Low Molecular Weight Oligosaccharides Research Articles

Protein microarray technology.

This review summarizes the major activities in the field of protein microarray technology. A short summary of the theoretical concepts of miniaturized ligand binding assays explains why such microspot assays represent the most sensitive approaches for capture-target assays. The main focus of this review is centered on the applications using miniaturized and parallelized protein binding assays which rely on the product formation between immobilized capture molecules and their corresponding target molecules which are present in the sample. These types of ligand binding assays are useful tools for protein identification, quantification and protein affinity studies. Protein identification and quantification assays have a great potential in the field of diagnostics and proteomics where many different protein markers which are present in complex samples have to be analyzed in parallel. Protein affinity assays can be used to analyze interactions between proteins such as antibodies, receptors or enzymes with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Different applications of protein microarray-based assays and their huge potential for diagnostic and proteomic approaches will be discussed.

Homopolysaccharides from lactic acid bacteria

Abstract In addition to heteropolysaccharides of complex structure, lactic bacteria produce a variety of homopolysaccharides containing only either d -fructose or d -glucose. These fructans and glucans have a common feature in being synthesized by extracellular transglycosylases (glycansucrases) using sucrose as glycosyl donor. The energy of the osidic bond of sucrose enables the efficient transfer of a d -fructosyl or d -glucosyl residue via the formation of a covalent glycosyl-enzyme intermediate. In addition to the synthesis of high molecular weight homopolysaccharides, glycansucrases generally catalyse the synthesis of low molecular weight oligosaccharides or glycoconjugates when efficient acceptors, like maltose, are added to the reaction medium. While the enzymatic synthesis of fructans (levan and inulin) is poorly documented at the molecular level, the field of Streptococcus and Leuconostoc glucansucrases (glucosyltransferases and dextransucrases) has been well studied, both at the mechanistic and gene structure levels. The nutritional applications of the corresponding polysaccharides and oligosaccharides account for this increasing interest.

13C NMR Studies of Hyaluronan. 2. Dependence of Conformational Dynamics on Chain Length and Solvent

The conformational dynamics of low molecular weight hyaluronan (HA) polymer and oligosaccharides have been assayed by 13C NMR T1 relaxation time measurements. For polymeric HA in aqueous solution, the average relaxation time for ring carbons was not significantly affected by changing the ionic strength or by changing counterion type. The average relaxation time of HA was found to be similar to that for chondroitin 4-sulfate or chondroitin 6-sulfate in aqueous solution. As has been observed previously for other polysaccharides, the segmental motions of HA chains studied at room temperature in aqueous solution appear to be dominated by viscous damping of the chain motions. Within a chain structure, there are variations in observed T1 that correspond to differences in relative mobility. The hydroxymethyl substituent group shows a difference in relaxation rate relative to the ring to which it is attached, depending on the ring configuration (glucose vs galactose). These differences correlate with relative rotational isomerization rates measured by ultrasonic relaxation. In short HA chains, the nonreducing and reducing terminal residues show much greater mobility than penultimate residues, and these are in turn more mobile than interior residues. In contrast, the more rigid hydrogen-bonded conformation of an HA tetrasaccharide in dimethyl sulfoxide solution shows less position-dependent variation in T1. These data are in accord with the dynamic nature of conformation-stabilizing hydrogen bonds for HA chains in aqueous solution.

Differential Biosynthesis of Polysialic Acid on Neural Cell Adhesion Molecule (NCAM) and Oligosaccharide Acceptors by Three Distinct α2,8-Sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III

Polysialylated neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Polysialylation of NCAM was shown to be achieved by two alpha2,8-polysialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which are moderately related to another alpha2,8-sialyltransferase, ST8Sia III. Here we describe that all three alpha2,8-sialyltransferases can utilize oligosaccharides as acceptors but differ in the efficiency of adding polysialic acid on NCAM. First, we found that ST8Sia III can form polysialic acid on the enzyme itself (autopolysialylation) but not on NCAM. These discoveries prompted us to determine if ST8Sia IV and ST8Sia II share the property of ST8Sia III in utilizing low molecular weight oligosaccharides as acceptors. By using a newly established method, we found that ST8Sia IV, ST8Sia II, and ST8Sia III all add oligosialic and polysialic acid on various sialylated N-acetyllactosaminyl oligosaccharides, including NCAM N-glycans, fetuin N-glycans, synthetic sialylated N-acetyllactosamines, and on alpha(2)-HS-glycoprotein. Our results also showed that monosialyl and disialyl N-acetyllactosamines can serve equally as an acceptor, suggesting that no initial addition of alpha2,8-sialic acid is necessary for the action of polysialyltransferases. Polysialylation of NCAM by ST8Sia IV and ST8Sia II is much more efficient than polysialylation of N-glycans isolated from NCAM. Moreover, ST8Sia IV and ST8Sia II catalyze polysialylation of NCAM much more efficiently than ST8Sia III. These results suggest that no specific acceptor recognition is involved in polysialylation of low molecular weight sialylated oligosaccharides, whereas the enzymes exhibit pronounced acceptor specificities if glycoproteins are used as acceptors.

Developments in plant breeding for improved nutritional quality of soya beans II. Anti-nutritional factors

Nutritional value of most plant materials is limited by the presence of numerous naturally occurring compounds which interfere with nutrient digestion and absorption. Although processing is employed widely in removal of these factors, selection of cultivars of soya beans with inherently low levels would have a considerable impact on efficiency of non-ruminant livestock production. The review considers the role of plant breeding in achieving this objective. The most abundant trypsin inhibitors are the Kunitz and the Bowman–Birk inhibitors, containing 181 and 71 amino acids respectively. The Kunitz inhibitor is present at a concentration of 1·4 g/kg of total seed contents and the Bowman–Birk inhibitor 1·6 g/kg. A large number of isoforms of the Bowman–Birk inhibitor have been described in soya bean cultivars and it has been shown that the general properties of the inhibitor are, in fact, attributable to different isoforms. Nulls for both Bowman–Birk and Kunitz trypsin inhibitors have been identified, allowing new low trypsin inhibitor cultivars to be produced. However, research into breeding for low trypsin inhibitor cultivars currently has limited application as trypsin inhibitors contribute a major proportion of the methionine content of soya beans. Trypsin inhibitors are thought to be involved in the regulation of and protection against unwanted proteolysis in plant tissues and also act as a defence mechanism against attack from diseases, insects and animals. Hence, in breeding programmes for low trypsin inhibitor cultivars, alternative protection for growing plants must be considered. Use of soya beans in non-ruminant animal feeds is limited by the flatulence associated with their consumption. The principal causes appear to be the low molecular weight oligosaccharides containing α-galactosidic and β-fructosidic linkages; raffinose and stachyose. Non-ruminants do not have the α-galactosidase enzyme necessary for hydrolysing the α-galactosidic linkages of raffinose and stachyose to yield readily absorbable sugars. Soya beans contain between 6·8 and 17·5 g of phytic acid/kg; a ring form of phosphorus (P) which chelates with proteins and minerals to form phytates not readily digested within the gut of non-ruminants. One approach for over-coming the effects of phytic acid is through synthesis of phytase in the seeds of transgenic plants. Currently, recombinant phytase produced in soya beans is not able to withstand the processing temperatures necessary to inactivate proteinaceous anti-nutritional factors present. Soya bean lectins have the ability to bind with certain carbohydrate molecules (N-acetyl-D-galactosamine and galactose) without altering the covalent structure. Lectins are present in raw soya bean at a concentration of between 10 and 20 g/kg. Purified soya bean agglutinin is easily inactivated by hydrothermal treatment but in complex diets binding with haptenic carbohydrates may confer protection against denaturation. The majority of research into soya bean lectins is carried out using laboratory animals so very limited information is available on their in vivo effects in farm animals. This review is concerned specifically with breeding but there are other means of improving nutritive value, for example processing which may alter protein structure and therefore functionality of proteinaceous anti-nutritional factors present.

Glucansucrases: mechanism of action and structure-function relationships.

Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood. However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca. 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function.

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells.

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by simian virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines. The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of 35SO4 were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons. These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.

Pustulan-Type Polysaccharides as a Constant Character of the Umbilicariaceae (Lichenized Ascomycotina)

Pustulan-Type Polysaccharides as a Constant Character of the Umbilicariaceae (Lichenized Ascomycotina)

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti.

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

Insect Chitinases: Molecular Biology and Potential Use as Biopesticides

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Absence of a hypocholesterolaemic action of chitosan in high‐serum‐cholesterol rabbits

AbstractIn rabbits fed a 0.9% cholesterol‐enriched (to 9 g kg−1) and 20 g kg−1 chitosan‐supplemented diet, total faecal bile acid and sterol levels increased 1.2‐1.4 times (cholic acid 5.0 times, and cholesterol 1.5 times) those in rabbits fed a cholesterol‐enriched (to 9g kg−1) diet. The serum cholesterol and triacylgiycerol levels of high‐serum‐cholesterol rabbits were affected neither by feeding with a 20 g kg−1 chitosan‐supplemented diet nor by intravenous injection with chitosan of low molecular weight (c3000) or chitosan oligosaccharides (300‐1162) daily. These data indicate that chitosan plays a hypocholesterolaemic action only in the intestine.

Characterization of N-linked oligosaccharides by electrospray and tandem mass spectrometry.

Electrospray and tandem mass spectrometry are used to characterize underivatized oligosaccharides that have been digested from asparagine side chains of glycoproteins. Oligosaccharides that contain sialic acids were detected with the best sensitivity in the negative-ion detection mode whereas those that do not contain sialic acid were detected with the best sensitivity in the positive-ion detection mode. The positive-ion abundances of oligosaccharides were greatly enhanced in electrospray mass spectra by adding 10 mM sodium acetate or ammonium acetate to the sample solvent. Tandem mass spectrometry was used to determine primary structural features of the oligosaccharides. Methodology that has been developed on branched high-mannose, hybrid, and complex carbohydrate standards was applied to a mixture of oligosaccharides that were digested with N-glycanase from the glycoprotein, ovalbumin. The composition and relative abundances of individual oligosaccharides obtained from the electrospray mass spectrum compare favorably to those obtained by anion-exchange chromatography/pulsed amperometric detection and by gel permeation chromatography of the oligosaccharides after radiolabelling the reducing end of the carbohydrates. The oligosaccharide content of ovalbumin was independently determined from the heterogeneity observed in the electrospray mass spectrum of the intact 44-kDa glycoprotein. Comparison of the oligosaccharide compositions determined before and after enzymatic digestion shows a selective digestion of high-mannose and low molecular weight oligosaccharides by N-glycanase.

Drought Stress Induces Changes in the Non-Structural Carbohydrate Composition of Wheat Stems

The effect of drought stress on the non-structural carbohydrate (NSC) composition and water relations of the wheat (Triticum aestivum L.) stem has been investigated. Five separate parts of the stem were sampled: the unsheathed portion of the peduncle (Stem 1a), the sheathed portion of the peduncle (Stem 1b), the penultimate internode (Stem 2), the lower internodes (Stem 3+4) and the flag-leaf-pulvinus (pulvinus). The NSC was analysed as two fractions, an ethanol-soluble carbohydrate (ESC) fraction containing mono- and di-saccharides and some low molecular weight oligosaccharides, and a water-soluble carbohydrate (WSC) fraction containing mostly fructans and some high molecular weight oligosaccharides. In Stems 1b, 2 and 3 +4, the imposition of drought midway through grain filling resulted in a shift in soluble carbohydrate from the WSC to the ESC fraction indicating hydrolysis of fructans. In Stem 2 on day 29 of grain filling, the WSC/ESC ratio was 7.6 � 1.5 in well watered plants in contrast to 0.5 � 0.1 in droughted plants on day 30, even though NSC concentration did not differ. The NSC content of Stem la and the pulvinus increased threefold under drought, although levels were significantly lower than in the rest of the stem. The WSC/ESC ratio in these tissues was low com- pared to the rest of the stem but still declined noticeably under drought stress. Turgor (P) was fully maintained in Stem 2 and the pulvinus of droughted plants. Under drought, P in the pulvinus was maintained at higher levels (1.9-2.4 MPa) than in Stem 2 (0.8-1.2 MPa).

Isolation and Characterization of Mucin from the Serum of Cystic Fibrosis Patients

Using monoclonal antibody 19-9, elevated levels of the sialyl Lea antigen (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3R) are detected in serum from most cystic fibrosis patients. We now report further characterization of the serum antigen and evidence that it is on a mucin glycoprotein and not on glycolipids. The antigen has an apparent molecular weight greater than 2 X 10(6) by gel filtration on Sephacryl S-400. On density gradient centrifugation, the antigen has a density of 1.54 g/ml in cesium chloride and 1.42 g/ml in cesium chloride/4 M guanidine HCl. Immunostaining with monoclonal antibody 19-9 of lipid extracts from cystic fibrosis patient serum and erythrocytes does not detect any antigen on glycolipids. The antigen was purified by gel filtration and density gradient centrifugation. After tritium labeling of the sialic acid residues, sodium dodecyl sulfate gel electrophoresis separates two subunits with apparent molecular weights of 200,000 and 400,000. All of the labeled sialic acid is released as low molecular weight oligosaccharides after mild alkaline borohydride degradation. The purified antigen contains fucose, galactosamine, glucosamine, and galactose but no mannose and is enriched in the amino acids threonine, serine, glycine, proline, and alanine. The purified antigen binds several antibodies recognizing epitopes common to many mucins. Thus, the physical, biochemical, and immunochemical properties of the purified antigen indicate that the sialyl Lea antigen is present on mucins in the serum of cystic fibrosis patients.

Structural studies on the galactan from the snail Helix pomatia

1. 1. The galactan of the snail Helix pomatia was subjected to two cycles of Smith-degradation and the resulting products were isolated by gel filtration and thin layer chromatography. 2. 2. The structures of the low molecular weight oligosaccharides were elucidated being identical to those obtained from Lymnaea stagnalis galactan. However, the quantities release differed significantly between the two species. 3. 3. The high molecular fractiosn comprising about 66% of the material were not obtained in a similar degradation of the Lymnaea stagnalis galactan. 4. 4. Thus the observed structural differences can explain easily the species-specific reactivity among the two polysaccharides seen earlier with lectins, enzymes and antibodies.

Isolation and characterization of a human cytomegalovirus glycoprotein containing a high content of O-linked oligosaccharides.

Several disulfide linked glycoprotein complexes were extracted from human cytomegalovirus with a non-ionic detergent and separated by anion exchange high performance liquid chromatography (HPLC). One complex had a molecular weight of 93,000 and was classified as gCII-93. Another complex had a molecular weight greater than 200,000 and was classified as gCII-200. Both complexes immunoprecipitated with a monoclonal antibody (9E10). A third set of complexes (classified as gC-I) immunoprecipitated with another monoclonal antibody (41C2). Isolated complexes were reduced, alkylated, and individual glycoproteins separated by gel-filtration HPLC. Glycoproteins with molecular weights of 50-52,000 from gCII-93 and gCII-200 appeared to be the same glycoprotein since they could be immunoprecipitated by 9 E 10 and had identical peptide maps. The amino sugar content of these glycoproteins was compared to that of higher molecular weight glycoproteins obtained from gCII-200 and to a glycoprotein with a molecular weight of 93,000 (gp93 (I] from gCI. Glycoproteins with molecular weights of 50-52,000 from gCII-93 and gCII-200 contained similar amounts of galactosamine (GalN), glucosamine (GlcN) and sialic acid. However, they contained 2-3 times more GalN than any other glycoprotein from gCII-200 and 10 times more GalN than was detected in gp93 (I). All glycoproteins from gCII-93 or gCII-200 also contained more sialic acid when compared to gp93 (I). GalN in these glycoproteins was present in O-linked oligosaccharides. This was demonstrated by release of low molecular weight oligosaccharides from high molecular weight glycopeptides by mild base hydrolysis and the conversion of GalN to galactosaminitol. Thus, gp52(II) appears to have a unique phenotype marked by a high amount of O-linked oligosaccharides.

Expression of mouse embryonic epitope TEC-2 on human carcinoma-derived cell lines and characterization of its glycoprotein carriers

Monoclonal antibody TEC-02, raised against mouse embryonal carcinoma cells, has been shown to react with murine preimplantation embryos and with a very limited number of adult mouse tissues. The target epitope, TEC-2, is a carbohydrate carried in mouse embryonal carcinoma cells by large glycoprotein-bound glycan. We report here the expression of TEC-2 epitope on human carcinoma-derived cell lines, HeLa and HS, and the properties of its carbohydrate carriers. Immunolabeling of Nonidet P-40 lysates of HeLa cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydispersed glycoconjugates of high molecular weight (mostly above 100 000). TEC-2 antigens detected by the two-site sandwich assay, in which the antigen is immobilized and detected with the same antibody, had a slightly higher molecular weight than those detected by the solid-phase assay. This suggests heterogeneity in the number of TEC-2 epitopes per carrier molecule. When the cells were lyzed by Triton X-114 and the detergent and aqueous phases were separated by warming and centrifugation, most of the TEC-2 antigenic activity was found in the aqueous phase. TEC-2 antigens isolated by indirect precipitation from [ 3H]galactose-labeled HeLa cells were degraded by extensive pronase digestion or mild alkaline treatment to glycopeptides or oligosaccharides of low molecular weight. Thus, TEC-2 epitope in human HeLa cells is carried by carbohydrates of only several monosaccharide units. TEC-02 antibody was also found to bind to Tamm-Horsfall glycoprotein isolated from human urine and its binding was enhanced by desialylation. Combined data indicate that TEC-02 antibody recognizes the GalNAcβ1 → 4Galβ1 → 4 structure which may be carried on different types of molecule, according to the site of their synthesis.

Severe heparin and pentosan polysulfate - induced thrombocytopenias : Clinical aspects - in vitro effects of pentosan polysulfate, low molecular weight (LMW) heparins and oligosaccharides

Severe heparin and pentosan polysulfate - induced thrombocytopenias : Clinical aspects - in vitro effects of pentosan polysulfate, low molecular weight (LMW) heparins and oligosaccharides

The effect of substrate modification on binding of porcine pancreatic alpha amylase: Hydrolysis of modified amylose containing d-allose residues

A modified amylose containing 10% of tritiated d-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified d-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that d-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of d-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of d-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.

The production of extracellular endo-α-mannanase by the mushroom Volvariella volvacea

Seven species of mushrooms were screened for yeast mannan hydrolysing enzyme. Low enzyme activity was identified in the culture filtrate of Volvariella volvacea, growing in starch–KNO3 medium under submerged conditions. Mannan (yeast) as a carbon source was found to stimulate enzyme production but not to support appreciable growth of the mushroom. Better enzyme production was achieved by developing a suitable fermentation medium containing mixed carbon sources (starch and mannan) and a suitable nitrogen source in proper concentrations. Some of the factors affecting liberation of reducing groups from yeast mannan by the culture filtrate enzyme were studied. Low molecular weight oligosaccharides, liberated from yeast mannan by the action of the enzyme, were isolated and identified as mannooligosaccharides. No mannose could be identified to be present in the enzymatic hydrolysate of mannan. Endo-α-mannase activity for this mushroom enzyme was suggested.