A Novel Multifunctional Arabinofuranosidase/Endoxylanase/β-Xylosidase GH43 Enzyme from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action To Produce Arabinose and Xylose from Cereal Arabinoxylan

Abstract

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, endoxylanase, and β-d-xylosidase activities. The α-l-arabinofuranosidase activity of PcAxy43B revealed a new property of GH43, via the release of both long-chain cereal arabinoxylan and short-chain arabinoxylooligosaccharide (AXOS), as well as release from both the C(O)₂ and C(O)₃ positions of AXOS, which is different from what has been seen for other arabinofuranosidases. PcAxy43B liberated a series of xylooligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endoxylanase activity. PcAxy43B produced xylose from xylobiose and reacted with p-nitrophenyl-β-d-xylopyranoside as a result of β-xylosidase activity. PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with the trifunctional endoxylanase/β-xylosidase/α-l-arabinofuranosidase PcAxy43A and the endoxylanase Xyn10C from 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 endoxylanase property of PcAxy43B. PcAxy43B is capable of hydrolyzing untreated cereal biomass, corn hull, and rice straw into XOSs and xylose. Hence, PcAxy43B, a 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/endoxylanase/β-d-xylosidase enzyme of 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 the trifunctional xylanolytic enzyme PcAxy43A and the endoxylanase Xyn10C from strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolyzing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

Keywords: Paenibacillus curdlanolyticus; carbohydrate-binding module; cereal arabinoxylan; cereal biomass; glycoside hydrolase family 43; multifunctional xylanolytic enzyme; α-l-arabinofuranosidase/endoxylanase/β-xylosidase activities.

FIG 1

(A) Schematic illustration of modular structures of PcAxy43B, PcAxy43B_36, PcCBM36, PcAxy43A, Xyn10C, and GH43B6 from P. curdlanolyticus B-6. SP, signal peptide module of a transmembrane protein; GH43_16, glycoside hydrolase family 43 subfamily 16 catalytic domain; GH43_35, glycoside hydrolase family 43 subfamily 35 catalytic domain; GH43_1, glycoside hydrolase family 43 subfamily 1 catalytic domain; GH10, glycoside hydrolase family 10 domain; CBM6, family 6 carbohydrate-binding module; CBM36, family 36 carbohydrate-binding module. (B) Amino acid sequence alignment of the GH43 catalytic domain of PcAxy43B from P. curdlanolyticus B-6 compared to other characterized GH43 subfamily 16 members, including the bifunctional endoxylanase/α-l-arabinofuranosidase XynD (GenBank accession number CAA40378.1) from P. polymyxa (16), the α-l-arabinofuranosidase BsAXHm23 (GenBank accession number CAB13699.1) from B. subtilis 168 (17), and the β-xylosidase Xyn43A (GenBank accession number ACZ98594.1) from C. ruminicola CGMCC 1.5065 (18). Conserved residues are highlighted in black, while essential catalytic residues are indicated by an asterisk. The four glucosyl substrate-binding subsites are numbered −3, −2, −1, and +1. The alignment was constructed by the Clustal Omega program on the EMBL-EBI server (https://www.ebi.ac.uk/Tools/msa/clustalo/). (C) A phylogenetic tree of the GH43 catalytic domain of PcAxy43B with all other characterized members of GH43 subfamily 16 (12 members) was constructed by the neighbor-joining method with bootstrap analysis of 1,000 replicates in MEGA 6.0 software (19). The number for each node is the bootstrap percentage. The scale for branch length is shown below the tree. Most members exhibited α-l-arabinofuranosidase activity, such as Axh43A (GenBank accession number BAL45491.1) from B. licheniformis SVD1, AXH (GenBank accession number AKF02745.1) from Bacillus sp. BP-7, BsAXHm23 (GenBank accession number CAB13699.1) from B. subtilis 168, CtAbf43A (GenBank accession number ABN52503.1) from H. thermocellum ATCC 27405, Abf43B (GenBank accession number CBL17514.1) from R. champanellensis 18P13, XynF (GenBank accession number AAB87371.1) from C. saccharolyticus, Csac_2411 (GenBank accession number ABP67988.1) from C. saccharolyticus DSM 8903, XynC (GenBank accession number AAB95326.1) from Caldicellulosiruptor sp. Rt69B.1, XynA (GenBank accession number AAD30363.1) from Caldicellulosiruptor sp. Tok7B.1, and Abf43E (GenBank accession number CBL17682.1) from R. champanellensis 18P13, while only one member of the β-xylosidase Xyn43A (GenBank accession number ACZ98594.1) from C. ruminicola CGMCC 1.5065 and one member of the bifunctional α-l-arabinofuranosidase/endoxylanase XynD from P. polymyxa have been reported.

FIG 2

Analysis of hydrolysis products of RAX, xylobiose, and A-X-A by PcAxy43B or PcAxy43B_36. (A and B) TLC patterns of the RAX hydrolysate by PcAxy43B (A) and PcAxy43B_36 (B). (C) TLC patterns of xylobiose by PcAxy43B_36. (D) TLC patterns of A-X-A by PcAxy43B for 1 h. Standards X1 to X6 are marked by M. Lane A, L-arabinose; lane X, d-xylose; lane A-X-A, diarabinosyl substituted at the C(O)₂ and C(O)₃ positions of xylose; lane A-X, monoarabinosyl substituted at the C(O)₂ or C(O)₃ position of xylose; lane 1, A-X-A hydrolysate products. The reactions on RAX, xylobiose, and A-X-A were carried out by incubating 1 μM enzyme with 10 mg · ml⁻¹ of each substrate in PBS-CaCl₂ buffer at 50°C with shaking at 200 rpm.

FIG 3

Analysis of hydrolysis products of BWX, XOSs, and cereal biomass by PcAxy43B. (A) TLC patterns of BWX hydrolysates. (B) HPLC profiles of X2 and X6 hydrolysates at 1 h. (C) TLC patterns of corn hull and rice straw hydrolysates at 18 h. Lanes CH and RS, control; lanes 1 and 2, reactions of the substrates incubated with PcAxy43B. Standards X1 to X6 are marked by M. Lane A, L-arabinose. The reactions were carried out by incubating 0.5 U of xylanase enzyme with 10 mg · ml⁻¹ of each polysaccharide or 2 mg · ml⁻¹ of each XOS in PBS-CaCl₂ buffer at 50°C with shaking at 200 rpm.

FIG 4

HPLC profiles of RAX hydrolysates by the combination of PcAxy43B, PcAxy43A, and Xyn10C. PcAxy43B was incubated in combination with PcAxy43A and/or Xyn10C at a ratio of 1:1 or 1:1:1 (on the basis of the enzyme concentration). The reaction was performed by incubating 1 μM each enzyme independently or in combination with RAX (10 mg · ml⁻¹) in PBS-CaCl₂ buffer at 50°C with shaking at 200 rpm for 1 h. Each standard sugar was used at 2 mg · ml⁻¹.