Xylobiose

A novel trifunctional, family GH10 enzyme from Acidothermus cellulolyticus 11B, exhibiting endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities.[Pubmed:29170828]

Extremophiles. 2018 Jan;22(1):109-119.

A novel, family GH10 enzyme, Xyn10B from Acidothermus cellulolyticus 11B was cloned and expressed in Escherichia coli. This enzyme was purified to homogeneity by binding to regenerated amorphous cellulose. It had higher binding on Avicel as compared to insoluble xylan due to the presence of cellulose-binding domains, CBM3 and CBM2. This enzyme was optimally active at 70 degrees C and pH 6.0. It was stable up to 70 degrees C while the CD spectroscopy analysis showed thermal unfolding at 80 degrees C. Xyn10B was found to be a trifunctional enzyme having endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities. Its activities against beechwood xylan, p-Nitrophenyl arabinofuranoside and p-Nitrophenyl acetate were found to be 126,480, 10,350 and 17,250 U mumol(-1), respectively. Xyn10B was highly active producing Xylobiose and xylose as the major end products, as well as debranching the substrates by removing arabinose and acetyl side chains. Due to its specific characteristics, this enzyme seems to be of importance for industrial applications such as pretreatment of poultry cereals, bio-bleaching of wood pulp and degradation of plant biomass.

High xylan recovery using two stage alkali pre-treatment process from high lignin biomass and its valorisation to xylooligosaccharides of low degree of polymerisation.[Pubmed:29433045]

Bioresour Technol. 2018 May;256:110-117.

In the present work, xylan from arecanut husk was extracted using 2 stage alkaline pretreatment process. In first step, biomass was incubated in alkali at different temperatures (25 degrees C, 50 degrees C and 65 degrees C), alkali concentrations (5%, 10%, 15% and 20% w/v), and incubation periods (8h, 16h and 24h) and evaluated for xylan recovery. It was observed that 40-52% of available xylan could be recovered using 10% alkali when incubated for 8-24h at 65 degrees C. Subsequently, the alkali pretreatment operating conditions which provided good xylan recovery were processed further using hydrothermal treatment to extract more xylan. For maximum xylan recovery (>90%), best operating conditions were identified when biomass was treated under hydrothermal treatment (1, 1.5 and 2h) with varying incubation periods (8, 16, 24h) and alkali concentrations (5%, 10%) using full factorial design. Incubating arecanut husk with 10% w/v NaOH, at 65 degrees C for a period of 8h, followed by hydrothermal treatment at 121 degrees C for 1h helped recover >94% xylan. In the next step, enzymatic hydrolysis process was optimized to recover maximum XOS (Optimized condition: 50 degrees C, pH 4 and 10U enzyme dose). The hydrolysate comprised of Xylobiose: 25.0+/-1.2g/100g xylan ( approximately 71% of XOS), xylotriose: 9.2+/-0.65g/100g xylan (26.2% of XOS) and xylotetrose: 0.9+/-0.04g/100g xylan (2% of XOS). The developed process enables to reduce alkali consumption for high recovery of xylan from biomass with relatively higher lignin content for its valorisation into a potential prebiotic oligosaccharide.

Fast automated online xylanase activity assay using HPAEC-PAD.[Pubmed:29184998]

Anal Bioanal Chem. 2018 Jan;410(1):57-69.

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, Xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Identification and characterization of the first beta-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10.[Pubmed:29499784]

Enzyme Microb Technol. 2018 May;112:72-78.

In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a beta-d-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40 degrees C. The enzyme was stable up to 40 degrees C over a wide pH range (3.1-8.9). rSWU43A activity was enhanced by Fe(2+) and Mn(2+) and inhibited by various metals (Ag(+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Ni(2+), and Zn(2+)), d-xylose, and l-arabinose. rSWU43A showed activity on p-nitrophenyl-beta-d-xylopyranoside and p-nitrophenyl-alpha-l-arabinofuranoside substrates, with specific activities of 0.09 and 0.06U/mg, respectively, but not on any xylosidic or arabinosidic polymers. rSWU43A efficiently degraded beta-1,3-xylooligosaccharides to produce xylose but showed little activity towards beta-1,4-Xylobiose, with specific activities of 1.33 and 0.003U/mg, respectively. These results demonstrate that SWU43A is a beta-1,3-d-xylosidase (EC 3.2.1.72), which to date has only been described in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first beta-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool for the structural elucidation and production of xylose from beta-1,3-xylan in seaweed cell walls.

Structure-based protein engineering of bacterial beta-xylosidase to increase the production yield of xylobiose from xylose.[Pubmed:29752942]

Biochem Biophys Res Commun. 2018 Jun 27;501(3):703-710.

Xylobiose consists of two molecules of xylose and has been highly recognized as a food supplement because it possesses high prebiotic functions. beta-xylosidase exhibits enzymatic activity to hydrolyze Xylobiose, and the enzyme can also catalyze the reverse reaction in the presence of high concentrations of xylose. Previously, beta-xylosidase from Bacillus pumilus IPO (BpXynB), belonging to GH family 43, was employed to produce Xylobiose from xylose. To improve the enzymatic efficiency, this study determined the high-resolution structure of BpXynB in a complex with Xylobiose and engineered BpXynB based on the structures. The structure of BpXynB deciphered the residues involved in the recognition of the Xylobiose. A site-directed mutation at the residue for Xylobiose recognition increased the yield of Xylobiose by 20% compared to a similar activity of the wild type enzyme. The complex structure of the mutant enzyme and Xylobiose provided the structural basis for a higher yield of the engineered protein. This engineered enzyme would enable a higher economic production of Xylobiose, and a similar engineering strategy could be applied within the same family of enzymes.

Xylobiose Prevents High-Fat Diet Induced Mice Obesity by Suppressing Mesenteric Fat Deposition and Metabolic Dysregulation.[Pubmed:29558403]

Molecules. 2018 Mar 20;23(3). pii: molecules23030705.

Obesity is a public concern and is responsible for various metabolic diseases. Xylobiose (XB), an alternative sweetener, is a major component of xylo-oligosaccharide. The purpose of this study was to investigate the effects of XB on obesity and its associated metabolic changes in related organs. For these studies, mice received a 60% high-fat diet supplemented with 15% d-xylose, 10% XB, or 15% XB as part of the total sucrose content of the diet for ten weeks. Body weight, fat and liver weights, fasting blood glucose, and blood lipids levels were significantly reduced with XB supplementation. Levels of leptin and adipokine were also improved and lipogenic and adipogenic genes in mesenteric fat and liver were down-regulated with XB supplementation. Furthermore, pro-inflammatory cytokines, fatty acid uptake, lipolysis, and beta-oxidation-related gene expression levels in mesenteric fat were down-regulated with XB supplementation. Thus, XB exhibited therapeutic potential for treating obesity which involved suppression of fat deposition and obesity-related metabolic disorders.

Biochemical and biophysical characterization of novel GH10 xylanase prospected from a sugar cane bagasse compost-derived microbial consortia.[Pubmed:29274424]

Int J Biol Macromol. 2018 Apr 1;109:560-568.

Environmental issues are promoting the development of innovative technologies for the production of renewable energy and "green products" from plant biomass residues. These technologies rely on the conversion of the plant cell wall (PCW) polysaccharides into simple sugars, which involve synergistic activities of different PCW degrading enzymes, including xylanases; these are widely applied in food and feed sectors, paper and textile industries, among others. We cloned, expressed and biochemically characterized a novel xylanase (Xyn10) from the GH10 identified in a metatranscriptome of compost-derived microbial consortia and determined its low-resolution SAXS molecular envelope in solution. Our results reveal that Xyn10 is a monomeric flexible globular enzyme, with high stability with a broad pH range from 4 to 10 and optimal activity conditions at pH 7 and 40 degrees C. Only 10% of activity loss was observed after the enzyme was incubated for 30h at 40 degrees C with a pH ranging from 5 to 10. Moreover, Xyn10 maintained 100% of its initial activity after incubation for 120h at 40 degrees C and 51% after incubation for 24h at 50 degrees C (pH=7.0). Xyn10 shows endocatalytic activity towards xylan and arabinoxylan, liberating xylose, Xylobiose, 1,2-alpha-d-methylglucuronic acid decorated xylotriose, and 1,3-alpha-l-arabinofuranose decorated Xylobiose and xylotriose oligosaccharides.