mannopentaose and galactomannan

Degradation of mannans is a key process in the production of foods and prebiotics. β-Mannanase is the key enzyme that hydrolyzes 1,4-β-D-mannosidic linkages in mannans. Heterogeneous expression of β-mannanase in Pichia pastoris systems is widely used; however, Saccharomyces cerevisiae expression systems are more reliable and safer. We optimized β-mannanase gene from Aspergillus sulphureus and expressed it in five S. cerevisiae strains. Haploid and diploid strains, and strains with constitutive promoter TEF1 or inducible promoter GAL1, were tested for enzyme expression in synthetic auxotrophic or complex medium. Highest efficiency expression was observed for haploid strain BY4741 integrated with β-mannanase gene under constitutive promoter TEF1, cultured in complex medium. In fed-batch culture in a fermentor, enzyme activity reached ~ 24 U/mL after 36 h, and production efficiency reached 16 U/mL/day. Optimal enzyme pH was 2.0-7.0, and optimal temperature was 60 °C. In studies of β-mannanase kinetic parameters for two substrates, locust bean gum galactomannan (LBG) gave K

Topics: Aspergillus; Batch Cell Culture Techniques; beta-Mannosidase; DNA, Fungal; Galactans; Galactokinase; Galactose; Gene Dosage; Gene Expression Regulation, Enzymologic; Hydrolysis; Industrial Microbiology; Mannans; Mannose; Oligosaccharides; Pichia; Plant Gums; Promoter Regions, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Trisaccharides

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-β-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 counter part Alicyclobacillus acidocaldarius ManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ≈ 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 β-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 β-mannanases and distinguishes them from counterparts of other families.

Topics: Alicyclobacillus; beta-Mannosidase; Binding Sites; Catalysis; Enzyme Activation; Galactose; Glycosides; Hydrolysis; Mannans; Mannosidases; Molecular Docking Simulation; Mutagenesis, Site-Directed; Oligosaccharides; Recombinant Proteins; Structural Homology, Protein; Substrate Specificity; Trisaccharides

Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-D-galacto-β(1 → 4)-D-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with (15)N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-D-galactopyranosyl doublets surrounded by regions of about four or more "naked" mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [6(1)-α(1 → 6)-D-galactosyl-β-D-mannotriaose and 6(3),6(4)-di-α(1 → 6)-D-galactosyl-β-D-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.

Topics: Amino Acid Motifs; Binding Sites; Carbohydrate Conformation; Drug Design; Galactose; Galectin 1; Humans; Magnetic Resonance Spectroscopy; Mannans; Models, Molecular; Oligosaccharides; Protein Binding; Protein Structure, Quaternary; Protein Structure, Tertiary; Trisaccharides

Carbohydrate-protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, "carbohydrate-binding modules" (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both the variable backbone and the side-chain decorations of heterogeneous ligands. "CBM29" modules, derived from a noncatalytic component of the Piromyces equi cellulase/hemicellulase complex, provide an example of this selective yet flexible recognition. They discriminate strongly against some polysaccharides while remaining relatively promiscuous toward both beta-1,4-linked manno- and cello-oligosaccharides. This feature may reflect preferential, but flexible, targeting toward glucomannans in the plant cell wall. The three-dimensional structure of CBM29-2 and its complexes with cello- and mannohexaose reveal a beta-jelly-roll topology, with an extended binding groove on the concave surface. The orientation of the aromatic residues complements the conformation of the target sugar polymer while accommodation of both manno- and gluco-configured oligo- and polysaccharides is conferred by virtue of the plasticity of the direct interactions from their axial and equatorial 2-hydroxyls, respectively. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components in the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote.

Topics: Binding Sites; Carbohydrate Sequence; Carbohydrates; Cellulase; Crystallography, X-Ray; Fungal Proteins; Galactose; Glycoside Hydrolases; Ligands; Mannans; Models, Molecular; Molecular Sequence Data; Oligosaccharides; Piromyces; Protein Structure, Tertiary; Recombinant Fusion Proteins; Substrate Specificity