Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

Abstract

cXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol ؊1 versus 94.7 U nmol ؊1 ) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (T m ), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions. X ylanases (endo-1,4-␤-xylanase; EC 3.2.1.8) hydrolyze the ␤-1,4 bond in the xylan backbone of hemicellulose, which yields short xylooligosaccharides or xylose. They are distributed in several glycoside hydrolase families (GH), such as GH5, GH7, GH8, GH10, GH11, and GH43, and most of them belong to GH10 and GH11 according to the CAZy (Carbohydrate Active Enzymes) database (1). Xylanase families differ in their physicochemical properties (molecular mass and isoelectric point [pI]), three-dimensional structures, and catalytic mechanisms (2). Enzymes from GH family 11 have a relatively low molecular weight and a high pI, and they possess a ␤-jelly roll secondary structure, a double displacement catalytic mechanism, and two glutamates acting as catalytic residues (3). In contrast, members of GH10 typically have a high molecular mass and a low pI and display an (␣/␤) 8 barrel fold. Xylanases have been found in many organisms, such as bacteria, fungi, algae, and protozoa (4). Xylanases from fungi or mesophilic bacteria generally share a low optimum temperature and poor thermal stability (5). Thermostable xylanases have been characterized from some thermophilic microorganisms, such as Clostridium thermocellum (6...

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

“…Deletion of the carbohydrate-binding modules (CBM) has effect on the thermo-stability of GH10 and GH11 xylanases. The truncated mutant XynATM1 harboring a GH11 catalytic module without CBM showed an improved thermal stability compared to XynA17. Moreover, DNA shuffling and error prone PCR were applied to engineer recombinant xylanase with improved thermo-stability181920.…”

Two Distinct α- l -Arabinofuranosidases in Caldicellulosiruptor Species Drive Degradation of Arabinose-Based Polysaccharides

“…The target genes with blunt ends were amplified from the genomic DNA of Caldicellulosiruptor sp. strain F32 and C. saccharolyticus using Kapa HiFi DNA polymerase (Kapa, USA) and cloned into pEASY blunt-E2 vector (TransGen, China), as previously described (22). The primers for cloning the genes are listed in Table 3.…”

“…To investigate synergistic effects of two arabinofuranosidases with other hemicellulases, xylanases XynA, XynA-TM, and XynB and xylosidase Xyl39B from Caldicellulosiruptor sp. strain F32 were produced as previously described (22). To determine the extent of synergy, different molar ratios (expressed in nanomoles) were used to obtain higher reducing ends from WAX.…”

“…Caldicellulosiruptor XynA is a typical GH11 xylanase, releasing short xylooligosaccharides (XOSs) such as xylobiose, xylotriose, and xylotetraose (22). DNS analysis indicated that XynA was synergistic with AbF51 and XynF (Fig.…”

“…To investigate the synergistic effects of the AbFs, analysis of the reducing saccharides that were released from insoluble WAX by combinations of three different endo-xylanases was performed by the 3,5-dinitrosalicylic acid (DNS) method (22). Different molar ratios were used to observe the synergistic effects of two AbFs with endo-xylanases ( Fig.…”