mannopentaose and mannotetraose

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

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-β-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.

Topics: Amino Acid Sequence; Aspergillus nidulans; beta-Mannosidase; Catalysis; Fungal Proteins; Mannans; Mannosidases; Molecular Sequence Data; Oligosaccharides; Phylogeny; Sequence Analysis, Protein

The glycolytic enzyme triosephosphate isomerase (TPI; EC 5.3.1.1) of Staphylococcus aureus is a candidate adhesion molecule for the interaction between the bacterium and the fungal pathogen Cryptococcus neoformans. TPI may recognize the mannan backbone of glucuronoxylomannan (GXM) of C. neoformans. We purified TPI from extracts of S. aureus surface proteins to investigate its binding by surface plasmon resonance analysis. The immobilized TPI reacted with GXM in a dose-dependent manner. Furthermore, the interactions between staphylococcal TPI and alpha-(1-->3)-mannooligosaccharides derived from GXM were examined. The oligosaccharides exhibited binding with TPI; however, monomeric mannose did not. Differences in the slopes of the sensorgrams were observed between oligosaccharides with an even number of residues versus those with an odd number. A heterogeneous ligand-parallel reaction model revealed the existence of at least two binding sites on TPI. The enzymic activities of TPI were inhibited in a dose-dependent manner by alpha-(1-->3)-mannooligosaccharides larger than triose. The binding of TPI and alpha-(1-->3)-mannotriose near the substrate-binding site was predicted in silico (AutoDock 3.05). An oligosaccharide of size equal to or greater than triose could bind to the site, affecting enzymic activities. Moreover, affinities were indicated, especially for biose and tetraose, to another binding pocket, which would not affect enzymic activity. These data suggest a novel role for TPI, in addition to glycolysis, on the surface of S. aureus.

Topics: Bacterial Adhesion; Binding Sites; Cryptococcus neoformans; Mannans; Mannose; Models, Biological; Oligosaccharides; Polysaccharides; Protein Binding; Staphylococcus aureus; Triose-Phosphate Isomerase; Trisaccharides

To obtain manno-oligosaccharides containing beta-1,2-linked nonreducing terminal groups from the mannan of Pichia pastoris IFO 0948 strain by acetolysis, an attempt was made to establish the reaction conditions under which cleavage of the alpha-1,6 linkage took place preferentially leaving manno-oligosaccharides composed largely of beta-1,2 linkages. By the action of an ordinary acetolysis medium, a 10/10/1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 13 h or at 25 degrees C for 120 h, the O-acetyl derivative of this mannan gave mannose, mannobiose, mannotriose, and mannopentaose. However, treatment of the same O-acetyl mannan with a 50/50/1 (v/v) acetolysis medium at 40 degrees C for 15 h gave a mannotetraose in addition to mannose, mannobiose, mannotriose, and mannopentaose. Use of a 100/100/1 (v/v) acetolysis medium at 40 degrees C for 36 h gave a more satisfactory result, a mixture of oligosaccharides, from mannose to mannopentaose, which contained more mannotetraose than mannopentaose. Because both mannotetraose and mannopentaose contained alpha-1,2 and beta-1,2 linkages, it was concluded that an acetolysis medium containing a low concentration of sulfuric acid, up to 0.5% (v/v), facilitates the preferential cleavage of the alpha-1,6 linkage, leaving manno-oligosaccharides containing the beta-1,2 linkage which was found to be labile to the action of the 10/10/1 (v/v) acetolysis medium.

Topics: Acetylation; Cell Wall; Chemical Phenomena; Chemistry; Hydrolysis; Magnetic Resonance Spectroscopy; Mannans; Mannose; Methylation; Oligosaccharides; Saccharomycetales; Sulfuric Acids