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In this study, an endophytic Bacillus sp. strain (K7) was isolated from the medicinally important ornamental plant, Jasminum officinale. Biochemical analyses were conducted to evaluate the nature of the extracted product, which displayed strong anticandidal activity against Candida albicans (CA) SC5314, as evident from the results obtained in agar-cup diffusion tests, phase-contrast microscopy, scanning electron microscopy and minimum inhibitory concentration assays. After confirming the presence of the gene clusters encoding the lipopeptides iturins and fengycin in the genome of K7, their corresponding molecular ions were identified using MALDI-TOF-MS. 3D structures of the lipopeptides were downloaded from specific databases and molecular docking was performed against a vital CA enzyme, exo-1,3-beta-glucanase, involved in cell wall remodelling, adhesion to polymer materials and biofilm formation. The docking score of iturins was found to be −8·6 and −8·2 kcal mol−1 and for fengycin it was −9·4 kcal mol−1, indicating a strong affinity of these cyclic lipopeptides towards exo-1,3-beta-glucanase. The combined in vitro and in silico anticandidal studies suggested that these secreted lipopeptides from Bacillus sp. may be used as potential therapeutics against opportunistic and complicated infections of CA.

Smut fungi comprise one of the largest groups of fungal plant pathogens causing disease in all cereal crops. They directly penetrate host tissues and establish a biotrophic interaction. To do so, smut fungi secrete a wide range of effector proteins, which suppress plant immunity and modulate cellular functions as well as development of the host, thereby determining the pathogen’s lifestyle and virulence potential. The conserved effector Erc1 (enzyme required for cell-to-cell extension) contributes to virulence of the corn smut Ustilago maydis in maize leaves but not on the tassel. Erc1 binds to host cell wall components and displays 1,3-β-glucanase activity, which is required to attenuate β-glucan-induced defense responses. Here we show that Erc1 has a cell type-specific virulence function, being necessary for fungal cell-to-cell extension in the plant bundle sheath and this function is fully conserved in the Erc1 orthologue of the barley pathogen Ustilago hordei.

Biological (or reductive) soil disinfestation (BSD or RSD) is a bioremediation process to control soil-borne plant pathogens using activities of indigenous bacteria in the soil. Three obligate anaerobic bacterial strains (TW1, TW10, and TB10), which were isolated from anoxic soil subjected to BSD treatments, were examined for their abilities to produce anti-fungal enzymes. All strains were affiliated with the different lineages of the genus Clostridium. The three strains decomposed β-1,3-glucans (curdlan and laminarin), and β-1,3-glucanase activities were detected from their culture supernatants with these glucans. The three strains also produced the enzyme with wheat bran as a growth substrate and killed the Fusarium pathogen (Fusarium oxysporum f. sp. spinaciae) in the anaerobic co-incubation conditions. Observation by fluorescence microscopy of the pathogen cells showed that the three strains had degraded the fungal cells in different manners upon co-incubation with wheat bran. When the three strains were cultivated with the dead cells or the cell wall samples prepared from the Fusarium pathogen, strain TW1 utilized these materials as easily decomposable substrates by releasing β-1,3-glucanase. When observed by fluorescence microscopy, it appeared that strain TW1 degraded the mycelial cell wall nearly thoroughly, with the septa remaining as undecomposed luminous rings. In contrast, the other two strains decomposed neither the dead cells nor the cell wall samples directly. The results indicate that the various anaerobic bacteria proliferated in the soil under the BSD treatments should play key roles as an organized bacterial community to eliminate fungal pathogens, namely by release of anti-fungal enzymes with different properties. Key points•Three clostridial strains isolated from BSD-treated soils produced β-1,3-glucanase.•All strains killed the Fusarium pathogen in the anaerobic co-incubation conditions.•One of the strains produced β-1,3-glucanase with the fungal cell wall as a substrate.•The strain degraded the cell wall almost completely, except for the mycelial septa.

BACKGROUND s-glucogallin (GG) is one of the major plant polyphenolic antioxidants that have been associated with positive effects on human health and are crucial in the developing defense mechanism against the risk of diseases. However, reports on the protective mechanism of GG in lens epithelial cells are limited. MATERIAL AND METHODS ARPE-19 cells (a human retinal epithelial cell line) were exposed to methylglyoxal (MG) with or without GG to illuminate the protective role of GG in counteracting the cataract signaling. RESULTS Cells predisposed to MG demonstrated an increase in oxidative stress with augmented (P

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β-mannanase/β-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported β-N-acetylglucosaminide/β-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Although peroxisomes play an essential role in viral pathogenesis, and viruses are known to change peroxisome morphology, the role of genotype in the peroxisomal response to viruses remains poorly understood. Here, we analyzed the impact of wheat streak mosaic virus (WSMV) on the peroxisome proliferation in the context of pathogen response, redox homeostasis, and yield in two wheat cultivars, Patras and Pamir, in the field trials. We observed greater virus content and yield losses in Pamir than in Patras. Leaf chlorophyll and protein content measured at the beginning of flowering were also more sensitive to WSMV infection in Pamir. Patras responded to the WSMV infection by transcriptional up-regulation of the peroxisome fission genes PEROXIN 11C (PEX11C), DYNAMIN RELATED PROTEIN 5B (DRP5B), and FISSION1A (FIS1A), greater peroxisome abundance, and activation of pathogenesis-related proteins chitinase, and β-1,3-glucanase. Oppositely, in Pamir, WMSV infection suppressed transcription of peroxisome biogenesis genes and activity of chitinase and β-1,3-glucanase, and did not affect peroxisome abundance. Activity of ROS scavenging enzymes was higher in Patras than in Pamir. Thus, the impact of WMSV on peroxisome proliferation is genotype-specific and peroxisome abundance can be used as a proxy for the magnitude of plant immune response.

Introduction The cell wall β-1,3-glucan of fungal pathogen Candida albicans is an attractive antifungal target. β-1,3-Glucan is the skeletal structure in the cell wall and the major scaffold for cell wall proteins. In previous studies using Saccharomyces cerevisiae , strong emulsification was detected by mixing cell wall proteins with oil. To date, there have been no reports of applying an emulsification phenomenon to assessing β-1,3-glucan synthesis inhibition. Objective The aim of this study was to clarify that emulsification is useful as an indicator for evaluating β-1,3-glucan synthesis inhibition in C. albicans. Methods At first, whether cell wall proteins released from cells by β-1,3-glucanase treatment worked as an effective emulsifier in C. albicans was examined. Next, whether emulsification occurred even in the culture supernatant brought about by treating with bioactive compounds, including β-1,3-glucan synthesis inhibitors, under osmotic protection was investigated. In addition, the release of cell wall proteins into the culture medium by treating with those compounds was examined. Finally, a simpler evaluation method using emulsion formation was examined for application to screening of inhibitors. Results Emulsification occurred by cell wall proteins obtained by treating with β-1,3-glucanase in C. albicans. In addition, cell wall proteins were released into the culture medium by treating with β-1,3-glucan synthesis inhibitors, resulting in emulsification. However, such phenomena were not observed in the case of other bioactive compounds. Furthermore, emulsification could be detected in the culture broth obtained by static culture on a small scale. Conclusions The obtained results strongly implied that emulsification results from decreased β-1,3-glucan levels in the cell wall. As emulsification can be simply evaluated by mixing the culture broth with oil, in the future application to the initial assessment and screening of β-1,3-glucan synthesis inhibitors is expected.

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2–5 (a hydrolysis yield of 97.75%), 3–6 (75.45%), and 3–7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Berberine hydrochloride (BH), an active component of Coptis chinensis and other plant taxa, has broad antimicrobial activity and may be useful for the treatment of Candida infections. In this study, the mechanisms underlying the inhibitory effect of BH against Candida albicans were evaluated, with a focus on the high-osmolarity glycerol mitogen-activated protein kinase (HOG-MAPK) pathway, which regulates multiple physiological functions. BH (256 and 64 μg ml

The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.

Enzymatic curdlan hydrolysis is gaining more attention for the value of oligo-β-glucans in many aspects. Currently, the triple-helical conformation of curdlan fiber was imposed to the structure of β-1,3-glucanase as its substrate without experimental evidence. Here, solution conformation of differently treated curdlan and each hydrolysis rate by a variety of β-1,3-glucanases were systematically examined. Results showed that different enzymes exhibited preferences over the trajectories of pH change that curdlan solution went through, and all enzymes hydrolyzed heat treated curdlan solution at their maximum rates where most of the higher ordered helices were diminished. Combined with molecular docking studies, a multi-step hydrolysis process was proposed. Recognition of triple-helical curdlan by their ancillary region of β-1,3-glucanase occurred before its unwinding into single- and double-helical forms, and the later ones fitted better to the catalytic cavity of the enzyme where the polysaccharides chain eventually got hydrolyzed into oligo-β-glucans.

The aim of this work was to evaluate the possibility of control of wilt disease caused by Fusarium andiyazi through chitosan (CS) and chitosan nanoparticles (CNPs). In the present study, the expression pattern of pathogenesis-related (PR) proteins genes such as PR-1, PR-2 (β-1,3-glucanase), PR-8 (chitinase), and PR-10 was analyzed using real-time RT-PCR. In vitro studies showed that among different concentrations (0.1–5.0 mg/ml), 5.0 mg/ml concentration of CS and CNPs produced maximum inhibition of radial mycelial growth, 54.8% and 73.81%, respectively. Also, upregulated expression of β-1,3-glucanase, chitinase, PR-1 and PR-10 genes were recorded with 1.48, 1.15, 1.15, and 1.41, fold expression in 24 hpi, respectively, in plants inoculated with CNPs. The most significant up-regulation was observed in transcript profile of SOD that showed 4.5-foldexpression, at 48 hpi. Therefore, our results confirmed that CS and CNPs induced up-regulation of PR-proteins and antioxidant genes might play a significant role for successful biocontrol.

Resistance is induced in cotton plants as the result of either viral infection or exogenous application of elicitors. Induced resistance can be evaluated by determining the production of β-1,3-glucanase and chitinase in plants as a biochemical parameter. The assays being used for the determination of chitinase and β-1,3-glucanase activity are laborious and not cost-effective, as the reducing sugars produced by the substrates colloidal chitin and laminarin are very expensive. The concentration of both substrates was standardized and reduced to 0.25% from 4% in a modified microplate assay, which appeared to be more effective. The amount of β-1,3-glucanase and chitinase produced was significant and determined by the new modified assay. The sensitivity of the microplate assay was significantly raised approximately one- to twofold.

Made available in DSpace on 2021-06-25T11:46:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-29 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, beta-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including beta-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii. Julio de Mesquita Filho Paulista State Univ, Grad Agr Microbiol Program, Agr & Veterinarian Fac, Sao Paulo, Brazil Univ Ctr Herminio Ometto Fdn FHO, Grad Program Biomed Sci, Sao Paulo, Brazil Sylvio Moreira Citriculture Ctr IAC, Rodovia Anhanguera,Km 158,CP 04, BR-13470970 Cordeiropolis, SP, Brazil Julio de Mesquita Filho Paulista State Univ, Grad Agr Microbiol Program, Agr & Veterinarian Fac, Sao Paulo, Brazil FAPESP: 2014/25067-3

The laminarans are biologically active water-soluble polysaccharide (1,3;1,6-β-D-glucans) of brown algae. These polysaccharides are an attractive object for research due to its relatively simple structure, low toxicity, and various biological effects. 1,3-β-D-glucanases are an effective tool for studying the structure of laminarans, and can also be used to obtain new biologically active derivatives. This review is to outline what is currently known about laminarans and enzymes that catalyze of their transformation. We focused on information about sources, structure and properties of laminarans and 1,3-β-D-glucanases, methods of obtaining and structural elucidation of laminarans, and biological activity of laminarans and products of their enzymatic transformation. It has an increased focus on the immunomodulating and anticancer activity of laminarans and their derivatives.

The fundamental and assorted roles of β-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on β-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/β)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical β-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of β-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of β-1,3-glucans, which can be exploited for biotechnological applications.

The induced resistance against plant pathogens via biocontrol agents is considered as an eco-friendly and promising strategy. In this study, the induced resistance against Magnaporthe oryzae (M. oryzae) in rice seedling by a new potential biocontrol agent Streptomyces JD211 (JD211) was evaluated. The effects of JD211 on defense-related enzymes activities and defense genes expression were investigated. The biocontrol efficacy of different JD211 concentrations was different, and the treatment of 10 g kg-1 JD211 achieved the highest biocontrol efficacy. Activities of catalase, phenylalanine ammonia-lyase (PAL) and β-1,3-glucanase significantly increased in the presence of JD211. The gene expression level of both PAL and pathogenesis related protein 1 increased when rice seedlings were inoculated with JD211 alone or co-inoculated with M. oryzae, and the expression level of chitinase gene was enhanced by JD211 in the later stage. All results suggested that JD211 could increase the rice resistance by stimulating a series of defense responses, which was the result of induced systemic resistance by JD211. This work will provide a new biocontrol agent against Magnaporthe oryzae in rice seedling.

β-1,3-glucan plays a role in Candida biofilm formation and survival of biofilm-forming Candida to stresses. In this study, we evaluated the antibiofilm activity of β-1,3-glucanase, which can degrade poly-β(1→3)-glucose of Candida albicans biofilms. Biofilm was dispersed by 55.96%. β-1,3-glucanase had no effect on Candida planktonic growth as well as adhesion. β-1,3-glucanase markedly enhanced the antifungal susceptibility of fluconazole and amphotericin B. The examination using confocal laser scanning microscopy and scanning electron microscope confirmed the antibiofilm activity of β-1,3-glucanase. Our findings demonstrate that β-1,3-glucanase may be useful as an antibiofilm agent.

Some β-glucans have attracted attention due to their functionality as an immunostimulant and have been used in processed foods. However, accurately measuring the β-glucan content of processed foods using existing methods is difficult. We demonstrate a new method, the Sodium hypochlorite Extracting and Enzymatic Digesting (SEED) assay, in which β-glucan is extracted using sodium hypochlorite, dimethyl sulfoxide, and 5 mol/L sodium hydroxide and then digested into β-glucan fragments using Westase which is an enzyme having β-1,6- and β-1,3 glucanase activity. The β-glucan fragments are further digested into glucose using exo-1,3-β-d-glucanase and β-glucosidase. We measured β-glucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds in various polysaccharide reagents and processed foods using our novel method. The SEED assay was able to quantify β-glucan with good reproducibility, and the recovery rate was90% for food containing β-glucan. Therefore, the SEED assay is capable of accurately measuring the β-glucan content of processed foods.

We investigated cell growth and activity of intra- and extracellular chitinase, β-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular β-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular β-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular β-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin.

β-1,3-glucan plays a role in non-albicans Candida species biofilm formation and survival of biofilm Candida to stresses. In this study, we evaluated the antibiofilm activity of β-1,3-glucanase, which can degrade poly-β(1 → 3)-glucose of non-albicans Candida species biofilms, on single and mixed species biofilm of non-albicans Candida species, including Candida tropicalis, Candida parapsilosis and Candida krusei. Biofilm by all tested species in microplate were dispersed more than 60%. β-1,3-glucanase also detached mixed species biofilm in microplate and on medical material surface. β-1,3-glucanase had no effect on Candida planktonic growth as well as adhesion. However, further biofilm formation was inhibited with β-1,3-glucanase added at 24 h after biofilm initiation. β-1,3-glucanase markedly enhanced the antifungal susceptibility of amphotericin B. The examination using confocal laser scanning microscopy and scanning electron microscope confirmed the antibiofilm activity of β-1,3-glucanase. Our findings demonstrate that β-1,3-glucanase may be useful as an antibiofilm agent.

Pythium insidiosum is an important oomycete due to its ability to infect humans and animals. It causes pythiosis, a disease of difficult treatment that occurs more frequently in humans in Thailand and in horses in Brazil. Since cell-wall components are frequently related to host shifts, we decided here to use sequences from the exo-1,3-β-glucanase gene (exo1), which encodes an immunodominant protein putatively involved in cell wall remodeling, to investigate the microevolutionary relationships of Brazilian and Thai isolates of P. insidiosum. After neutrality ratification, the phylogenetic analyses performed through Maximum parsimony (MP), Neighbor-joining (NJ), Maximum likelihood (ML), and Bayesian analysis (BA) strongly supported Thai isolates being paraphyletic in relation to those from Brazil. The structure recovered by these analyses, as well as by Spatial Analysis of Molecular Variance (SAMOVA), suggests the subdivision of P. insidiosum into three clades or population groups, which are able to explain almost 81% of the variation encountered for exo1. Moreover, the two identified Thai clades were almost as strongly differentiated between each other, as they were from the Brazilian clade, suggesting an ancient Asian subdivision. The derived positioning in the phylogenetic tree, linked to the lower diversity values and the recent expansion signs detected for the Brazilian clade, further support this clade as derived in relation to the Asian populations. Thus, although some patterns presented here are compatible with those recovered with different molecular markers, exo1 was revealed to be a good marker for studying evolution in Pythium, providing robust and strongly supported results with regard to the patterns of origin and diversification of P. insidiosum.

A novel β-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67 %) with a glycoside hydrolase family 17 β-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8 % recovery yield. The protein’s molecular mass was approximately 65 kDa, and its maximum activity appeared at pH 5.0 and temperature 45 °C. Heavy metal ions Fe2+, Mn2+, Cu2+, Co2+, Ag+, and Hg2+ had inhibitory effects on Ccglu17A, but Ba2+ promoted the enzyme’s activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing β-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84 mg/mL and Vmax of 10.7 μmol glucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-β-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.

Highlight Axillary buds uniquely regulate gibberellin (GA) pathway genes, enabling them to stay inhibited but simultaneously poised for growth. Decapitation promotes expression of GA-inducible 1,3-β-glucanase genes that function to reinvigorate symplasmic connections to the stem., Axillary buds (AXBs) of hybrid aspen (Populus tremula×P. tremuloides) contain a developing dwarfed shoot that becomes para-dormant at the bud maturation point. Para-dormant AXBs can grow out after stem decapitation, while dormant AXBs pre-require long-term chilling to release them from dormancy. The latter is mediated by gibberellin (GA)-regulated 1,3-β-glucanases, but it is unknown if GA is also important in the development, activation, and outgrowth of para-dormant AXBs. The present data show that para-dormant AXBs up-regulate GA receptor genes during their maturation, but curtail GA biosynthesis by down-regulating the rate-limiting GIBBERELLIN 3-OXIDASE2 (GA3ox2), which is characteristically expressed in the growing apex. However, decapitation significantly up-regulated GA3ox2 and GA4-responsive 1,3-β-glucanases (GH17-family; α-clade). In contrast, decapitation down-regulated γ-clade 1,3-β-glucanases, which were strongly up-regulated in maturing AXBs concomitant with lipid body accumulation. Overexpression of selected GH17 members in hybrid aspen resulted in characteristic branching patterns. The α-clade member induced an acropetal branching pattern, whereas the γ-clade member activated AXBs in recurrent flushes during transient cessation of apex proliferation. The results support a model in which curtailing the final step in GA biosynthesis dwarfs the embryonic shoot, while high levels of GA precursors and GA receptors keep AXBs poised for growth. GA signaling, induced by decapitation, reinvigorates symplasmic supply routes through GA-inducible 1,3-β-glucanases that hydrolyze callose at sieve plates and plasmodesmata.

Made available in DSpace on 2019-10-06T17:11:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Antimicrobial Photodynamic Therapy (aPDT) has been proposed as a means to treat Candida infections. However, microorganisms in biofilms are less susceptible to aPDT than planktonic cultures, possibly because the matrix limits the penetration of the photosensitizer. Therefore, the goals here were: (1) to target biofilm matrix components of a fluconazole-susceptible (S) and a fluconazole-resistant (R) C. albicans (Ca) strains using the hydrolytic enzymes β-glucanase and DNase individually or in combination; (2) to apply the best enzyme protocol in association with aPDT mediated by Photodithazine® (PDZ); (3) to verify under confocal microscope the penetration of PDZ in biofilms pre-treated or not with DNase at different periods of incubation. CaS and CaR 48h-old biofilms were incubated with the hydrolytic enzymes (5 min) and evaluated by cell viability, biomass, and matrix components. DNase showed the best outcomes by significantly reducing extracellular DNA (eDNA) and soluble proteins from the matrix of both strains; and water-soluble polysaccharides from CaR matrix. Subsequently, 48h-old biofilms were incubated with DNase for 5 min, followed by incubation with PDZ for 20 min and exposure to LED light (660 nm, 50 J/cm²). Controls were biofilms treated only with aPDT without DNase, PDZ only, PDZ + DNase, light only, light + DNase, and biofilm without treatment. Pre-treatment with DNase allowed PDZ penetration into deeper biofilm layers, and the aPDT effect was enhanced, showing a significant reduction of the cell viability (p = 0.000) and eDNA amounts (p ≤ 0.047). DNase affected the matrix composition improving the penetration of the photosensitizer, thereby, improving the effectiveness of subsequent aPDT. Department of Dental Materials and Prosthodontics São Paulo State University (Unesp) School of Dentistry Department of Dental Materials and Prosthodontics São Paulo State University (Unesp) School of Dentistry FAPESP: # 2013/07276-1 FAPESP: # 2014/18804-1

β‐1,3‐d‐glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water‐soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi‐angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith‐degraded β‐1,3‐d‐glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide–hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order‐disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation. Locked until 12.06.2020 due to copyright restrictions. This is the peer reviewed version of an article, which has been published in final form at [DOI:10.1002/bip.23315 ]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Phytophthora is considered one of the most destructive genus for many agricultural plant species worldwide, with a strong environmental and economic impact. Phytophthora cinnamomi is a highly aggressive Phytophthora species associated with the forest decline and responsible for the ink disease in chestnut trees (Castanea sativa Miller), a culture which is extremely important in Europe. This pathogenicity occurs due to the action of several enzymes like the hydrolysis of 1,3-β-glucans at specific sites by the enzyme endo-1,3-β-d-glucosidase. The aim of this work to analyze the heterologous expression in two microorganisms, Escherichia coli and Pichia pastoris, of an endo-1,3-β-d-glucosidase encoded by the gene ENDO1 (AM259651) from P. cinnamomi. Different plasmids were used to clone the gene on each organism and the real-time quantitative polymerase chain reaction was used to determine its level of expression. Homologous expression was also analyzed during growth in different carbon sources (glucose, cellulose, and sawdust) and time-course experiments were used for endo-1,3-β-d-glucosidase production. The highest expression of the endo-1,3-β-d-glucosidase gene occurred in glucose after 8 h of induction. In vivo infection of C. sativa by P. cinnamomi revealed an increase in endo-1,3-β-d-glucosidase expression after 12 h. At 24 h its expression decreased and at 48 h there was again a slight increase in expression, and more experiments in order to further explain this fact are underway. info:eu-repo/semantics/publishedVersion

A novel β-1,3-glucanase from Arca inflata was purified using chromatography methods. It was determined as a glycoprotein comprising 23.65% carbohydrate content with O-linked glycan and showed specific activity of 90.01 ± 1.2 U/mg against laminarin. The optimal pH and temperature for the activity of the glucanase were 6.0 and 40 °C, respectively. The affinity parameter of the glucanase using laminarin was determined as Kd = 13.09 μM. The activity of the glucanase was 27 ± 2.6% enhanced by 2-mM Mn2+ ions and inhibited by 40–50% using 2-mM Zn2+, Cu2+, or Ba2+ ions. The glucanase showed an endo-type cleavage mode and hydrolyzed laminarin into glucoses, disaccharides, trioligosaccharides, and tetraoligosaccharides. Otherwise, the glucanase exhibited immune-enhancing effects via significantly increasing the phagocytic activity of macrophages and inducing the release of nitric oxide, tumor necrosis factor α, and interleukin-6 in RAW264.7 cells. It might be used as a bifunctional additive for the food industry.

Aluminum (Al) toxicity is a primary limiting factor for crop production in acid soils. Callose deposition, an early indicator and likely a contributor to Al toxicity, is induced rapidly in plant roots under Al stress. SbGlu1, encoding a β-1,3-glucanase for callose degradation, showed important roles in sorghum Al resistance, yet its regulatory mechanisms remain unclear. The STOP1 transcription factors mediate Al signal transduction in various plants. Here, we identified their homolog in sweet sorghum, SbSTOP1, transcriptionally activated the expression of SbGlu1. Moreover, the DNA sequence recognized by SbSTOP1 on the promoter of SbGlu1 lacked the reported cis-acting element. Complementation lines of Atstop1 with SbSTOP1 revealed enhanced transcription levels of SbGlu1 homologous gene and reduced callose accumulation in Arabidopsis. These results indicate, for the first time, that SbSTOP1 is involved in the modulation of callose deposition under Al stress via transcriptional regulation of a β-1,3-glucanase gene. SbSTOP1 directly activities the transcription of a β-1,3-glucanase gene, which confers reduced callose deposition and increased Al tolerance in plants.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the β-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

Among three MAPK disruptants of Neurospora crassa, Δmak-1 was sensitive and Δmak-2 was hypersensitive to micafungin, a beta-1,3-glucan synthase inhibitor, than the wild-type or Δos-2 strains. We identified six micafungin-inducible genes that are involved in cell wall integrity (CWI) and found that MAK-1 regulated the transcription of non-anchored cell wall protein gene, ncw-1, and the beta-1,3-endoglucanase gene, bgt-2, whereas MAK-2 controlled the expression of the glycosylhydrolase-like protein gene, gh76-5, and the C4-dicarboxylate transporter gene, tdt-1. Western blotting analysis revealed that, in the wild-type strain, MAK-1 was constitutively phosphorylated from conidial germination to hyphal development. In contrast, the phosphorylation of MAK-2 was growth phase-dependent, and micafungin induced the phosphorylation of unphosphorylated MAK-2. It should be noted that the phosphorylation of MAK-1 was virtually abolished in the Δmak-2 strain, but was significantly induced by micafungin, suggesting functional cross talk between MAK-1 and MAK-2 signalling pathway in CWI.

In order to investigate the effect of mating type and ploidy on enzymatic activity and fermentation performance in yeast with multiple δ-integrated foreign genes, eight ploidy series strains were constructed. The initial haploid strain BGL-a was shown to contain about 19 copies of the bgl1 gene. In rich media containing 2% (w/v) sugar the specific activities of BGL-aα were lower than those of BGL-aa or BGL-αα, which indicates the existence of mating type effects. While the maximum OD660 decreased with rising ploidy, the biomass yield showed no significant difference between the eight strains and the specific activities (expressed as U/mL or U/mg DCW) showed little to no variation. When cellobiose was used as the carbon source and β-glucosidase substrate, β-glucosidase was expressed more quickly and at higher levels than in glucose-containing media. The maximum specific activitiy values obtained were 19.07U/mL and 19.39U/mL for BGL-αα and BGL-aa, repsectively. The anaerobic biomass and ethanol-producing performance in rich media containing 10% cellobiose showed no significant difference among the eight strains. Their maximal ethanol concentrations and corresponding yields ranged from 40.27 to 43.46g/L and 77.56 to 83.71%, respectively. When the acid- and alkali-pretreated corncob (10% solids content) was used, the diploid BGL-aα fermented the best. When urea was used as the only supplemented nutrient, the ethanol titer and yield were 35.65g/L and 83.69%, respectively, while a control experiment using industrial Angel yeast with exogenous β-glucosidase addition gave values of 37.93g/L and 89.04%. The combined effects of δ-integration of bgl1, ploidy and mating type result in BGL-aa or BGL-αα being the optimal choice for enzyme production and BGL-aα being more suitable for cellulosic ethanol fermentation. These results provide valuable information for future yeast breeding and utilization efforts.