Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the characterization of a multimodular ß-1,4-xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular ß-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50 °C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Accessing carboxylesterase diversity from termite hindgut symbionts through metagenomics.

A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in Escherichia coli and the purified enzymes exhibited temperature optima of between 40-50°C. Substrate-profiling studies revealed that the characterised enzymes were 'true' carboxylesterases based on their preferences for short to medium chain length p-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches.