Complete Genome Sequence of ?-1,3-Glucanase-Producing Strain Paracoccus mutanolyticus RSP-02.
ABSTRACT: A mutanase (?-1,3 glucanase)-producing bacterial strain of Paracoccus mutanolyticus was isolated from soil samples rich in cellulosic waste. Here, we report the whole-genome sequencing and annotation of P. mutanolyticus, which has a genome size of around 3.5 Mb and the potential to degrade water-insoluble ?-1,3 glucans with an overall G+C content of 67.4%.
Project description:Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.
Project description:This study reports that a high concentration of the endo-?-1,3-glucanase ENG (200??g ml<sup>-1</sup>) induced heat-inactivated stipe wall extension of <i>Coprinopsis cinerea</i>, whereas a high concentration of the extracellular ?-glucosidase BGL2 (1,000??g ml<sup>-1</sup>) did not; however, in combination, low concentrations of ENG (25??g ml<sup>-1</sup>) and BGL2 (260??g ml<sup>-1</sup>) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ?-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60?min; the stipe cell wall extension induced by a high concentration of ?-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ?-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ?-1,6 branched ?-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ?-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.<b>IMPORTANCE</b> We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom <i>Coprinopsis cinerea</i> In this study, we explored that ?-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ?-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ?-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ?-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ?-glucans during stipe elongation growth.
Project description:Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.
Project description:Mutanases are enzymes that have the ability to cleave ?-1,3 linkages in glucan polymer. In the present investigation, mutanase enzyme purified from the culture filtrate of <i>Paracoccus mutanolyticus</i> was evaluated for <i>Streptococcal</i> biofilm degradation and antimicrobial activity against pathogenic fungi along with enzyme kinetics, activation energies, pH and thermal stability. Biochemical and molecular characterization depicted that the enzyme showed optimum activity at pH 5.5 and at 50 °C. It displayed Michaelis-Menten behaviour with a K<sub>m</sub> of 1.263?±?0.03 (mg/ml), V<sub>max</sub> of 2.712?±?0.15 U/mg protein. Thermal stability studies denoted that it required 55.46 and 135.43 kJ mol<sup>-1</sup> of energy for activation and deactivation in the temperature range of 30-50 °C and 50-70 °C respectively. Mutanase activity was enhanced ~?50 and 75% by Fe<sup>2+</sup> and EDTA, respectively, while presence of Hg<sup>2+</sup> and Mn<sup>2+</sup> inhibit >?90% of its activity. This enzyme has a molecular mass of 138 kDa and showed monomeric nature by Zymography. Scanning electron microscopy analysis of mutanase treated <i>Streptococcal</i> cells revealed cleavage of linkages among the cells and complete separation of cells, indicating its potential in dentistry as an anticaries agent in the prophylaxis and therapy of dental caries. In addition, antifungal activity of mutanase against <i>Colletotrichum capsici</i> MTCC 10147 and <i>Cladosporium cladosporioide</i> MTCC 7371 revealed that the enzyme has potential towards biological control of phytopathogens which could be used as an alternative bio-control agent against chemical pesticides in the future.
Project description:A novel β-(1,3)-glucanase gene designated <i>lamC</i>, cloned from <i>Corallococcus</i> sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on β-(1,3)(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward β-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel β-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.<b>IMPORTANCE</b> In this report of a bacterial β-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the β-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-β-(1,3)/(1,6)-glucanase and endo-β-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.
Project description:<h4>Unlabelled</h4>The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes ?-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall ?-glucans, which results in enhanced binding to the Dectin-1 ?-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-?) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed ?-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In ?-glucan-producing Histoplasma strains, Eng1 acts in concert with ?-glucan to minimize ?-glucan exposure: ?-glucan provides a masking function by covering the ?-glucan-rich cell wall, while Eng1 removes any remaining exposed ?-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed ?-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes.<h4>Importance</h4>The success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a ?-glucanase, Eng1, which reduces recognition of fungal cell wall ?-glucans. We demonstrated that the Eng1 ?-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed ?-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host ?-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall ?-glucans by ?-glucan and enzymatic removal of exposed ?-glucans by the Eng1 ?-glucanase.
Project description:The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as ?-1,3-glucanase. In this study, a novel endo-type ?-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited ?-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of ?-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
Project description:Recently, in order to improve the resistance of flax plants to pathogen infection, transgenic flax that overproduces ?-1,3-glucanase was created. ?-1,3-glucanase is a PR protein that hydrolyses the ?-glucans, which are a major component of the cell wall in many groups of fungi. For this study, we used fourth-generation field-cultivated plants of the Fusarium -resistant transgenic line B14 to evaluate how overexpression of the ?-1,3-glucanase gene influences the quantity, quality and composition of flax fibres, which are the main product obtained from flax straw.Overproduction of ?-1,3-glucanase did not affect the quantity of the fibre obtained from the flax straw and did not significantly alter the essential mechanical characteristics of the retted fibres. However, changes in the contents of the major components of the cell wall (cellulose, hemicellulose, pectin and lignin) were revealed. Overexpression of the ?-1,3-glucanase gene resulted in higher cellulose, hemicellulose and pectin contents and a lower lignin content in the fibres. Increases in the uronic acid content in particular fractions (with the exception of the 1 M KOH-soluble fraction of hemicelluloses) and changes in the sugar composition of the cell wall were detected in the fibres of the transgenic flax when compared to the contents for the control plants. The callose content was lower in the fibres of the transgenic flax. Additionally, the analysis of phenolic compound contents in five fractions of the cell wall revealed important changes, which were reflected in the antioxidant potential of these fractions.Overexpression of the ?-1,3-glucanase gene has a significant influence on the biochemical composition of flax fibres. The constitutive overproduction of ?-1,3-glucanase causes a decrease in the callose content, and the resulting excess glucose serves as a substrate for the production of other polysaccharides. The monosaccharide excess redirects the phenolic compounds to bind with polysaccharides instead of to partake in lignin synthesis. The mechanical properties of the transgenic fibres are strengthened by their improved biochemical composition, and the increased antioxidant potential of the fibres supports the potential use of transgenic flax fibres for biomedical applications.
Project description:Tannerella forsythia and Fusobacterium nucleatum are dental plaque bacteria implicated in the development of periodontitis. These two species have been shown to form synergistic biofilms and have been found to be closely associated in dental plaque biofilms. A number of genetic loci for TonB-dependent membrane receptors (TDR) for glycan acquisition, with many existing in association with genes coding for enzymes involved in the breakdown of complex glycans, have been identified in T. forsythia In this study, we focused on a locus, BFO_0186-BFO_0188, that codes for a predicted TDR-SusD transporter along with a putative ?-glucan hydrolyzing enzyme (BFO_0186). This operon is located immediately downstream of a 2-gene operon that codes for a putative stress-responsive extracytoplasmic function (ECF) sigma factor and an anti-sigma factor. Here, we show that BFO_0186 expresses a ?-glucanase that cleaves glucans with ?-1,6 and ?-1,3 linkages. Furthermore, the BFO_0186-BFO_0188 locus is upregulated, with an induction of ?-glucanase activity, in cobiofilms of T. forsythia and F. nucleatum The ?-glucanase activity in mixed biofilms in turn leads to an enhanced hydrolysis of ?-glucans and release of glucose monomers and oligomers as nutrients for F. nucleatum In summary, our study highlights the role of T. forsythia ?-glucanase expressed by the asaccharolytic oral bacterium T. forsythia in the development of T. forsythia-F. nucleatum mixed species biofilms, and suggest that dietary ?-glucans might contribute in plaque development and periodontal disease pathogenesis.IMPORTANCE The development of dental plaque biofilm is a complex process in which metabolic, chemical and physical interactions between bacteria take a central role. Previous studies have shown that the dental pathogens T. forsythia and F. nucleatum form synergistic biofilms and are closely associated in human dental plaque. In this study, we show that ?-glucanase from the periodontal pathogen T. forsythia plays a role in the formation of T. forsythia-F. nucleatum cobiofilms by hydrolyzing ?-glucans to glucose as a nutrient. We also unveiled that the expression of T. forsythia ?-glucanase is induced in response to F. nucleatum sensing. This study highlights the involvement of ?-glucanase activity in the development of T. forsythia-F. nucleatum biofilms and suggests that intake of dietary ?-glucans might be a contributing risk factor in plaque development and periodontal disease pathogenesis.
Project description:Endo-β-1,3-glucanase plays an essential role in the deconstruction of β-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-β-1,3-glucanase (GluY) with a ricin-type β-trefoil lectin domain (RICIN)-like domain from <i>Cellulosimicrobium funkei</i> HY-13 was identified and biocatalytically characterized. The recombinant enzyme (rGluY: 57.5 kDa) displayed the highest degradation activity for laminarin at pH 4.5 and 40 °C, while the polysaccharide was maximally decomposed by its C-terminal truncated mutant enzyme (rGluYΔRICIN: 42.0 kDa) at pH 5.5 and 45 °C. The specific activity (26.0 U/mg) of rGluY for laminarin was 2.6-fold higher than that (9.8 U/mg) of rGluYΔRICIN for the same polysaccharide. Moreover, deleting the C-terminal RICIN domain in the intact enzyme caused a significant decrease (>60%) of its ability to degrade β-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of β-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against <i>Candida albicans</i> in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of <i>C. albicans</i> by rGluY (approximately 1.8 μM) was approximately 80% of the fungal growth. The superior anti-fungal activity of rGluY suggests that it can potentially be exploited as a supplementary agent in the food and pharmaceutical industries.