80°C. Error bar represents the standard deviation between three independent measurements.