Endo-beta-N-acetylglucosaminidases acting on carbohydrate moieties of glycoproteins: purification and properties of the two enzymes with different specificities from Clostridium perfringens.

Two endo-β-N-acetylglucosaminidases (C_I and C_I) acting on carbohydrate moieties of glycoproteins were highly purified from the culture fluid of Clostridium perfringens. C_I had the substrate specificity indistinguishable from that of endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae. C_II showed the specificity similar to that of endo-β-N-acetylglucosaminidase H from Streptomyces griseus but is distinct from the streptomyces enzyme with respect to the relative activity toward ovalbumin glycopeptides and Unit A glycopeptides of thyroglobulin. Both enzymes from C. perfringens were most active at neutral pH and were inhibited by p-chloromercuriphenylsulfonate.     

Glycopeptide elicitors of stress responses in tomato cells: N-linked glycans are essential for activity but act as suppressors of the same activity when released from the glycopeptides

Induction of ethylene, an early symptom of the stress response in tomato (Lycopersicon esculentum [L.] Mill) cells, was used as a bioassay to purify elicitor activity from yeast extract. The purified elicitor preparation consisted of small glycopeptides (mean relative molecular weight of approximately 2500) and induced ethylene biosynthesis and phenylalanine ammonia-lyase activity half-maximally at 15 nanograms per milliliter. Elicitor activity was partially abolished by pronase and almost completely by endo-β-N-acetylglucosaminidase H, α-mannosidase, or periodate. The oligosaccharides released upon treatment with endo-β-N-acetylglucosaminidase H competitively inhibited the elicitor activity of the glycopeptides. This suppressor activity was abolished by periodate oxidation and α-mannosidase treatment. The suppressors were chromatographically separated into four active fractions with sizes corresponding to 7 to 10 monosaccharides. They consisted predominantly of mannose and contained also N-acetylglucosamine and glucose. The suppressors had no effect on the response of the tomato cells to a different elicitor, derived from cell walls of Phytophthora megasperma f. sp. glycinea. This strongly suggests that different recognition sites exist for different elicitors in tomato cells, and that the oligosaccharide suppressors act specifically on the perception of just one elicitor. The hypothesis is put forward that the suppressors bind to one of the elicitor recognition sites nonproductively, i.e. without producing a signal, thereby preventing induction of the stress responses by the corresponding elicitor.     

Purification and Properties of β-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced β-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher β-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had β-N-acetylgalactosaminidase activity equivalent to about 40% of its β-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for β-N-acetylglucosaminidase activity and 5.5 to 6.5 for β-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase

On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase.     

A Novel β-N-Acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: Gene Cloning,Expression, and Assignment to Family 3 of the Glycosyl Hydrolases

A β-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an M_r of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical β-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent K_m and V_max values for p-nitrophenyl-β-N-acetylglucosamine were 425.7 μM and 24.8 μmol min⁻¹ mg of protein⁻¹, respectively.Streptomycetes are gram-positive, mycelial soil bacteria with a high G+C content. In addition to having the ability to synthesize a wide variety of antibiotics and chemotherapeutic agents, they produce extracellular hydrolytic enzymes to obtain nutrients and energy by solubilizing polymeric compounds in soil. These enzymes include proteases, nucleases, lipases, and a variety of enzymes that hydrolyze different types of polysaccharides such as cellulose, chitin, and xylan (13). This last class of enzymes has received considerable attention not only from the standpoint of the utilization of renewable resources but also from that of basic research. Among actinomycetes, Streptomyces spp. make up one group regarded as particularly efficient in the breakdown of chitin (10). Following cellulose, chitin is the second most abundant polymer (β-1,4-linked polymer of N-acetylglucosamine) in nature. Efficient degradation of chitin by microorganisms is achieved by the concerted action of chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) (1, 19, 20).We have been studying the chitinolytic system of Streptomyces thermoviolaceus OPC-520 to clarify the roles of individual enzymes involved in chitin degradation, the relationship between structure and function, and the regulation of gene expression. When S. thermoviolaceus OPC-520 is cultivated in the presence of chitin, this strain secretes three different chitinases and only one β-N-acetylglucosaminidase and the production is repressed by glucose (unpublished data). Previously, we purified and characterized a major chitinase (Chi40) produced by the strain, which shows a high optimum temperature (70 to 80°C), high optimum pH (pH 8.0 to 10.0), and heat stability (22), and recently reported the cloning and expression of the Chi40 gene (23).While a number of chitinase genes have been isolated from a wide variety of organisms, including bacteria, fungi, insects, plants, and animals, examples of cloning of the β-N-acetylglucosaminidase gene involved in a chitinolytic system are few. To understand the role of β-N-acetylglucosaminidase in chitin degradation by strain OPC-520, its relationship to similar proteins isolated from other sources, and the regulatory system involved in the induction of the enzyme, we have isolated and expressed the gene encoding β-N-acetylglucosaminidase. Here we report the molecular cloning and biochemical characterization of a β-N-acetylglucosaminidase, designated NagA, from S. thermoviolaceus OPC-520. This novel enzyme, which is clearly different from the N-acetylglucosaminidases so far reported, is assigned to family 3 of the glycosyl hydrolases on the basis of sequence comparison. This is the first report of a β-N-acetylglucosaminidase gene isolated from the genus Streptomyces.     

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

γ-Aminobutyric acid type A (GABA_A) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA_A receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABA_A receptors by glycosylation.     

Identification of Amino Acid Residues Required for the Substrate Specificity of Human and Mouse Chondroitin Sulfate Hydrolase (Conventional Hyaluronidase-4)

Human hyaluronidase-4 (hHYAL4), a member of the hyaluronidase family, has no hyaluronidase activity, but is a chondroitin sulfate (CS)-specific endo-β-N-acetylgalactosaminidase. The expression of hHYAL4 is not ubiquitous but restricted to placenta, skeletal muscle, and testis, suggesting that hHYAL4 is not involved in the systemic catabolism of CS, but rather has specific functions in particular organs or tissues. To elucidate the function of hyaluronidase-4 in vivo, mouse hyaluronidase-4 (mHyal4) was characterized. mHyal4 was also demonstrated to be a CS-specific endo-β-N-acetylgalactosaminidase. However, mHyal4 and hHYAL4 differed in the sulfate groups they recognized. Although hHYAL4 strongly preferred GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-containing sequences typical in CS-D, where GlcUA represents d-glucuronic acid, mHyal4 depolymerized various CS isoforms to a similar extent, suggesting broad substrate specificity. To identify the amino acid residues responsible for this difference, a series of human/mouse HYAL4 chimeric proteins and HYAL4 point mutants were generated, and their preference for substrates was investigated. A combination of the amino acid residues at 261–265 and glutamine at 305 was demonstrated to be essential for the enzymatic activity as well as substrate specificity of mHyal4.     

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons

The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N-acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase β-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by β-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons.     

Studies on N-glycosylation by elongating tissues and membranes from pea stems

Glucosamine and mannose were incorporated into oligosaccharides linked to either polar membrane-lipids or to asparagine residues of endogenous proteins in apical growing tissues of the etiolated pea stem. The glycolipids were subject to turnover in pulse-chase tests and protein-linked oligosaccharides accumulated with time, as expected for a precursor-product relationship. The newly formed glycoproteins were hydrolyzed by endo-β-N-acetylglucosaminidase H to oligosaccharides in the same size range as those released by dilute acid from the lipid-linked oligosaccharides formed during the pulse. The glycoproteins were also partly degraded to free N-acetylglucosamine by β-N-acetylhexosaminidase. Affinity of the carbohydrate moiety of the protein for concanavalin A increased between the beginning and the end of the chase, indicating processing following core glycosylation.

The addition of UDP-N-acetyl-[¹⁴C]glucosamine plus external peptide acceptors (derived from carboxymethylated α-lactalbumin) to membrane preparations from the pea stem resulted in peptide glycosylation at the expense of lipid-linked oligosaccharide. Glycosylation of endogenous protein acceptors did not take place via lipid intermediates but directly from the sugar nucleotide substrate. Tunicamycin inhibited glycosyltransfer to both glycolipids and added peptides, but not to endogenous protein. It is concluded that limiting factors for N-glycosylation by pea membranes in vitro could include the unavailability of endogenous acceptors or the inability to fully elongate and internalize lipid precursors, but is not due to any limitation in capacity for N-glycosylation.

     

Preferential secretion of R-type alpha-amylase molecules in rice seed scutellum at high temperatures

Exposure of fresh scutella excised from 4-day-old rice seedlings to higher temperatures, (40-42°C), drastically reduced the biosynthesis of α-amylase as determined by the incorporation of [³⁵S]methionine into the immunoprecipitable product. However, the intracellular transport and extracellular secretion of the enzyme molecules were enhanced at high temperatures, indicating that the biosynthesis and secretion of α-amylase are distinguishable in their temperature dependency. At the higher temperature regime (40°C), the complex-type α-amylase isoform, resistant to hydrolytic digestion by endo-β-N-acetylglucosaminidase H (Endo-β-H) was predominantly secreted, whereas at lower temperatures (15°C), the isoform susceptible to Endo-β-H attack was the major molecular form secreted.     

Elimination of Differences in the Mobility of Flax Isoperoxidases on PAGE by Digestion with alpha-Mannosidase

In the flax (Linum usitatissimum) genotype Stormont cirrus, anodic peroxidases from the genotroph S migrate more slowly on PAGE and SDS-PAGE than the corresponding peroxidases from the genotroph L. When purified isoperoxidases S2 and L2 were digested with α-mannosidase, the difference in mobility was eliminated. Treatment with α-fucosidase and β-xylosidase also altered the mobility of S2 and L2, but affected the sensitivity to the action of endo-β-N-acetylglucosaminidase H of only S2. Our results suggest differences in posttranslational processing of the carbohydrate moiety between S and L isoperoxidases. These differences were also found in other S and L glycoenzymes (anodic acid phosphatases) as well as in the peroxidases of other flax genotypes.     

Biochemical Studies on “Bakanae” Fungus. Part 68. Synthesis of Substances related to Gibberellins

Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-β-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-β-N-acetylglucosaminidases of other microorganisms: ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins.     

Galactinol Synthase is an Extravacuolar Enzyme in Tubers of Japanese Artichoke (Stachys sieboldii)

Galactinol synthase (GS, UDP-α-d-galactose:1l-myo-inositol-1-O- α-d-galactopyranosyltransferase) is a key enzyme in the biosynthetic pathway of the raffinose family of oligosaccharides. The subcellular location of GS was studied in the parenchyma of stachyose-storing tubers of Japanese artichoke (Stachys sieboldii) by isolation of protoplasts and vacuoles. A comparison of the activities of GS, malate dehydrogenase, and alcohol dehydrogenase (extravacuolar markers) and α-mannosidase and β-N-acetylglucosaminidase (vacuolar markers) in parenchyma protoplasts with those of vacuoles isolated from them showed that GS was an extravacuolar enzyme.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Substrate specificity of mammalian endo-beta-N-acetylglucosaminidase: study with the enzyme of rat liver

The substrate specificity of mammalian endo-β-N-acetylglucosaminidase was studied in detail by using rat liver enzyme. The enzyme hydrolytically cleaves the N,N′-diacetylchitobiose moiety of Manα1 → 6 (Manα1 → 3)Manβ1 → 4GlcNacβ1 → 4R in which R represents either GlcNac → Asn or N-acetylglucosamine. The enzyme can hardly act on the sugar chains with Fucα1 → 3 or 6GlcNac → Asn or N-acetylglucosaminitol as their R residues. The sugar chains substituted at C-3 and C-6 positions of the Manα1 → 6 residue and at C-2 position of the Manα1 → 3 residue by other sugars are also cleaved by the enzyme. The sugar chains substituted at C-4 position of the β-mannosyl residue and at C-2 position of the Manα1 → 6 residue by other sugars are hydrolyzed at one place lower rate. The specificity of the mammalian endo-β-N-acetylglucosaminidase indicates that the enzyme is responsible for the formation of most of the oligosaccharides excreted in the urine of patients with congenital exoglycosidase deficiencies and also explains why large amount of glycopeptides are excreted in the urine of fucosidosis patients.     

The release of oligosaccharides from glycoproteins by endo-β-N-acetylglucosaminidase of Flavobacterium sp.

Endo-β-N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.     

Structural Basis and Catalytic Mechanism for the Dual Functional Endo-β-N-Acetylglucosaminidase A

Endo-β-N-acetylglucosaminidases (ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A) in 3 forms, one in native form, one in complex with Man₃GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues – E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as “gate-keepers”. Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.     

Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033.     

Lysis of Yeast Cell Walls: Glucanases from Bacillus circulans WL-12

Endo-β-(1 → 3)- and endo-β-(1 → 6)-glucanases are produced in high concentration in the culture fluid of Bacillus circulans WL-12 when grown in a mineral medium with bakers' yeast cell walls as the sole carbon source. Much lower enzyme levels were found when laminarin, pustulan, or mannitol was the substrate. The two enzyme activities were well separated during Sephadex G-100 chromatography. The endo-β-(1 → 3)-glucanase was further purified by diethylaminoethyl-cellulose and hydroxyapatite chromatography, whereas the endo-β-(1 → 6)-glucanase could be purified further by diethylamino-ethyl-cellulose and carboxymethyl cellulose chromatography. The endo-β-(1 → 3)-glucanase was specific for the β-(1 → 3)-glucosidic bond, but it did not hydrolyze laminaribiose; laminaritriose was split very slowly. β-(1 → 4)-Bonds in oat glucan in which the glucosyl moiety is substituted in the 3-position were also cleaved. The kinetics of laminarin hydrolysis (optimum pH 5.0) were complex but appeared to follow Michaelis-Menten theory, especially at the lower substrate concentrations. Glucono-δ-lactone was a noncompetitive inhibitor and Hg²⁺ inhibited strongly. The enzyme has no metal ion requirements or essential sulfhydryl groups. The purified β-(1 → 6)-glucanase has an optimum pH of 5.5, and its properties were studied in less detail. In contrast to the crude culture fluid, the two purified β-glucanases have only a very limited hydrolytic action on cell wall of either bakers' yeast or of Schizosaccharomyces pombe. Although our previous work had assumed that the two glucanases studied here are responsible for cell wall lysis, it now appears that the culture fluid contains in addition a specific lytic enzyme which is eliminated during the extensive purification process.     

Activation of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae by various sugars

Endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae was activated by the addition of glucose, mannose, N-acetylglucosamine, and β-allose. While the enzyme did not appear to be significantly affected by the addition of galactose or N-acetylgalactosamine. These results indicate that the C-4 and C-6 positions of the monosaccharide are the most important for enzyme activation. Moreover, the enzyme was activated by the addition of disaccharides such as cellobiose, gentiobiose, and di-N-acetylchitobiose, but not by polysaccharides such as starch and yeast mannan. In the presence of N-acetylglucosamine, the enzyme activation occurred well over pH 4.0 and the K_m value of the enzyme for (Man)₆(GlcNAc)₂-Asn-dansyl changes from 1.2 mM to 3.2 mM.