c Few members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-␤-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilic Alicyclobacillus sp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counterpart Alicyclobacillus acidocaldarius ManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose Ϸ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of ␤-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support ؊4 to ؊1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 ␤-mannanases and distinguishes them from counterparts of other families.A major component of hemicellulose in the plant cell walls of softwood, plant seeds, and beans is ␤-1,4-mannan, including mannan polysaccharides, glucomannan, galactomannan, and galactoglucomannan. It is composed of a backbone of ␤-1,4-linked mannose or a combination of glucose and mannose with side chains of ␣-1,6-linked galactose residues (1). The mannan-degrading enzymes have biotechnological applications in various areas, such as feed manufacturing (2), paper processing (3), and coffee extract treatment (4). Mannooligosaccharides, the major hydrolysis products of mannan, are beneficial as animal nutrition additives due to their potential prebiotic properties (5). The complete degradation of mannan polysaccharides into monomers requires an enzyme system including ␤-mannanase (EC 3.2.1.78), ␤-mannosidase (EC 3.2.1.25), ␤-glucosidase (EC 3.2.1.21), and ␣-galactosidase (EC 3.2.1.22). Of these, ␤-mannanase randomly hydrolyzes the ␤-D-1,4-mannopyranoside linkages and plays the major role in mannan degradation.␤-Mannanases are widely distributed in various organisms, including bacteria, yeasts, filamentous fungi, and plants. Based on the amino acid sequence and structural similarities of catalytic domains, ␤-mannanases are grouped into three glycoside hydrolase (GH) families in the Carbohydrate-Active enZYmes (CAZy) database (6), i.e., 5, 26, and 113. GH 1...

A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0–9.0 (incubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the same microbial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.