hemoglobin-randwick and galactomannan

Many glycoside hydrolases involved in deconstruction of cellulose and xylan from the excellent plant cell wall polysaccharides-degrader Caldicellulosiruptor bescii have been cloned and analyzed. However, far less is known about the enzymatic breakdown of mannan, an important component of hemicellulose. We herein cloned, expressed and purified the first β-mannosidase CbMan2A from C. bescii. CbMan2A is thermophilic, with an optimal temperature of 80 °C. CbMan2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with CbMan5A, an endo-mannanase from the same bacterium, in releasing mannose from β-1,4-mannan. Thus CbMan2A forms the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how CbMan2A may assist C. bescii in mannan utilization is presented. In addition, CbMan2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, CbMan2A represents an excellent model for investigation of the binding of GH2 β-mannosidases to galactomannan.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Mannosidase; Cloning, Molecular; Escherichia coli; Firmicutes; Galactose; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mannans; Mannose; Molecular Sequence Data; Protein Binding; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Temperature

Genes for the enzymes that make plant cell wall hemicellulosic polysaccharides remain to be identified. We report here the isolation of a complementary DNA (cDNA) clone encoding one such enzyme, mannan synthase (ManS), that makes the beta-1, 4-mannan backbone of galactomannan, a hemicellulosic storage polysaccharide in guar seed endosperm walls. The soybean somatic embryos expressing ManS cDNA contained high levels of ManS activities that localized to Golgi. Phylogenetically, ManS is closest to group A of the cellulose synthase-like (Csl) sequences from Arabidopsis and rice. Our results provide the biochemical proof for the involvement of the Csl genes in beta-glycan formation in plants.

Topics: Amino Acid Sequence; Arabidopsis; Catalytic Domain; Cellulose; Cyamopsis; Databases, Nucleic Acid; Expressed Sequence Tags; Galactose; Gene Expression; Gene Library; Genes, Plant; Glucosyltransferases; Glycine max; Golgi Apparatus; Mannans; Mannose; Mannosyltransferases; Molecular Sequence Data; Multigene Family; Oryza; Phylogeny; Plants, Genetically Modified; Protein Structure, Tertiary; Seeds; Transformation, Genetic

Galactomannan was coupled to a protein carrier for the preparation of monoclonal antibodies. The monoclonal antibodies generated bound to galactomannans from different sources as well as to glucomannan and galactoglucomannan. One monoclonal antibody, BGM C6, was characterised and found to be specific for (1-->4)-beta-linked mannopyranosyl residues; it had a binding affinity estimated at 1x10(-6) M for the (1-->4)-beta-linked mannohexaose. BGM C6 was used in immunogold labelling studies to locate galactomannans in the endosperm walls of normal coconuts (Cocos nucifera L.) and those of the mutant makapuno at two different developmental stages. The pattern and intensity of antibody labelling varied for each type of coconut at the mature and immature stages, indicating differences in the galactomannan composition of the endosperm walls.

Topics: Antibodies, Monoclonal; Cocos; Galactose; Immunohistochemistry; Mannans; Molecular Structure; Mutation; Protein Transport; Seeds