Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome

Abstract

The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan degrading enzymes including Pm25, a multi-modular Glycoside Hydrolase (GH) family 10. Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two Carbohydrate Binding Modules (CBMs) from family 4. The genes upstream of pm25 are susC-susD-unk suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization loci. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture, and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating its importance in glycan foraging for the gut microbiota. In order to understand its unusual multi-modularity and the possible role of the CBMs, a detailed characterization of the full length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific towards the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria. IMPORTANCE: Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Bacteroidetes, one of the main phyla in Rumen and Human gut microbiota, have been shown to encode polysaccharide utilization loci (PUL) dedicated to the degradation of xylan. Here we present the biochemical characterization of a xylanase encoded by a bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multi-modular enzyme of which sequence is interrupted by the insertion of two CBM from family 4. Our results show that not only this enzyme resemble homologues that were shown to be important for xylan degradation in rumen or human diet but show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilisation system. This study shed light on a better understanding toward xylan degradation and plant cell wall deconstruction which can conduct to several applications in food, feed and bioeconomy.

In vitro bioprocessing of corn as poultry feed additive by the influence of carbohydrate hydrolyzing metagenome derived enzyme cocktail

“…This result is lower than a previous study which found K m of 0.248 mM for the novel xylanase from fungi 52 . The K cat and K cat /K m values of the PersiXyn8 were higher than the results reported for xylanase from gut metagenome using beechwood xylan as substrate 53 . The PersiXyn8 was also active in the presence of various metal ions and inhibitors increasing the number of applications for the enzyme 49 , 54 .…”

PACER: a novel 3D plant cell wall model for the analysis of non-catalytic and enzymatic responses

“…One of its open reading frames encodes a multimodular GH10, designated P. militaris 25 ( Pm 25). This enzyme is characterized by a discontinuous organization that includes the insertion of two tandem CBM4 domains (CBM4-1 and CBM4-2) within the catalytic GH10 xylanase domain (Table 1 ) [ 44 ].…”

“…This technique allows the visualization of enzyme penetration and migration along a constructed cellulose composite, and impacts of non-catalytic proteins and protein modules on enzyme migration. Three lines of investigation were pursued for proof of concept of the PACER assay: (1) comparative analysis of three endo -1,4-β-xylanases to assess the impact of protein size and presence of a CBM on enzyme migration through a formulated cellulose/azo-xylan matrix; (2) evaluation of two loosenins for their potential to increase xylanase migration through the formulated matrix, and (3) characterization of Pm 25, an endo -1,4-β-xylanase recently discovered from termite gut metagenome [ 44 ]. By simultaneously measuring product release and migration of enzymes through defined cellulosic materials, the PACER assay can uncover determinants of enzyme accessibility not detected through measuring release of soluble products alone.…”

Unlocking the potential of insect and ruminant host symbionts for recycling of lignocellulosic carbon with a biorefinery approach: a review

“…Therefore, a in depth characterization of full length and truncated variants of Pm 25 gene (Xyn10C-like enzyme- part of xylan utilization locus) was performed in order to understand the CBM role with unusual multimodularity. Results indicate that the CBM would act synergistically to improve the enzyme activity and its is more specific towards the xylan hydrolysis [ 44 ]. In turn, the metagenomic of gut symbionts sever as a strategy in novel enzyme identification that are potential with high enzyme activities.…”