Functional screening of a Caatinga goat (Capra hircus) rumen metagenomic library reveals a novel GH3 β-xylosidase

Abstract

Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-β-D-glucopyranoside (pNPG), 4-nitrophenyl-β-D-xylopyranoside (pNPX) and 4-nitrophenyl-β-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with β-glucosidase, β-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular β-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 μmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most β-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, β-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol.

Fig 1

Schematic of Clone 11 insert obtained in a functional screen of a goat rumen metagenomic library for hydrolysis of the glucoside esculin. The Clone 11 insert has 3,185 bp, and a total of 3 ORFs related to carbohydrate metabolism are present; ORF1 and 2 are complete and ORF3 is partial. Results for Blastp analysis of each ORF are shown in gray boxes. ORF2 (118 to 2,544 bp), which showed similarity to a beta-glucosidase from an unknown Prevotella ceaebacterium, was named BGL11 and chosen to be investigated.

Fig 2

Molecular phylogenetic tree of BGL11 obtained with the software MEGA6, using the maximum likelihood method and bootstrap analysis (1,000 replicates). The percentage of trees in which the associated taxa clustered together is shown next to the branches. In addition to BGL11 (indicated by the arrow), a total of 30 protein sequences similar to BGL11 and retrieved from NCBI are presented in the tree. A beta-glucosidase from Xanthomonas campestris (CEM59903.1) was used as outgroup. Each sequence presented shows the species of origin followed by the corresponding Genbank or NCBI identity. Branch lengths are proportional to the number of substitutions per site (bar at the bottom).

Fig 3

Molecular phylogenetic tree of BGL11 obtained with the software MEGA 6 using the maximum likelihood method and boostrap analysis (1,000 replicates). The percentage of trees in which the associated taxa clustered together is shown next to the branches. In addition to BGL11 (indicated by arrow), the tree shows 34 beta-xylosidase protein sequences whose biochemical characterization has been reported. Each sequence presented shows the species of origin, followed by the protein name, and the Genbank number. Different glycoside hydrolase families (GH1, GH3, GH30, GH39, GH43, GH52) are separated by boxes. Branch lengths are proportional to the number of substitutions per site (bar at the bottom).

Fig 4

Alignment of BGL11 and the closest phylogetically biochemically characterized related sequences. Sequences used in alignment were: BGL11 (this study), Caulobacter crescentus XynB5 (Genbank ID CCNA03149), Corynebacterium alkanolyticum xylD (Genbank ID AJY53618.1) and Rumen Metagenome from Chinese yaks RuBGX1 (Genbank ID GQ324952.1) in this order of presentation. Software Geneious was used to generate this picture, using databanks Pfam, PHOBIUS, PRINTS, SIGNALP_EUK, SMART and Superfamily.

Fig 5

SDS-PAGE analysis of purified BGL11. Lane M, PageRuler™ Plus Prestained 10–250 kDa Protein Ladder (Thermo Scientific™); Lane BGL11, purified protein with the size of 90 kDa.

Fig 6

Biochemical characteristics of BGL11. Effects of pH (A), and temperature (B) and thermal stability at 40°C and 50°C (C) of the purified BGL11 relative activity. The assays were performed in triplicate, error bars represent standard deviation.