cellulase and galactomannan

Spent coffee grounds (SCG) are a promising raw material for galactomannan (GalM) production based upon its enrichment in galactomannan polysaccharides. In this work, SCG was pretreated by autohydrolysis for maximumly improving GalM extractability by endo-mannanase. The GalM in the prehydolyzate (GalM-PH) and enzymatic hydrolyzate (GalM-EH) were obtained by ethanol precipitation and characterized. Under the optimized autohydrolysis conditions, 50.1% of GalM in pretreated SCG was converted into free GalM in enzymatic hydrolyzate. Compositional analysis results revealed that GalM-PH was comprised of 81.7% galactomannan, higher than that of GalM-EH (76.4%). The molecular weight of GalM-PH and GalM-EH were 44.5 kDa and 28.0 kDa, respectively. Antioxidant assays indicated that both GalM-EH and GalM-PH could scavenge 2,2-diphenyl-1-picryl-hydrazyl radicals and hydroxyl radicals. Immunological and prebiotics analysis showed all GalM preparations exhibited pronounced activities for proliferating the probiotics and proliferating the Macrophages cell for NO production, in which the GalM-EH outperformed the GalM-PH. These results imply that the GalM extracted from SCG are the bioactive substances that can be used as antioxidant, prebiotics, and immunostimulants.

Topics: Antioxidants; beta-Mannosidase; Cellulase; Coffee; Ethanol; Galactose; Humans; Hydrolysis; Mannans; Polysaccharides; Waste Products

In our previous study using a fluorescently labeled cohesin biomarker, we detected and identified a putative cellulosomal mannanase belonging to the glycosyl hydrolase family 26 from Clostridium cellulovorans in xylan-containing cultures. In this study, a mannanase gene, manB from C. cellulovorans, was expressed in Escherichia coli. The optimal pH of a purified enzyme was around pH 7.0 and the optimal temperature was 40 °C. The purified mannanase B (ManB) showed high hydrolytic activity toward galactomannan. An assembly of ManB with mini-CbpA, which contains a carbohydrate-binding module that provides proximity to insoluble substrates, increased the activity toward galactomannan [locust bean gum (LBG) and guar gum] 1.7- and 2.0-fold over those without mini-CbpA. We tested the synergistic effects on galactomannan (LBG and guar gum) degradation using cellulosomal mannanase ManB with cellulosomal endoglucanase E, which was predicted to have mannanase activity in C. cellulovorans as a cellulolytic complex. When assembled with the mini-CbpA, the mixture of endoglucanase E (EngE) and ManB at a molar ratio of 1:2 showed the highest synergistic effect (2.4-fold) on LBG. The mixture at a ratio of 1:3 showed the highest synergistic effect (2.8-fold) on guar gum. These synergistic actions indicated that ManB assembled with mini-CbpA hydrolyzed insoluble galactomannan, which in turn promoted soluble galactomannan degradation by EngE.

Topics: Bacterial Proteins; beta-Mannosidase; Cellulase; Clostridium cellulovorans; Escherichia coli; Galactans; Galactose; Gene Expression Regulation, Bacterial; Mannans; Mannose-6-Phosphate Isomerase; Multienzyme Complexes; Nucleotidyltransferases; Plant Gums; Xylans

Endoglucanases are important enzymes for biomass conversion and other industrial processes. Determining the specificity of endoglucanases from various glycoside hydrolase families is of interest for bioinformatic functional prediction and substrate-tailored enzyme development. To do so, we characterized approximately 30 endoglucanases from six glycoside hydrolase families. For p-nitrophenyl cellobioside and lactoside, only family 7 enzymes showed significant activity. For xyloglucan, both family 7 and 12 enzymes showed significant activity. For xylan and arabinoxylan, only family 7 enzymes showed significant activity. For mannan and galactomannan, both family 5 and 9 enzymes showed significant activity. The difference in specificity was preliminarily attributed mainly to the structural difference of the enzymes' active sites. For family 7 endoglucanases, difference in thermal stability might affect their performance in hydrolyzing various (hemi)cellulose substrates. Phylogenetic analysis on the subfamily distribution of family 5 endoglucanases (in relation with mannanases) suggested that their mannanase side-activity might be the remnant of an ancestral multi-function enzyme. Similar analysis was also made with the xyloglucanase or arabionxylans side-activity of family 12 and 7 endoglucanases. The apparent dependence of the specificity on family (primary/tertiary structure) might assist us in better understanding the structure-function relationship of the enzymes, and developing more versatile biocatalysts for the utilization of biomass.

Topics: Biocatalysis; Cellulase; Cellulose; Enzyme Stability; Galactose; Mannans; Multigene Family; Phylogeny; Polysaccharides; Substrate Specificity; Temperature; Xylans

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

The Clostridium josui aga27A gene encodes the cellulosomal alpha-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly. The catalytic domain is highly homologous to those of various alpha-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants. The recombinant Aga27A alpha-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose.

Topics: alpha-Galactosidase; Amino Acid Sequence; Animals; Base Sequence; Cellulase; Clostridium; DNA, Bacterial; Galactose; Humans; Mannans; Mice; Molecular Sequence Data; Mutagenesis; Organelles; Sequence Analysis, DNA; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Substrate Specificity

Sclerotium rolfsii CBS 191.62 was cultivated on a number of carbon (C) sources, including mono- and disaccharides, as well as on polysaccharides, to study the formation of different mannan-degrading enzyme activities. Highest levels of mannanase activity were obtained when alpha-cellulose-based media were used for growth, but formation of mannanase could not be enhanced by employing galactomannan as the only carbon source. Although both xylanase and cellulase formation was almost completely repressed when S. rolfsii was grown on more readily metabolizable carbohydrates, including glucose or mannose, considerable amounts of mannanase activity were secreted under these growth conditions. Enhanced mannanase production only commenced when glucose was depleted in the medium. The maximal mannanase activity of 240 IU/mL obtained in a laboratory fermentation is remarkable. Mannanase activity formed under these derepressed conditions could be mainly attributed to one major, acidic mannanase isoenzyme with a pI value of 2.75.

Topics: Basidiomycota; beta-Mannosidase; Biodegradation, Environmental; Cellulase; Cellulose; Culture Media; Fermentation; Galactose; Glucose; Isoenzymes; Mannans; Mannosidases; Xylan Endo-1,3-beta-Xylosidase; Xylosidases