Studies on the Glucanase of Sclerotinia libertiana

The digestion of yeast cells with the glucanase of Sclerotinia was found to be significantly increased by pretreating the cells with papaya lysozyme, as well as by pretreating with dilute sodium hydroxide solution. Defatted chlorella cells were digested to a certain extent with the glucanase alone. Pretreatment with lipolytic enzyme slightly stimulated the digestion of yeast cells by glucanase, but this effect was not found with the yeast cells treated by soaps or the defatted chlorella cells. Egg white lysozyme had no effect on digestion of yeast cells. The effect of papaya lysozyme seemed to have relation with the liberation of hexosamine compounds from the yeast-cell walls. It is suggested that, in normal cells, the glucan is present to form a further complex structure with certain other component, becoming insusceptible to the action of glucanase.     

Purification and Properties of Lytic β-Glucanase from an Arthrobacter Bacterium

A glucanase was isolated from a culture fluid of an Arthrobacter bacterium. The purified enzyme preparations consisted of the glucanase components having the same enzymatic activity. The enzyme was stable in a broad pH range, but lost its activity rapidly at above 60°C. Optimum pH values were found to be 5.5~6.5.

The glucanase attacked the following glucan preparations and liberated a relatively small amount of reducing power: Saccharomyces cerevisiae glucan, Candida albicans glucan, Saccharomyces fragilis glucan, pachyman, curdlan and laminaran. The most prominent sugar spot on the chromatogram of the digest from yeast glucan was identified with laminan-pentaose, and the other faint spots with a series of laminaridextrins. The β-1,6 glucosidic bonds in yeast glucan were not hydrolyzed and concentrated in a soluble fraction which was found near the origin of the chromatogram.     

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.     

Lytic β-1,3 Glucanase from Arthrobacter: Pattern of Action

The action pattern of lytic β-1,3 glucanase (glucanase I) from Arthrobacter which liberates predominantly laminaripentaose from various β-glucans has been studied. The enzyme was not active on short linear laminaridextrins, but was active on an enzymatically synthesized, linear β-1,3 oligoglucan preparation. Any intactness of the glucose residues of the chain ends of a substrate did not seem to be necessary for the action of the enzyme. The results of determination of laminaripentaose during a relatively early phase of the reaction suggested that about half of the reducing power liberated in the medium might be explained by the formation of the sugar. It seems that the formation of laminaripentaose relates to the initial attack of glucanase 1 on β-1,3 glucan chains.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Continuous-culture studies of synthesis and regulation of extracellular beta(1-3) glucanase and protease enzymes from Oerskovia xanthineolytica

This article describes the synthesis and regulation of beta(1-3)glucanase and protease enzymes from the cell lytic system of Oerskovia xanthineolytica LL-G109 in continuous culture using different concentrations of carbon source (glucose) and inducer (glucan). These two enzyme activities are the main components of a lytic system capable of lysing and disrupting whole yeast cells; it is subject to catabolite repression by glucose and is induced by yeast glucan. Peaks of beta(1-3)glucanase and protease activity are obtained at dilution rates of between 0.05 and 0.15 h(-1). The glucanase-protease ratio is very high compared to other strains. At dilution rates above 0.15 h(-1) all activities are similar to those obtained in batch culture. The lytic enzyme system appears to contain several beta(1-3)glucanase enzymes. In continuous culture both productivity and enzyme concentrations are greatly in creased when compared to batch culture, 11- and 4.4-fold, respectively.     

Lysis of Yeast Cell Wall by Enzymes from Streptomycetes

This paper deals with yeast cell-wall lytic enzymes formed by Streptomyces with regard to the connection with the cell-wall structure.

In the first place, 29 organisms of β-glucanase-producing Streptomycetes were selected among 777 strains belonging to genus Streptomyces by means of a cylinder-plate method employing the yeast glucan as a substrate. As for these organisms, the depolymerizing activity against the yeast glucan was considered to be mainly due to β-1,3-glucanase activity. Against the heat-treated cell of bakers’ yeast, the crude enzymes merely showed poor lytic activities, however, in the combined employment with some protease preparations, especially with an alkaline protease from St. satsumaensis nov. sp., a remarkable increase of the lytic activities was demonstrated. On the other hand, the intact cell wall of bakers’ yeast, or both the heat-treated and the intact cells of Sacch. cerevisiae 18.29 strain were dissolved very easily by a sole action of β-glucanase or of protease, respectively. In consequence, it seemed that the lysis occurred with different mechanisms in response to differences of substrates. On this subject, the results of investigations and discussions were described in special measure. In addition, the possibility, that some other enzymes than β-glucanase or protease might concern to the lysis of the cell wall, was also investigated and discussed.     

Lyticase: Endoglucanase and Protease Activities That Act Together in Yeast Cell Lysis

Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a β-1,3-glucanase with a molecular weight of 55,000. The K_m for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of β-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.     

Microbial Utilization of Benzenesulfonic Acid

An enzyme which degrades yeast glucan and yeast cells in the logarithmic phase of growth (log yeast cells) and produces protoplasts from log yeast cells has been crystallized from the culture filtrate of a strain belonging to Fungi Imperfecti.

Analyses by ultracentrifugation and disc gel electrophoresis showed the crystalline enzyme to be homogeneous. Its molecular weight was found to be 24,500. The hydrolysis of laminarin, pachyman and yeast glucan was catalysed by the enzyme to produce a mixture of laminaridextrins. The conversion of log yeast cells to protoplasts was obtained by the addition of only this enzyme, the addition of mercaptoethanol or phosphomannanase to the enzyme promoted the conversion.     

Cloning and expression in Escherichia coli of the gene for an Arthrobacter beta-(1----3)-glucanase.

When inserted in the correct orientation at the BamHI site of plasmid YRp7, an 8.6-kilobase BamHI fragment of Arthrobacter sp. strain YCWD3 DNA gave Escherichia coli HB101 cells harboring the recombinant plasmid pBX20 the ability to lyse bakers' yeast cell walls or bakers' yeast glucan in agar medium. An extract of the transformed E. coli cells contained an endo-beta-(1----3)-glucanase with the same activity pattern as that of glucanase I produced by Arthrobacter sp. strain YCWD3. Although part of the glucanase activity was contributed by apparently defective molecules, two protein species were found which had high lytic activity on yeast cell walls and adsorbed to microcrystalline cellulose, and both had a single constituent polypeptide with a molecular weight of about 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these properties the protein species were indistinguishable from those glucanase I protein species of Arthrobacter sp. strain YCWD3 which we believe are nearly the intact molecule. We conclude that the cloned fragment of Arthrobacter sp. strain YCWD3 DNA contains the structural gene for glucanase I. A recombinant plasmid obtained by subcloning a PstI fragment of pBX20 into pBR322 caused the transformed E. coli cells to produce apparently defective glucanase molecules only. This observation serves as additional supporting evidence for our conclusion.     

Purification,Crystallization, and Properties of an Endo β-1,3-Glucanase from Rhizopus chinensis R-69

An endo β-l,3-glucanase was purified in crystalline form from a culture filtrate of Rhizopus chinensis R-69. Molecular weight of the enzyme was determined to be 23,000 by molecular sieve chromatography and the mode of action of the enzyme was suggested to be a less random type of β-1,3-glucanase. Km and V_max of the enzyme for laminarin are 3.4 g/liter and 1541. U., respectively. The enzyme does not decompose the cell walls of living yeast; it decomposes, however, the preparation of yeast glucan.     

Defense responses elicited in pine cell suspension cultures

To understand mechanisms of disease resistance in pine trees, we took advantage of the fact that suspension cultured cells exhibit many of the defense responses that are characteristic of intact tissues. In this study, we measured constitutive and elicitor-induced levels of ethylene production, chitinase activity and glucanase activity in cells of loblolly pine (Pinus taeda L). Increased ethylene production was induced similarly by a live fungus (Ophiostoma minus Hedgc. H.P. Sydow) and chitosan, a general elicitor. Culture age, relative to the most recent transfer, affected the constitutive level of all defense responses. Culture age also had a pronounced effect on the ability of the cells to produce ethylene and cellular chitinase, but not on secreted chitinase, cellular glucanase, secreted glucanase, or lignification. In older cultures, elicitation induced a 4- to 10-fold increase in ethylene production and a 2-fold increase in cellular chitinase, secreted chitinase and cellular glucanase. Chitosan elicitation did not affect secreted glucanase. The overall regulation of the defense response in pine cells appears complex, but individual components of the response can be differentially induced in cell cultures under appropriate experimental conditions.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.     

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation

Water‐insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α‐1,3‐glucanase) and dextranase (α‐1,6‐glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi‐functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE‐SUMOstar Amp plasmid vector. The resultant his‐tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non‐adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol‐sulfuric acid method, and reducing sugars were quantified by the Somogyi–Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose‐dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.     

beta-d-Glucan of Avena Coleoptile Cell Walls

A specific glucanase was used to liberate a noncellulosic beta-d-glucan from isolated cell walls of Avena sativa coleoptile tissue. Cell walls of this tissue contain as much as 7 to 9 mg of glucan/100 mg of dry wall. Because of the specific action pattern of the enzyme, a linkage sequence of.. 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc.. is indicated and the predominance of trisaccharide and tetrasaccharide as hydrolytic products suggests a rather regular repeating pattern in the polysaccharide. The trisaccharide and the tetrasaccharide are tentatively identified as 3-O-beta-cellobiosyl-d-glucose and 3-O-beta-cellotriosyl-d-glucose, respectively. Recovery of these oligosaccharides following glucanase treatment of native wall material was feasible only after wall-bound glucosidases were inactivated. In the absence of enzyme inactivation the released fragments were recovered as glucose. The beta-d-glucan was not extracted from walls by either hot water or protease treatment.Cell walls prepared from auxin-treated Avena coleoptile segments yielded less glucan than did segments incubated in buffer suggesting an auxin effect on the quantity of this wall component. No IAA-induced change in the ratio of the trisaccharide and tetrasaccharide could be detected, suggesting no shift in the 1,3 to 1,4 linkage ratio. While the enzyme acts directly on the beta-d-glucan, no elongation response was apparent when Avena sections were treated with the purified glucanase. The presence of the glucan was not associated with any wound response which could be attributed to the preparation of coleoptile segments. The relationship of glucan metabolism to auxin growth responses is discussed.     

Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and β-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and β-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose.     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H₂O₂-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.     

Purification and Properties of Lytic, β-1, 3-Glucanase from Flavobacterium dormitator var. glucanolyticae

An endo β-1, 3-glucanase which is able to disrupt the cells of living yeast has been purified in homogeneous state from the culture filtrate of Flavobacterium dormitator var. glucanolyticae. The molecular weight of the enzyme was estimated to be 17,000 ~ 22,000. The mode of enzyme action has been suggested to be a “random” type of β-1, 3-glucanase. The enzyme preferes larger chains saccharides as substrate for its action, however, smaller oligosaccharides such as laminaritriose and laminaribiose are also decomposed by the enzyme. The Km values of the enzyme for laminarin, laminarihexaose, and laminaritetraose were determined to be 0.26, 1.18, and 2.00 g/liter, respectively. The ability of this enzyme to disrupt the cells of living yeast is its remarkable point, since endo β-1, 3-glucanase of a smaller oligosaccharide-producing type from most sources has been recognized to be inactive (or very weakly active) on living yeast cells.