Purification and substrate specificity of an endo-dextranase of Streptococcus mutans K1-R

An extracellular endo-dextranase has been isolated from Streptococcus mutans K1-R. Incubation of cell-free culture fluid with sucrose permitted the removal of a large proportion of the extracellular d-glucosyltransferases by irreversible adsorption onto the insoluble glucans that these enzymes synthesize from sucrose. The remaining d-glucosyltransferases were separated from dextranase by precipitation with ammonium sulphate, chromatography on hydroxylapatite and DEAE-cellulose, followed by filtration on Ultrogel. The major products of action of the purified dextranase on (1→6)-α-d-glucans were isomaltotriose (IM₃), isomaltotetraose (IM₄), and isomaltopentaose (IM₅). Further hydrolysis of IM₄ and IM₅ occurred after prolonged incubation with excess of enzyme, to give d-glucose, IM₂, and IM₃. The relative rate of hydrolysis of isomaltose saccharides fell sharply with decreasing chainlength from IM₁₂ to IM₅. The hydrolysis of dextrans containing 96% or more of (1→6)-α-d-glucosidic linkages, expressed as apparent conversion into IM₃, was virtually complete, and substrates such as Streptococcus sanguis glucan, containing sequences of (1→6)-α-d-glucosidic linkages, were also effectively hydrolyzed. Dextranase activity towards the soluble glucan of Streptococcus mutans was limited, and there was no action on the insoluble glucan synthesized by S. mutans sucrose 3-d-glucosyltransferase.     

Purification,characterization, and action-pattern studies on the endo-(1 → 3)-β-d-glucanase from Rhizopus arrhizus QM 1032

The extracellular (1 → 3)-β-d-glucanase [1 → 3)-β-d-glucan glucanohydrolase, EC 3.2.1.6] produced by Rhizopus arrhizus QM 1032 was purified 305-fold in 70% overall yield. This preparation was found to be homogeneous by ultracentrifugation (sedimentation velocity and studies), electrophoresis on acrylamide gel with normal, sodium dodecyl sulfate, and urea-acetic acid gels, and upon isoelectric focusing. The amino acid composition of the enzyme has been determined and it possesses a carbohydrate moiety composed of mannose and galactose (in the ratio ≈5:1) that is linked to the protein through a 2-acetamido-2-deoxyglucose residue. The molecular number was confirmed by electrophoresis on gels of sodium dodecyl sulfate. The enzyme does not posses subunit structure. It hydrolyzes it substrates with retention of configuration and possesses transglycosylating ability. The rates of hydrolysis of a wide variety of substrates were determined, and its action pattern on a series of oligosaccharides containing mized (1 → 3-, (1 → 4)-, and (1 → 6)-β-d-glucopyranosyl residues was investigated. The enzyme favors stretches of β-d-(1 → 3) linkages, but it can hydrolyze β-d-(1 → 4) linkages that are flanked on the non-reducing side with stretches of β-d-(1 → 3) links. The enzyme will not act on (1 → 6)-β-d-glucosyl linkages located in stretches of β-d-(1 → 3) and will not act on (1 → 3) β-d-glycosidic linkages involving sugars other than d-glucose.     

Lysis of yeast cell-walls: non-lytic and lytic (1→6)-β-D-glucanases from Bacillus circulans WL-12

Two types of extracellular (1→6)-β-D-glucanases are produced by Bacillus circulans WL-12, and these enzymes are differentiated by their ability to lyse yeast cell-walls. The non-lytic (1→6)-β-D-glucanase was isolated by a combination of Sephadex G-100, Bio-Gel P-100, and DEAE-Bio-Gel A chromatography. The purified enzyme was eloctrophoretically homogeneous and had a molecular weight of 52,000. For the substrate pustulan, the enzyme exhibited the following kinetic properties: pH, 5.0; K_m, 0.83 mg of pustulan/ml; V_max, 104 microequivalents of D-glucose released/min/mg of protein. Pustulan was hydrolysed by an endo-mechanism, producing D-glucose and gentiobiose as preponderant final products. The non-lytic enzyme was specific for the (1→6)-β-D-glucosidic linkage and did not hydrolyse branched, (1→3)-β-D-linked glucans containing (1→6)-interchain linkages. In contrast, the lytic (1→6)-β-D-glucanase produced D-glucose, gentiobiose, and gentiotriose as the final products of pustulan hydrolysis, and exhibited significant activity on branched (1→3)-β-D-glucans having (1→6)-interchain linkages. In these cases, the major products were gentiobiose and D-glucose, suggesting an ability of the lytic enzyme to cleave some (1→3)-linkages surrounding a (1→6)-branch-point. This latter property may explain the ability of this enzyme to weakly lyse yeast cell-walls.     

Isolation and some properties of extracellular d-glucosyltransferases and d-fructosyltransferases from Streptococcus mutans serotypes c, e, and f

Extracellular d-glucosyltransferases (GTase) and d-fructosyltransferases (FTase) were isolated from Streptococcus mutans IB (serotype c), B14 (e), and OMZ175 (f) by chromatofocusing, followed by hydroxyapatite column chromatography. The GTases isolated from serotypes c, e, and f are basic proteins (pI 7.4). The serotype c and e enzymes have two protein components having M_r 173 000 and 158 000 and the enzyme of the serotype f one component having M_r 156 000. The GTases of all the serotypes showed a K_m value for sucrose of 10–14mm and an optimum pH 5.5–6.0 for enzyme activity, and their activities were enhanced by the presence of primer Dextran T10. The α-d-glucans synthesized by the purified GTases are water soluble and primarily consist of (1→6)-α-d-glucosidic linkage (41–66 mol/100 mol) and α-d-(1→3,6)-branch linkage (6–20 mol/100 mol), but significant proportions of α-d-(1→3), α-d-(1→4), and α-d-(1→3,4) linkages (11, 6, and 14 mol/100 mol, respectively) were detected in the serotype c α-d-glucan. The isolated FTases of the serotypes c, e, and f are acidic enzymes (pI 4.6) and consist of two components having M_r 84 000 and 76 000 for the serotype c enzyme, and 106 000 and 84 000 for the serotypes e and f enzymes, respectively. The K_m value for sucrose was 6, 10, and 17mm for the serotypes c, e, and f enzymes, respectively, and the optimum pH of enzymic activity 5.5–6.0. Reactivity with Concanavalin A, susceptibility to acid hydrolysis, and paper chromatography of the hydrolyzates suggested that the water-soluble β-d-fructans synthesized by the purified FTases were of the inulin-type and had chemical structures somewhat different among the serotypes.     

Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

Isolation and Characterization of a Novel Endo-β-galactofuranosidase from Bacillus sp.

A soil bacterium capable of growing on a polysaccharide containing β(1→6)galactofuranoside residues derived from the acidic polysaccharide of Fusarium sp. as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as a Bacillus sp. The bacterium produced β- galactofuranosidase inductively in the culture media. The most effective inducer for the β-galactofuranosidase production was a polysaccharide containing β(1→5) or β(1→6)-linked galactofuranoside residues, but gum arabic, gum guar, gum ghati, arabinogalactam, araban, and pectic acid did not induce the enzyme. The enzyme had three different molecular weight forms. The low molecular-weight form was purified by a combination of Toyopearl HW-55 and DEAE-Toyopearl 650S column chromatographies, and preparative polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 67,000 by SDS–polyacrylamide gel electrophoresis. The enzyme was most active at pH 6 and 37°C, and was stable between pH 4 to 8 at 5°C. The action of the enzyme was inhibited by the addition of Cd²⁺, Co²⁺, Hg²⁺, Zn²⁺, iodoacetic acid, and EDT A. The purified enzyme cleaved β(1→5) and β(1→6)-linked galactofuranosyl chains. Based upon the mode of liberation of galactofuranosyl residues from pyridylamino β(1→6)-linked galactofuranoside oligomers, the enzyme can be classified as an endo-β-galactofuranosidase that randomly hydrolyzes the linkage.     

Mammalian-lung galactan

Exopolysaccharides of Agrobacterium tumefaciens and Rhizobium meliloti, containing d-glucose, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 6:1:1:1.5, were analysed by methylation. They were found to contain the following main structural units (all β-glycosidic): chain residues of (1→3)-linked d-glucose (24%), (1→3)-linked d-galactose (15%), (1→4)-linked d-glucose (20%), and (1→6)-linked d-glucose (18%); (1→4,1→6)-linked branching residues of d-glucose (12%), and terminal d-glucose residues substituted at positions 4 and 6 by pyruvate (11%). Uronic acid-containing exopolysaccharides of Rhizobium leguminosarum, R. phaseoli, and R. trifolii contained d-glucose, d-glucuronic acid, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 5:2:1:2:3. Methylation gave identical patterns of methylated sugar components, from which the following structural elements were deduced: chain residues of (1→3)-linked d-glucose substituted at positions 4 and 6 by pyruvate (13%), (1→4)-linked d-glucose (32%), and (1→4)-linked d-glucuronic acid (20%); (1→4,1→6)-linked branching residues of d-galactose and/or d-glucose (13%), and terminal d-glucose and/or d-galactose residues substituted at positions 4 and 6 by pyruvate (13%).     

Intracellular α-Amylase of Streptococcus mutans

Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

An apparatus for safe and convenient handling of anhydrous, liquid hydrogen fluoride at controlled temperatures and reaction times. Application to the generation of oligosaccharides from polysaccharides

β-d-Gal-(1 → 4)-β-d-GlcNAc-OC₆H₄NO₂-p (p-nitrophenyl N-acetyl-β-lactosaminide) and β-d-Gal-(1 → 6)-β-d-GlcNAc-OC₆H₄NO₂-p (p-nitrophenyl N-acetyl-β-isolactosaminide) were regioselectively synthesized from lactose and p-nitrophenyl 2-acetamido-2-deoxy-glucopyranoside, employing transglycosylation by the β-d-galactosidase from Bacillus circulans and by controlling the concentration of organic solvent in the reaction system. The (1 → 4)-linked disaccharide was formed exclusively when the concentration of organic solvent was high, whereas the (1 → 6)-linked isomer was produced with a low concentration. Further utilization of the transglycosylation by the enzyme led to the regioselective formation of β-d-Gal-(1 → 4)-d-GalNAc and β-d-Gal-(1 → 4)-β-d-GalNAc-OC₆H₄NO₂-p. With the enzyme, β-d-galactosyl transfer occurred preferentially at the O-4 position of GlcNAc and GalNAc, regardless of the configuration of the hydroxyl group.     

Characterization of d-galactosyl-β1→4-l-rhamnose phosphorylase from Opitutus terrae

We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45 °C and at pH 6.0–7.0. The values of k_cat and K_m of the phosphorolytic activity of the enzyme on GalRha were 60 s^?1 and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms.     

Synthesis and properties of some O-(2,2-dialkoxyethyl)glycolaldehydes

β-d-Mannosidase (β-d-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β-d-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α-d-mannosidase, β-d-galactosidase, 2-acet-amido-2-deoxy-d-glucosidase, (1→4)-β-d-mannanase, and (1→4)-β-d-glucanase activities, almost devoid of α-d-galactosidase activity, and contaminated with <0.02% of β-d-glucosidase activity. The purified enzyme had the same K_m for borohydride-reduced β-d-manno-oligosaccharides of d.p. 3–5 (12.5mm). The initial rate of hydrolysis of (1→4)-linked β-d-manno-oligosaccharides of d.p. 2–5 and of reduced β-d-manno-oligosaccharides of d.p. 3–5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β-d-mannopyranosides were readily hydrolysed. β-d-Mannobiose was hydrolysed at a rate ～25 times that of 6¹-α-d-galactosyl-β-d-mannobiose and 6³-α-d-galactosyl-β-d-mannotetraose, and at ～90 times the rate for β-d-mannobi-itol.     

Calcification of Selected Strains of Streptococcus mutans and Streptococcus sanguis

Nine strains of cariogenic Streptococcus mutans and two strains of Streptococcus sanguis were tested for their ability to form hydroxyapatite. The cells were examined by X-ray diffraction and electron microscopy for apatite crystals after growth in a synthetic calcification medium. Each of the test isolates, except for one strain of S. sanguis, produced intracellular mineral. Two strains of S. mutans formed both intra- and extracellular crystals. There was no apparent relationship between calcifiability and serotype.     

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.     

Comparative studies on beta-glucan hydrolases. Isolation and characterization of an exo(1 yields 3)-beta-glucanase from the snail, Helix pomatia.

An exo-β-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-200, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades β-(1 → 3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1→3; 1→4)-β-glucans are also extensively degraded and β-(1→6)- and β-(1→4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-β-glucanases, reported previously, in the broadness of its substrate specificity. The K_m values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50–55 °C.     

Cleavage of oligosaccharides by rat kidney sialidase Influence of substrate structure

The specificity of the sialidase activity present in rat kidney cortex (12 000 × g pellet) was studied with various tritiated oligosaccharidic substrates: (i) αNeuAc2 → 3βGall → 4Glc-itol[³H], αNeuAc2 → 6βGall → 4Glc-itol[³H] and αNeuAc2 → 8αNeuAc2 → 3βGall → 4Glc-itol[³H] from bovine colostrum; (ii) α-NeuAc2 → 6βGall → 4βGlcNAc-itol[³H], αNeuAc2 → 3βGal1 → 4βGlcNAcl → 2αManl → 3βMan1 → 4GlcNAc-itol[³H]. αNeuAc2 → 6βGall → 4βGlcNAcl → 2αManl α 3(βGall → 4GlcNAcl → 2αManl → 6)βManl → 4GlcNAc-itol [³H]et αNeuAc2 → 6βGall → 4βGlcNAcl → 2αManl-3(αNeuAc2 → 6βGall → 4βGlcNAcl → 2αManl → 6)βManl 4GlNAc-itol[³H] isolated from the urine of a patient with mucolipidosis I. The enzyme cleaves α2 → 3 and α2 → 8 linkages at a greater rate than the α2 → 6 bonds. Its activity decreases with the length of the oligosaccharidic chain. Substitution of a glucose moiety by Nacetylglucosamine results in diminished activity. The specificity of rat kidney sialidase differs from that reported for other mammalian of viral sialidases.     

A d-glucose- and d-xylose-tolerant GH1 β-glucosidase from Cellulosimicrobium funkei HY-13, a fibrolytic gut bacterium of Eisenia fetida

The GluM gene (1491-bp) coding for a β-glucosidase comprising a single catalytic glycoside hydrolase family 1 domain from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium funkei HY-13, was cloned and over-expressed in Escherichia coli BL21. The recombinant histidine-tagged enzyme (rGluM: 56 kDa) displayed the highest cleavage activity toward p-nitrophenyl (pNP)-β-d-glucopyranoside at pH 5.0 and 40 °C. The β-glucosidase activity of rGluM was enhanced over 1.8-fold of its original activity in the presence of 1 mM Ca²⁺, Ni²⁺, Mn²⁺, and Co²⁺ ions, respectively, while it was highly sensitive to 5 mM N-bromosuccinimide and 1 mM Hg²⁺. The susceptibility of some pNP-sugar derivatives and d-cellobiose to rGluM was evaluated to be in the order of pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > d-cellobiose > pNP-β-d-cellobioside > pNP-β-d-mannopyranoside. The k_cat/K_m values of rGluM toward pNP-β-d-glucopyranoside, pNP-β-d-galactopyranoside, and d-cellobiose were 302.28, 179.73, and 6.40 mM^-1 s^-1, respectively. At a concentration below 1.0 M, d-galactose was a potent activator of rGluM with β-glucosidase activity enhanced by approximately 160% in a dose-dependent manner. Moreover, the d-glucose (< 400 mM) and d-xylose (≤ 700 mM) stimulation of rGluM suggests that it can be exploited as a potential biocatalyst to generate d-glucose molecules in d-cellobiose degradation.     

Comparative studies of asparagine-linked oligosaccharide structures of rat liver microsomal and lysosomal beta-glucuronidases

The sugar chains of microsomal and lysosomal β-glucuronidases of rat liver were studied by endo-β-N-acetylglucosaminidase H digestion and by hydrazinolysis. Only a part of the oligosaccharides released from microsomal β-glucuronidase was an acidic component. The acidic component was not hydrolyzed by sialidase and by calf intestinal and Escherichia coli alkaline phosphatases, but was converted to a neutral component by phosphatase digestion after mild acid treatment indicating the presence of a phosphodiester group. The neutral oligosaccharide portion of microsomal enzyme was a mixture of five high mannose-type sugar chains: (Manα1 → 2)_0~4 [Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc]. In contrast, lysosomal enzyme contains only Manα1 → 6 (Manα1 → 3) Manα1 → 6(Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc. The result indicates that removal of α1 → 2-linked mannosyl residues from (Manα1 → 2)₄[Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn] starts already in the endoplasmic reticulum of rat liver.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

Further study of the structure of lentinan,an anti-tumor polysaccharide from Lentinus edodes

The structure of lentinan, an anti-tumor polysaccharide from Lentinus edodes, has been further investigated. Periodate oxidation, Smith degradation, methylation analysis, and bioassay were the principal methods used. These studies showed that a branched molecule having a backbone of (1→3)-β-d-glucan and side chains of both β-d-(1→3)- and β-d-(1→6)-linked d-glucose residues, together with a few internal β-d-(1→6)-linkages, is present.