Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass

Background Xylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the β-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis. Results xynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50–55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold. Conclusion The supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.

Two Bifunctional β-Xylosidase /α-L-Arabinofuranosidases From GH43 With Different Structures in the xylan Degradation Strain of Paenibacillus Physcomitrellae XB Displayed the Similar xylo-oligosaccharides Degradation Ability

BackgroundThe strain Paenibacillus physcomitrellae XB isolated from moss of Physcomitrella patens was found have the xylan degradation ability, but its degradation characteristics and the related mechanism has not been revealed.ResultsIn this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability under the different substrates of corncob xylan (CCX), oat spet xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). Genomic analysis showed that ~ 38 genes were related to xylan degradation, and quantitative real time RT-PCR showed that two glycoside hydrolase family 43 genes (Pph_0602 and Pph_2344) were up-regulated on 1% CCX and xylose. Substrate-specific experiments with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both of them exhibited β-xylosidase activity toward chromogenic substrate p-nitrophenyl–D-xylopyranoside and α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside, indicating at least bifunctionality. Combined their degradation features on the natural substrates of different xylans with the hydrolytic products separated by thin-layer chromatography and high-performance anion exchange chromatography profiles, it was found that both Ppxyl43A and Ppxyl43B were with the similar degradation ability on xylo-oligosaccharides (like CCX, OSX, xylohexaose and xylobiose). Both of them even could hydrolyze xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX to produce xylooligosaccharides or xylose, suggesting they have no endo-xylanase activity and mainly hydrolyze xylo-oligosaccharides by β-xylosidase activity. Moreover, the kinetic parameters of β-xylosidase and α-L-arabinofuranosidase of both two proteins indicated their affinity with all the detected natural substrate (CCX) and chromogenic substrates were nearly similar. In addition, despite having no signal peptides, both of them might export outside the cell by the nonconventional pathways. However, Ppxy143B exhibited wider temperature and pH ranges, higher pH and thermostability, and was less influenced by metal ions than Ppxyl43A. Given its enzymatic characteristics and predicted structure, it is likely that the C-terminus domain (GH43_C2) of Ppxyl43B enhances the stability of the two enzymes and also restricts the substrates’ or metal ions’ access to the active sites.ConclusionsPpxyl43A and Ppxyl43B were β-xylosidase/α-L-arabinofuranosidase bifunctional enzymes with different structures from Paenibacillus physcomitrellae XB and exhibited similar xylo-oligosaccharides hydrolyse ability, which would be useful in the further lignocellulosic biomass conversion.

Effect of Oligosaccharide Degree of Polymerization on the Induction of Xylan-Degrading Enzymes by Fusarium oxysporum f. sp. Lycopersici

Xylan is one of the most abundant carbohydrates on Earth. Complete degradation of xylan is achieved by the collaborative action of endo-β-1,4-xylanases and β-d-xylosidases and a number of accessories enzymes. In filamentous fungi, the xylanolytic system is controlled through induction and repression. However, the exact mechanism remains unclear. Substrates containing xylan promote the induction of xylanases, which release xylooligosaccharides. These, in turn, induce expression of xylanase-encoding genes. Here, we aimed to determine which xylan degradation products acted as inducers, and whether the size of the released oligomer correlated with its induction strength. To this end, we compared xylanase production by different inducers, such as sophorose, lactose, cellooligosaccharides, and xylooligosaccharides in Fusarium oxysporum f. sp. lycopersici. Results indicate that xylooligosaccharides are more effective than other substrates at inducing endoxylanase and β-xylosidases. Moreover, we report a correlation between the degree of xylooligosaccharide polymerization and induction efficiency of each enzyme. Specifically, xylotetraose is the best inducer of endoxylanase, xylohexaose of extracellular β-xylosidase, and xylobiose of cell-bound β-xylosidase.

Investigating the multi-modularity of a GH10 Xylanase found in termite gut metagenome

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.

Investigation of Microorganisms Deteriorating Ancient Ola Leaf Manuscripts

Ola leaf manuscripts from Sri Lanka date back to several centuries. While they have been well preserved over the last century, their condition has worsened in recent years when black dots caused by microorganisms started occurring on their surface. In this study, the current state of preservation and the factors causing deterioration are examined using microscopy techniques. Microscopic images clearly show that the manuscripts are contaminated by microorganisms which penetrated deeply into the carrier material, destroying the internal structure. A Penicillium griseofulvum strain was recognized as the most active microorganism in xylan degradation. Sri Lanka’s climate provides favorable conditions for the growth of these fungi. Therefore, it is suggested that temperature and humidity of the archival space should be better controlled in order to ensure the Ola leaf manuscripts’ long-term preservation.

Unraveling the xylanolytic potential of Acidobacteria bacterium AB60 from Cerrado soils

ABSTRACT The presence of genes for glycosyl hydrolases in many Acidobacteria genomes indicates an important role in the degradation of plant cell wall material. Acidobacteria bacterium AB60 was obtained from Cerrado oligotrophic soil in Brazil, where this phylum is abundant. The 16S rRNA gene analyses showed that AB60 was closely related to the genera Occallatibacter and Telmatobacter. However, AB60 grew on xylan as carbon source, which was not observed in Occallatibacter species; but growth was not detected on medium containing carboxymethyl cellulose, as observed in Telmatobacter. Nevertheless, the genome analysis of AB60 revealed genes for the enzymes involved in cellulose as well as xylan degradation. In addition to enzymes involved in xylan degradation, α-l-rhamnosidase was detected in the cultures of AB60. Functional screening of a small-insert genomic library did not identify any clones capable of carboxymethyl cellulose degradation, but open reading frames coding α-l-arabinofuranosidase and α-l-rhamnosidase were present in clones showing xylan degradation halos. Both enzymes act on the lateral chains of heteropolymers such as pectin and some hemicelluloses. These results indicate that the hydrolysis of α-linked sugars may offer a metabolic niche for slow-growing Acidobacteria, allowing them to co-exist with other plant-degrading microbes that hydrolyze β-linked sugars from cellulose or hemicellulose backbones.