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1.
Biochemistry (Mosc) ; 88(1): 152-161, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37068877

RESUMO

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.


Assuntos
Fosfatase Ácida , Glucana Endo-1,3-beta-D-Glucosidase , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Parede Celular/metabolismo , Glucanos/metabolismo , Manganês/metabolismo , Polímeros , Polifosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo
2.
PLoS Pathog ; 18(1): e1010192, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34995333

RESUMO

Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes ß-glucan in the fungal cell wall. C. albicans ß-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how ß-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates ß-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated ß-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated ß-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.


Assuntos
Candida albicans/patogenicidade , Candidíase/imunologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Virulência/fisiologia , beta-Glucanas/imunologia , Animais , Candida albicans/imunologia , Candida albicans/metabolismo , Candidíase/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , beta-Glucanas/metabolismo
3.
Biomolecules ; 11(8)2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34439747

RESUMO

Endo-ß-1,3-glucanase plays an essential role in the deconstruction of ß-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-ß-1,3-glucanase (GluY) with a ricin-type ß-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei 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 ß-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of ß-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against Candida albicans in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of C. albicans 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.


Assuntos
Actinobacteria/metabolismo , Antifúngicos/farmacologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Polissacarídeos/química , Antifúngicos/química , Candida albicans/metabolismo , Catálise , Clonagem Molecular , Glucanos/química , Concentração de Íons de Hidrogênio , Hidrólise , Filogenia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Temperatura , beta-Glucanas/química
4.
Int J Biol Macromol ; 186: 424-432, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34246678

RESUMO

An endo-ß-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of ß-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-ß-gentiobiosyl-d-glucose and hydrolyzed barley ß-glucan to mainly produce 3-O-ß-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley ß-glucan and E. bicyclis laminarin. Further study reveals that 3-O-ß-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley ß-glucan and 3-O-ß-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that ß-1,4-linkage or -1,6-linkage at non-reducing end of ß-glucotrioses had enhancing effect on antioxidant activity compared to ß-1,3-linkage. Furthermore, 3-O-ß-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley ß-glucan, indicating that ß-1,4-linkage at reducing end of ß-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to ß-1,3-linkage.


Assuntos
Antioxidantes/farmacologia , Aspergillus nidulans/enzimologia , Celobiose/análogos & derivados , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glucanos/metabolismo , Glucose/farmacologia , Hordeum , beta-Glucanas/metabolismo , Antioxidantes/metabolismo , Compostos de Bifenilo/química , Catálise , Celobiose/biossíntese , Glucose/análogos & derivados , Glucose/metabolismo , Hordeum/química , Hidrólise , Estrutura Molecular , Picratos/química , Relação Estrutura-Atividade , Especificidade por Substrato , beta-Glucanas/isolamento & purificação
5.
Microb Cell Fact ; 20(1): 126, 2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34217291

RESUMO

BACKGROUND: The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital. RESULTS: Here we examined the efficiency and reproducibility of 12 recombinant endo-ß(1,3)-D-glucanases for specifically degrading the cell wall ß(1,3)-D-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, ß(1,3)-D-glucan and ß(1,6)-D-glucan. We found that the GH16 endo-ß(1,3)-D-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade ß(1,3)-D-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-ß(1,3)-D-glucanase no longer commercially available. Moreover, other recombinant ß(1,3)-D-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides. CONCLUSIONS: The discovery of a reliable and efficient recombinant endo-ß(1,3)-D-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall ß(1,3)-D-glucan. This new commercial endo-ß(1,3)-D-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis.


Assuntos
Parede Celular/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura , Parede Celular/química , Glucana Endo-1,3-beta-D-Glucosidase/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/química , beta-Glucanas/metabolismo
6.
Plant Sci ; 302: 110700, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33288013

RESUMO

This work presents the biochemical, cytochemical and molecular studies on two groups of PR proteins, ß-1,3-glucanases and chitinases, and the arabinogalactan proteins (AGP) during the early stages of androgenesis induction in two breeding lines of rye (Secale cereale L.) with different androgenic potential. The process of androgenesis was initiated by tillers pre-treatments with low temperature, mannitol and/or reduced glutathione and resulted in microspores reprogramming and formation of androgenic structures what was associated with high activity of ß-1,3-glucanases and chitinases. Some isoforms of ß-1,3-glucanases, namely several acidic isoforms of about 26 kDa; appeared to be anther specific. Chitinases were well represented but were less variable. RT-qPCR revealed that the cold-responsive chitinase genes Chit1 and Chit2 were expressed at a lower level in the microspores and whole anthers while the cold-responsive Glu2 and Glu3 were not active. The stress pre-treatments modifications promoted the AGP accumulation. An apparent dominance of some AGP epitopes (LM2, JIM4 and JIM14) was detected in the androgenesis-responsive rye line. An abundant JIM13 epitopes in the vesicles and inner cell walls of the microspores and in the cell walls of the anther cell layers appeared to be the most specific for embryogenesis.


Assuntos
Quitinases/fisiologia , Glucana Endo-1,3-beta-D-Glucosidase/fisiologia , Mucoproteínas/fisiologia , Proteínas de Plantas/fisiologia , Secale/metabolismo , Quitinases/metabolismo , Produção Agrícola/métodos , Flores/crescimento & desenvolvimento , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Mucoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Reprodução/fisiologia , Secale/enzimologia , Secale/fisiologia , Estresse Fisiológico
7.
Int J Mol Sci ; 21(21)2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33167499

RESUMO

Glucan linked to proteins is a natural mega-glycoconjugate (mGC) playing the central role as a structural component of a yeast cell wall (CW). Regulation of functioning of non-covalently bound glucanosyltransglycosylases (ncGTGs) that have to remodel mGC to provide CW extension is poorly understood. We demonstrate that the main ncGTGs Bgl2 and Scw4 have phosphorylated and glutathionylated residues and are represented in CW as different pools of molecules having various firmness of attachment. Identified pools contain Bgl2 molecules with unmodified peptides, but differ from each other in the presence and combination of modified ones, as well as in the presence or absence of other CW proteins. Correlation of Bgl2 distribution among pools and its N-glycosylation was not found. Glutathione affects Bgl2 conformation, probably resulting in the mode of its attachment and enzymatic activity. Bgl2 from the pool of unmodified and monophosphorylated molecules demonstrates the ability to fibrillate after isolation from CW. Revealing of Bgl2 microcompartments and their mosaic arrangement summarized with the results obtained give the evidence that the functioning of ncGTGs in CW can be controlled by reversible post-translational modifications and facilitated due to their compact localization. The hypothetical scheme of distribution of Bgl2 inside CW is represented.


Assuntos
Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Sequência de Aminoácidos/genética , Antifúngicos/metabolismo , Genes Fúngicos/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glucanos/metabolismo , Glucosidases/metabolismo , Glucosiltransferases/fisiologia , Glicosilação , Conformação Molecular , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transferases/metabolismo
8.
Mar Drugs ; 18(9)2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32947865

RESUMO

Endo-1,3-ß-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-ß-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-ß-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-ß-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-ß-glucanases, which facilitated development of a unique endo-1,3-ß-glucanase tool in the synthesis of novel glycosides.


Assuntos
Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Oligossacarídeos/metabolismo , Pectinidae/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Glucana Endo-1,3-beta-D-Glucosidase/química , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/isolamento & purificação , Glucanos/metabolismo , Glicosilação , Hidrólise , Pectinidae/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
9.
Enzyme Microb Technol ; 140: 109625, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32912685

RESUMO

Endo-ß-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast ß-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Glucana Endo-1,3-beta-D-Glucosidase/química , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/isolamento & purificação , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metais/química , Modelos Moleculares , Oligossacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura
10.
Molecules ; 25(10)2020 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-32429524

RESUMO

Cucumber mosaic cucumovirus (CMV) is a deadly plant virus that results in crop-yield losses with serious economic consequences. In recent years, environmentally friendly components have been developed to manage crop diseases as alternatives to chemical pesticides, including the use of natural compounds such as glycine betaine (GB) and chitosan (CHT), either alone or in combination. In the present study, the leaves of the cucumber plants were foliar-sprayed with GB and CHT-either alone or in combination-to evaluate their ability to induce resistance against CMV. The results showed a significant reduction in disease severity and CMV accumulation in plants treated with GB and CHT, either alone or in combination, compared to untreated plants (challenge control). In every treatment, growth indices, leaf chlorophylls content, phytohormones (i.e., indole acetic acid, gibberellic acid, salicylic acid and jasmonic acid), endogenous osmoprotectants (i.e., proline, soluble sugars and glycine betaine), non-enzymatic antioxidants (i.e., ascorbic acid, glutathione and phenols) and enzymatic antioxidants (i.e., superoxide dismutase, peroxidase, polyphenol oxidase, catalase, lipoxygenase, ascorbate peroxidase, glutathione reductase, chitinase and ß-1,3 glucanase) of virus-infected plants were significantly increased. On the other hand, malondialdehyde and abscisic acid contents have been significantly reduced. Based on a gene expression study, all treated plants exhibited increased expression levels of some regulatory defense genes such as PR1 and PAL1. In conclusion, the combination of GB and CHT is the most effective treatment in alleviated virus infection. To our knowledge, this is the first report to demonstrate the induction of systemic resistance against CMV by using GB.


Assuntos
Betaína/farmacologia , Quitosana/farmacologia , Cucumis sativus/efeitos dos fármacos , Cucumovirus/efeitos dos fármacos , Resistência à Doença/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Antioxidantes/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Catalase/genética , Catalase/metabolismo , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Quitinases/genética , Quitinases/metabolismo , Clorofila/metabolismo , Cucumis sativus/genética , Cucumis sativus/metabolismo , Cucumis sativus/virologia , Cucumovirus/crescimento & desenvolvimento , Cucumovirus/patogenicidade , Ciclopentanos/metabolismo , Resistência à Doença/genética , Giberelinas/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Ácidos Indolacéticos/metabolismo , Lipoxigenase/genética , Lipoxigenase/metabolismo , Oxilipinas/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
11.
Int J Biol Macromol ; 152: 516-524, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112847

RESUMO

We previously reported endo-ß-1,3-glucanase ENG in combination with ß-glucosidase BGL2 at low concentration induced stipe cell wall extension. This study further explored ENG could be replaced by endo-ß-1,3(4)-glucanase ENG16A in combination with BGL2 to induce stipe cell wall extension; similarly, BGL2 could be replaced by ß-glucosidase BGL1 to cooperate with ENG to induce stipe cell wall extension. However, ENG could not be replaced by exo-ß-1,3-glucanase EXG in combination with BGL2 to induce stipe cell wall extension, although EXG alone released higher level of soluble sugars from the stipe cell walls during the reconstituted wall extension than that released from the stipe cell walls by a combination of ENG16A or ENG and BGL2 or BGL1, which was different from chitinase-mediated stipe cell wall extension. These results indicate endo-ß-1,3-glucanases loosen the stipe cell wall, whereas exo-ß-1,3-glucanases and ß-glucosidases play a synergistic role to maintain a low and efficient concentration of endo-ß-1,3-glucanases for stipe cell wall extension. Furthermore, ENG was expressed at a very high level in the matured pilei, in contrast, ENG16A was expressed at a very high level in the elongating apical stipe. Therefore, ENG16A might be involved in stipe elongation growth, while ENG might participate in autolysis of pilei.


Assuntos
Agaricales/enzimologia , Parede Celular/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , beta-Glucanas/metabolismo , Metabolismo dos Carboidratos , Quitina/química , Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , RNA Mensageiro/metabolismo , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
12.
Mol Biol Rep ; 47(2): 935-942, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31741259

RESUMO

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-ß-glucans at specific sites by the enzyme endo-1,3-ß-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-ß-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-ß-D-glucosidase production. The highest expression of the endo-1,3-ß-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-ß-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.


Assuntos
Glucana Endo-1,3-beta-D-Glucosidase/genética , Phytophthora/genética , Clonagem Molecular/métodos , Glucana 1,3-beta-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glucosidases/genética , Glucosidases/metabolismo , Phytophthora/metabolismo , Doenças das Plantas , Reação em Cadeia da Polimerase em Tempo Real/métodos
13.
J Infect Dis ; 220(4): 657-665, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31100118

RESUMO

Glucan is the major cell wall component of Pneumocystis cysts. In the current study, we have characterized Pneumocystis Bgl2 (EC 3.2.1.58), an enzyme with glucanosyltransferase and ß-1,3 endoglucanase activity in other fungi. Pneumocystis murina, Pneumocystis carinii, and Pneumocystis jirovecii bgl2 complementary DNA sequences encode proteins of 437, 447, and 408 amino acids, respectively. Recombinant P. murina Bgl2 expressed in COS-1 cells demonstrated ß-glucanase activity, as shown by degradation of the cell wall of Pneumocystis cysts. It also cleaved reduced laminaripentaose and transferred oligosaccharides, resulting in polymers of 6 and 7 glucan residues, demonstrating glucanosyltransferase activity. Surprisingly, confocal immunofluorescence analysis of P. murina-infected mouse lung sections using an antibody against recombinant Bgl2 showed that the native protein is localized primarily to the trophic form of Pneumocystis in both untreated mice and mice treated with caspofungin, an antifungal drug that inhibits ß-1,3-glucan synthase. Thus, like other fungi, Bgl2 of Pneumocystis has both endoglucanase and glucanosyltransferase activities. Given that it is expressed primarily in trophic forms, further studies are needed to better understand its role in the biology of Pneumocystis.


Assuntos
Antifúngicos/farmacologia , Caspofungina/farmacologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Pneumocystis/enzimologia , Sequência de Aminoácidos , Animais , Ligante de CD40/genética , Células COS , Parede Celular/enzimologia , Chlorocebus aethiops , Glucana Endo-1,3-beta-D-Glucosidase/antagonistas & inibidores , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucanos/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumocystis/genética , Pneumocystis/imunologia , Pneumonia por Pneumocystis/imunologia , Proteínas Recombinantes , Alinhamento de Sequência
14.
J Ind Microbiol Biotechnol ; 46(6): 769-781, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30806871

RESUMO

Enzymatic degradation of the ß-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-ß-D-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis ß-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.


Assuntos
Euglena gracilis/enzimologia , Citometria de Fluxo/métodos , Glucanos/análise , Escherichia coli/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Hidrólise , Proteômica
15.
Protein Pept Lett ; 25(8): 734-739, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29972101

RESUMO

BACKGROUND: Oligosaccharides are of great value in drug discovery programs which address a wide range of therapeutic strategies in medical specialties. However, owing to difficulties in oligosaccharide synthesis by conventional methods, oligosaccharide assembly using enzymes has been explored. The transglycosylases have been demonstrated to be effective for the oligosaccharide synthesis. Further studies are required to improve the specificity and activity of transglycosylases. There is an additional approach to use mutated glycosidase which transforms into glycosyltransferase with a decreased hydrolytic activity. The substitution of catalytic residue in glycosidase results in the loss of hydrolytic activity. During the reaction with glucanase, reaction of water with the substrate - enzyme intermediate results in the production of a hydrolyzed sugar. When the water molecule is replaced by a competing sugar, a new glycoside linkage is formed as a result of transglycosylation. OBJECTIVE: In this article, we evaluated the transglycosylation activity of endo-1,3-ß-glucanase mutant, E119G, toward laminarioligosaccharides under various pH and temperature conditions, in comparison with those of the wild-type enzyme. We also analyzed the effect of glucose and laminaribiose on the transglycosylation activity. METHOD: In this article, we generated the E119G mutant of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans DK-1. The residue, Glu119, would act as a nucleophile in the reaction and affect the balance between hydrolysis and transglycosylation. The enzymatic activities of wild-type and E119G were estimated by detecting the products obtained from laminarioligosaccharides as substrates. We also analyzed the effect of reaction conditions such as temperature and pH on the enzymatic activity of E119G toward laminaritriose. We further analyzed the enzymatic activity of E119G toward laminaritriose in the presence of glucose or laminaribiose to investigate whether these additional molecules could accelerate the transglycosylation activity. RESULTS: The purified E119G mutant of endo-1,3-ß-glucanase was properly folded, and exhibited the secondary structure, similar to that of wild-type. The E119G mutant exhibited enhanced transglycosylation activity and decreased hydrolytic activity, relative to the wild-type. The hydrolytic as well as transglycosylation activities of E119G decreased with the decrease in temperature, however, the ratio of transglycosylation products increased. The temperature-dependent degree of reduction in hydrolytic activity was higher than that in the transglycosylation activity. The enzymatic activities were similar within the range of pH 4.0 - 7.4, while those at pH 8.0 and 8.5 were slightly decreased. The enzymatic activity of E119G toward laminaritriose in the presence of glucose was ineffective, while the addition of laminaribiose evidently increased the transglycosylation products such as laminaritetraose and laminaripentaose. CONCLUSION: A mutation of catalytic residue, Glu119 to Gly, in endo-1,3-ß-glucanase from Cellulosimicrobium cellulans exhibited transglycosylation activity on laminarioligosaccharides. The combination of laminaribiose and laminaritriose as a substrate enhanced the transglycosylation activity. According to the structural information previously reported, laminaritriose mainly binds to the enzyme at the subsites from -1 to -3 and forms a link with laminaribiose, which transiently binds to the subsites +1 and +2. To increase the amount of transglycosylation product, the reaction was found to be effective at low temperature.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Domínio Catalítico/fisiologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Actinobacteria/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Dissacarídeos/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/química , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucose/metabolismo , Glicosilação , Mutação/genética , Especificidade por Substrato
16.
Yakugaku Zasshi ; 138(6): 837-842, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29863056

RESUMO

 Only 4 classes of antifungal agents comprising 9 compounds are effective against deep mycosis in Japan, and it has been difficult to develop new antifungal specific agents. Micafungin, which has been used as an antifungal agent since 2002, inhibits ß-1,3-glucan synthesis in fungal cell walls, thereby killing yeast and filamentous fungi with no septum. In this study, we constructed a pYES2-BGL2 vector to overexpress ß-1,3-glucanase (BGL2) in yeast (Saccharomyces cerevisiae INVSc1) and evaluated the synergy between BGL2 overexpression and conventional antifungal agents. The recombinant yeast was incubated in SC-Ura medium, which contained galactose to induce BGL2 overexpression. The recombinant yeast with induced BGL2 overexpression was also frozen and crushed to obtain crude protein for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which revealed robust BGL2 overexpression compared with that in the control yeast without the expression vector. Therefore, we considered that we successfully constructed the recombinant yeast to express more BGL2. Further, 3 conventional antifungal agents (amphotericin B, micafungin, and miconazole) were more effective against the recombinant yeast than against the control yeast. From this result, it is suggested that BGL2 overexpression has an enhancing effect on conventional antifungal agents. Hence, glucanase-inducing compounds could act as novel antifungal drugs by augmenting the effectiveness of conventional antifungal agents.


Assuntos
Antifúngicos/farmacologia , Expressão Gênica , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Recombinação Genética , Saccharomyces cerevisiae/genética
17.
BMC Genomics ; 19(1): 232, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29614953

RESUMO

BACKGROUND: Sugarcane bagasse has been proposed as a lignocellulosic residue for second-generation ethanol (2G) produced by breaking down biomass into fermentable sugars. The enzymatic cocktails for biomass degradation are mostly produced by fungi, but low cost and high efficiency can consolidate 2G technologies. A. fumigatus plays an important role in plant biomass degradation capabilities and recycling. To gain more insight into the divergence in gene expression during steam-exploded bagasse (SEB) breakdown, this study profiled the transcriptome of A. fumigatus by RNA sequencing to compare transcriptional profiles of A. fumigatus grown on media containing SEB or fructose as the sole carbon source. Secretome analysis was also performed using SDS-PAGE and LC-MS/MS. RESULTS: The maximum activities of cellulases (0.032 U mL-1), endo-1,4-ß--xylanase (10.82 U mL-1) and endo-1,3-ß glucanases (0.77 U mL-1) showed that functional CAZymes (carbohydrate-active enzymes) were secreted in the SEB culture conditions. Correlations between transcriptome and secretome data identified several CAZymes in A. fumigatus. Particular attention was given to CAZymes related to lignocellulose degradation and sugar transporters. Genes encoding glycoside hydrolase classes commonly expressed during the breakdown of cellulose, such as GH-5, 6, 7, 43, 45, and hemicellulose, such as GH-2, 10, 11, 30, 43, were found to be highly expressed in SEB conditions. Lytic polysaccharide monooxygenases (LPMO) classified as auxiliary activity families AA9 (GH61), CE (1, 4, 8, 15, 16), PL (1, 3, 4, 20) and GT (1, 2, 4, 8, 20, 35, 48) were also differentially expressed in this condition. Similarly, the most important enzymes related to biomass degradation, including endoxylanases, xyloglucanases, ß-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and ß-glucosidases, were also identified in the secretome. CONCLUSIONS: This is the first report of a transcriptome and secretome experiment of Aspergillus fumigatus in the degradation of pretreated sugarcane bagasse. The results suggest that this strain employs important strategies for this complex degradation process. It was possible to identify a set of genes and proteins that might be applied in several biotechnology fields. This knowledge can be exploited for the improvement of 2G ethanol production by the rational design of enzymatic cocktails.


Assuntos
Aspergillus fumigatus/crescimento & desenvolvimento , Celulose/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica/métodos , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Celulases/genética , Celulases/metabolismo , Cromatografia Líquida , Frutose/química , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Saccharum/metabolismo , Análise de Sequência de RNA/métodos , Espectrometria de Massas em Tandem , Xilosidases/genética , Xilosidases/metabolismo
18.
Chem Commun (Camb) ; 54(37): 4677-4680, 2018 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-29675537

RESUMO

Surface engineering of individual living cells is a promising field for cell-based applications. However, engineering individual cells with controllable thickness by chemical methods has been rarely studied. This article describes the development of a new cytocompatible chemical strategy to decorate individual living cells. The thicknesses of the crosslinked shells could be conveniently controlled by the irradiation time, visible light intensity, or monomer concentration. Moreover, the lag phase of the yeast cell division was extended and their stability against lysis was improved, which could also be tuned by controlling the shell thickness.


Assuntos
Engenharia Celular/métodos , Polietilenoglicóis/química , Polietilenoimina/química , Divisão Celular/efeitos dos fármacos , Parede Celular/metabolismo , Difusão , Glucana Endo-1,3-beta-D-Glucosidase/química , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Luz , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Polietilenoglicóis/síntese química , Polietilenoglicóis/efeitos da radiação , Polietilenoimina/síntese química , Polietilenoimina/efeitos da radiação , Polimerização , Saccharomyces cerevisiae/química
19.
FEMS Yeast Res ; 18(1)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29272386

RESUMO

A cell wall (CW) provides a protective barrier for a yeast cell and is a firm structure that nevertheless dynamically changes during cell's growth. Bgl2p is a non-covalently anchored glucanosyltransglycosylase in the CW of the yeast Saccharomyces cerevisiae. The mode of its anchorage is poorly understood, while its association with CW components is tight and resistant to 1-h treatment with 1% SDS at 37°C. In order to demarcate the potential structural block responsible for incorporation of Bgl2p into the CW, bioinformatics analysis of its sequence was performed, and a conservative structural region was identified in the C-terminal region of Bgl2p, which was absent in its homologues in S. cerevisiae, the Scw4p and Scw10p. Deletion of this region disrupted the incorporation of Bgl2p into the CW and led to release of this protein through the CW into the culture medium. Two left-handed polyproline-II helices were identified in the C-terminal region of the structure model of a wild-type Bgl2p. These helices potentially formed binding sites, which were absent in the truncated protein. Using immune fluorescence microscopy, we demonstrated that C-truncated Bgl2p was exported into culture medium and lost its ability to form fibrils described earlier. It was also shown that the C-terminal truncation of Bgl2p led to a more severe decrease of cell survivability in extreme conditions than BGL2 deletion.


Assuntos
Parede Celular/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular/genética , Biologia Computacional/métodos , Genótipo , Glucana Endo-1,3-beta-D-Glucosidase/química , Glucana Endo-1,3-beta-D-Glucosidase/genética , Modelos Moleculares , Conformação Molecular , Mutação , Fenótipo , Domínios Proteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
20.
Enzyme Microb Technol ; 108: 34-41, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29108625

RESUMO

A novel ß-1,3-glucanase gene (PaBglu50A) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli. The deduced amino acid sequence of PaBglu50A showed the highest identity of 34% with the ß-agarase belonging to glycoside hydrolase (GH) family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45°C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40°C. The Km and Vmax of PaBglu50A for curdlan and laminarin were 94.4mgml-1 and 23.4µmolmin-1mg-1, 3.65mgml-1 and 8.89µmolmin-1mg-1, respectively. All characterized members of GH family 50 were only active towards agarose so far. However, the recombinant protein PaBglu50A did not display activity towards agarose but showed activity towards water-insoluble curdlan and laminarin. The hydrolysis products for curdlan supported this protein to be an endo-ß-1,3-glucanase, making a significant difference from the reported enzymes of GH family 50. These results suggested that PaBglu50A is the first endo-type ß-1,3-glucanase (EC 3.2.1.39) in GH family 50.


Assuntos
Proteínas de Bactérias/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Genes Bacterianos , Glucana Endo-1,3-beta-D-Glucosidase/classificação , Glucana Endo-1,3-beta-D-Glucosidase/genética , Hidrólise , Cinética , Filogenia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , beta-Glucanas/metabolismo
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