A Novel Multifunctional Arabinofuranosidase/Endo-xylanase/β-Xylosidase GH43 from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action to Produce Arabinose and Xylose from Cereal Arabinoxylan

The PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6) and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-L-arabinofuranosidase, endo-xylanase and β-D-xylosidase activities. α-L-Arabinofuranosidase of PcAxy43B revealed the new property of GH43, which released arabinose from the short-chain arabinoxylo-oligosaccharide (AXOS) and cereal arabinoxylan, and from both sides of the xylose residues of AXOS, which usually obstruct the action of xylanolytic enzymes. The PcAxy43B liberated series of xylo-oligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endo-xylanase activity. The PcAxy43B produced xylose from xylobiose and reacted with p -nitrophenyl-β-D-xylopyranoside as a result of β-xylosidase activity. The PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with a trifunctional endo-xylanase/β-xylosidase/α-L-arabinofuranosidase PcAxy43A and an endo-xylanase Xyn10C from the strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endo-xylanase property of PcAxy43B. The PcAxy43B is capable of hydrolysing untreated cereal biomass, corn hull and rice straw into XOSs and xylose. Hence, PcAxy43B, the significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-L-arabinofuranosidase/endo-xylanase/β-D-xylosidase enzyme of the glycoside hydrolase family 43. It is effective in releasing arabinose, xylose and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with trifunctional xylanolytic enzyme PcAxy43A and endo-xylanase Xyn10C from the strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolysing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

Thermoxylanolytic and Thermosaccharolytic Potential of a Heat Adapted Bacterial Consortium Developed From Goat Rumen Contents

Thermoactive xylanases have important applications in the industrial deconstruction of lignocellulosic plant biomass, due to their sustained activity even at high temperature conditions of industrial bioreactors. We herein report the development of a thermoactive xylanolytic microbial consortium from the semi-digested contents of goat rumen and characterization of the xylanolytic enzyme cocktail produced by it. The consortium exhibited maximum endoxylanase activity at pH6 and at 60°C. Zymogram analysis revealed the production of multiple xylanases. The xylanase cocktail was stable over a pH range of 5–9 after pre-incubation for 3 h. It retained 74% activity after pre-incubation (60°C) for 50 min. It’s activity was enhanced in presence of β-mercaptoethanol, NH4+, Mg2⁺ and Ca2⁺, whereas Hg2⁺ had an inhibitory effect. The xylanolytic cocktail was further utilized for the saccharification of alkali pre-treated rice straw and mushroom spent rice straw. Saccharification was studied quantitatively using the dinitrosalicylic acid method and qualitatively using scanning electron microscopy. Results indicated the potential of the xylanolytic cocktail for the saccharification of rice straw and highlighted the significance of chemical and/or biological pre-treatment in improving the accessibility of the substrate towards the xylanase cocktail.

Improved Production of Xylanase in Pichia pastoris and Its Application in Xylose Production From Xylan

Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.