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1.
Pestic Biochem Physiol ; 159: 34-40, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400782

RESUMO

The present study investigated the insecticidal activity of the different organic extracts from the entomopathogenic fungi, Cladosporium cladosporioides, Metarhizium anisopliae, Purpureocillium lilacinum, and Trichoderma longibrachiatum towards cotton aphid, Aphis gossypii. The methanol extracts from the mycelia and spores of C. cladosporioides and P. lilacinum exhibited the highest insecticidal activity against A. gossypii compared with other extracts, which LC50 values were recorded to be 57.60 and 94.18 ppm, respectively. The major constituents identified in both methanol extracts by GC-MS analysis were linoleic acid and palmitic acid. The methanol extracts of C. cladosporioides and P. lilacinum caused a voluminous increase in the total carbohydrates content of A. gossypii adults, while the total protein content was significantly decreased by both extracts. The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly reduced by methanol extracts. The P. lilacinum extract caused a considerable reduction in the activity of glutathione-S-transferase (GST), α- and ß-esterase by 28.9, 27.9 and 23.4%, respectively. Both extracts induced a significant increase in phenoloxidase and chitinase activity of A. gossypii adults. These results suggest that C. cladosporioides and P. lilacinum methanol extracts could be used as a promising approach for the management of A. gossypii in many economically crops.


Assuntos
Afídeos/efeitos dos fármacos , Gossypium/parasitologia , Inseticidas/farmacologia , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Animais , Quitinases/genética , Quitinases/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Resistência a Inseticidas/genética , Dose Letal Mediana , Metanol/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo
2.
Pestic Biochem Physiol ; 158: 166-174, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378353

RESUMO

Pesticidal properties of Bacillus thuringiensis and its associated toxic proteins is an ever-growing science with potential implications in biological pest management. In the present study 80 Bacillus thuringiensis isolates native to Uttarakhand Himalayas were evaluated for chitinolytic activity and potent ones (11 isolates) were further subjected to multiphasic characterization for their antifungal, insecticidal and synergistic properties with selected chemical insecticides. Although all the 11 potent isolates were biologically active, only three isolates (VLBt27, VLBt109 and VLBt238) showed >90% inhibition in radial growth of 3 out of 4 tested plant pathogenic fungi (Rhizoctonia solani, Fusarium oxysporum, Alternaria pori and Pyricularia oryzae). The key antagonism was manifested in the form of disruptions in growing tips and uneven mycelial thickenings. In insect bioassays (against Helicoverpa armigera, Mythimna separata and Thysanoplusia orichalcea), no considerable direct mortality was observed. However, the larval weight reduction was prominent in four isolates (VLBt27, VLBt38, VLBt109 and VLBt135) which accounts to >75% in first instar larvae of H. armigera. Joint action of these four isolates with chemical insecticides showed an overall additive interaction against Brevicoryne brassicae and synergism against H. armigera. All the isolates were compatible with tested insecticides at their field recommended doses except for chlorpyriphos with around 130 kDa protein as chitinase. The study identified VLBt27 and VLBt109, two native isolates of B. thuringiensis with potential antagonistic activity and synergism as well. These isolates have possible implications as single strategy against two diverse pest problems (pathogenic fungi and phytophagous insect) of agriculture with a view of reduced pesticide application.


Assuntos
Bacillus thuringiensis/fisiologia , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Animais , Quitinases/metabolismo , Controle Biológico de Vetores
3.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
4.
World J Microbiol Biotechnol ; 35(8): 114, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332537

RESUMO

N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6 h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with ß-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.


Assuntos
Acetilglucosamina/biossíntese , Aeromonas caviae/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Hidrólise , Espectroscopia de Ressonância Magnética , Peso Molecular , Temperatura Ambiente , Viscosidade , Difração de Raios X
5.
Adv Exp Med Biol ; 1142: 131-151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31102245

RESUMO

Chitin, a structural polysaccharide of ß-1,4-linked N-acetyl-D-glucosamine residues, is the second most abundant natural biopolymer after cellulose. The metabolism of chitin affects the global carbon and nitrogen cycles, which are maintained by marine and soil-dwelling bacteria. The degradation products of chitin metabolism serve as important nutrient sources for the chitinolytic bacteria. Chitinolytic bacteria have elaborate enzymatic systems for the degradation of the recalcitrant chitin biopolymer. This chapter introduces chitin degradation and utilization systems of the chitinolytic bacteria. These bacteria secrete many chitin-degrading enzymes, including processive chitinases, endo-acting non-processive chitinases, lytic polysaccharide monooxygenases, and N-acetyl-hexosaminidases. Bacterial chitinases play a fundamental role in the degradation of chitin. Enzymatic properties, catalytic mechanisms, and three-dimensional structures of chitinases have been extensively studied by many scientists. These enzymes can be exploited to produce a range of chitin-derived products, e.g., biocontrol agents against many plant pathogenic fungi and insects. We introduce bacterial chitinases in terms of their reaction modes and structural features.


Assuntos
Bactérias/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Oxigenases de Função Mista
6.
Adv Exp Med Biol ; 1142: 153-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31102246

RESUMO

Chitin is one of the most important carbohydrates of the fungal cell wall, and is synthesized by chitin synthases. Chitin can be degraded by chitinases, which are important virulence factors in pathogenic fungi. Knowledge about the biosynthesis and degradation of chitin, and the enzymes responsible, has accumulated in recent years. In this review, we analyze the amino acid sequences of chitin synthases from several typical fungi. These enzymes can be divided into seven groups. While the different chitin synthases from a single fungus share a low degree of similarity, the same type of chitin synthase from different fungi shows high similarity. The number of chitinase genes in fungi display wide variation, from a single gene in Schizosaccharomyces pombe, to 36 genes in Trichoderma virens. Chitinases from different fungi can be divided into four groups. The functions of chitin synthases and chitinases in several typical fungi are summarized, and the crystal structures of chitinases and chitinase modification are also discussed.


Assuntos
Quitina Sintase/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Fungos/metabolismo , Sequência de Aminoácidos , Parede Celular , Conformação Proteica
7.
Int J Med Microbiol ; 309(3-4): 252-257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31079999

RESUMO

Plants are always found together with bacteria and other microbes. Although plants can be attacked by phytopathogenic bacteria, they are more often engaged in neutral or mutualistic bacterial interactions. In the soil, plants associate with rhizobia or other plant growth promoting rhizosphere bacteria; above ground, bacteria colonise plants as epi- and endophytes. For mounting appropriate responses, such as permitting colonisation by beneficial symbionts while at the same time fending off pathogenic invaders, plants need to distinguish between the "good" and the "bad". Plants make use of proteins containing the lysin motif (LysM) for perception of N-acetylglucosamine containing carbohydrate structures, such as chitooligosaccharides functioning as symbiotic nodulation factors or bacterial peptidoglycan. Moreover, plant hydrolytic enzymes of the chitinase family, which are able to cleave bacterial peptidoglycan or chitooligosaccharides, are essential for cellular signalling induced by rhizobial nodulation factors during symbiosis as well as bacterial peptidoglycan during pathogenesis. Hence, LysM receptors seem to work in concert with hydrolytic enzymes that fine-tune ligand availability to either allow symbiotic interactions or trigger plant immunity.


Assuntos
Quitinases/metabolismo , Interações entre Hospedeiro e Microrganismos , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Receptores de Superfície Celular/metabolismo , Bactérias/química , Bactérias/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Lisina , Peptidoglicano/metabolismo , Proteínas de Plantas/química , Receptores de Superfície Celular/química , Transdução de Sinais
8.
J Appl Microbiol ; 127(2): 556-564, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102427

RESUMO

AIMS: To assess phylogenetic and genotypic diversity of Metarhizium anisopliae lineage within diverse agroecosystems in the Karnataka State of India and to compare their chitinase activity and pathogenicity against insect pest of field crops subterranean termite, Odontotermes obesus. METHODS AND RESULTS: Three phylogenetic and 27 microsatellite markers were used to assess the genetic diversity of M. anisopliae lineage within multiple agroecosystems. Multilocus phylogeny of the Metarhizium isolates identified two species: Metarhizium pingshaense and Metarhizium guizhouense. Multilocus phylogeny and microsatellite markers resolved two phylogenetic species of M. pingshaense, Mp_1 and Mp_2, and one phylogenetic species of M. guizhouense, Mg_1. Phylogenetic species, Mp_2 and Mg_1, were detected with one genotype each and Mp_1 with eleven genotypes. Metarhizium pingshaense GKVK 02_16 isolate caused significantly high mortality of O. obesus in bioassays and detected with high chitinase activity. CONCLUSIONS: The study revealed phylogenetic and genotypic diversity of M. anisopliae lineage in agroecosystems of Karnataka State. Findings of pathogenicity and chitinase activity suggest that M. pingshaense GKVK 02_16 isolate provides effective control of O. obesus. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation provided an understanding of the genetic diversity and biocontrol efficiency of M. anisopliae lineage in agroecosystem. These data will serve as a resource in the future pest management strategies at a regional scale.


Assuntos
Variação Genética , Isópteros , Metarhizium , Controle Biológico de Vetores , Agricultura , Animais , Quitinases/metabolismo , Ecossistema , Genótipo , Índia , Metarhizium/classificação , Metarhizium/enzimologia , Metarhizium/genética , Metarhizium/isolamento & purificação , Repetições de Microssatélites , Filogenia
9.
Mol Plant Microbe Interact ; 32(9): 1121-1133, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31039081

RESUMO

ChiIV3, a chitinase of pepper (Capsicum annuum), stimulates cell death in pepper plants. However, there are only scarce reports on its role in resistance against bacterial wilt disease such as that caused by Ralstonia solanacearum and their transcriptional regulation. In this study, the silencing of ChiIV3 in pepper plants significantly reduced the resistance to R. solanacearum. The transcript of ChiIV3 was induced by R. solanacearum inoculation (RSI) as well as exogenous application of methyl jasmonate and abscisic acid. The bioinformatics analysis revealed that the ChiIV3 promoter consists of multiple stress-related cis elements, including six W-boxes and one MYB1AT. With the 5' deletion assay in the ChiIV3 promoter, the W4-box located from -640 to -635 bp was identified as the cis element that is required for the response to RSI. In addition, the W4-box element was shown to be essential for the binding of the ChiIV3 promoter by the WRKY40 transcription factor, which is known to positively regulate the defense response to R. solanacearum. Site-directed mutagenesis in the W4-box sequence impaired the binding of WRKY40 to the ChiIV3 promoter. Subsequently, the transcription of ChiIV3 decreased in WRKY40-silenced pepper plants. These results demonstrated that the expression of the defense gene ChiIV3 is controlled through multiple modes of regulation, and WRKY40 directly binds to the W4-box element of the ChiIV3 promoter region for its transcriptional regulation.


Assuntos
Capsicum , Quitinases , Resistência à Doença , Ralstonia solanacearum , Fatores de Transcrição , Capsicum/enzimologia , Capsicum/genética , Capsicum/microbiologia , Quitinases/genética , Quitinases/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Humanos , Mutagênese Sítio-Dirigida , Doenças das Plantas/microbiologia , Proteínas de Plantas , Ligação Proteica/genética , Ralstonia solanacearum/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Food Chem ; 289: 103-111, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955591

RESUMO

The effect of three combinations of bioprocessing methods by lactic acid fermentation, cell wall hydrolyzing enzymes and phytase on the biochemical (protein, fat, carbohydrate composition) and technofunctional properties (protein solubility, emulsifying and foaming properties) of wheat bran protein isolates were evaluated. The bioprocessing increased the protein (up to 80%) and fat content (up to 22.8%) in the isolates due to the degradation of starch and soluble pentosans. Additional proteins, globulin 3A and 3C, chitinase, ß-amylase and LMW glutenins, were identified from the electrophoretic pattern of the protein isolate bioprocessed with added enzymes. Generally, the bioprocessed protein isolate had lower protein solubility and stronger net charge in pH below 7, when compared to the protein isolate made without bioprocessing. The emulsifying properties of the protein isolates were not affected by bioprocessing. However, the foaming stability of the protein isolates was nearly doubled by bioprocessing with cell wall hydrolyzing enzymes and phytase.


Assuntos
Fibras na Dieta/metabolismo , Proteínas de Plantas/química , Quitinases/química , Quitinases/isolamento & purificação , Quitinases/metabolismo , Eletroforese em Gel de Poliacrilamida , Hidrólise , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Estabilidade Proteica , Solubilidade , Amido/metabolismo , beta-Amilase/química , beta-Amilase/isolamento & purificação , beta-Amilase/metabolismo
11.
Carbohydr Res ; 478: 1-9, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31005672

RESUMO

Chitooligosaccharides (COS), the depolymerization products of chitin, have many potential applications in agriculture and medicine since they induce immunostimulating effects and disease protective responses. Most of their biological activities require degrees of polymerization (DP) larger than the tetrasaccharide, but structurally well-defined COS with DP larger than six are difficult to produce due to their high insolubility and complex isolation from chitin hydrolysates. Enzymatic synthesis by exploiting the transglycosylation activity of chitinases offers a potential strategy for the assembly of oligomers in the range of bioactive DPs. We here explore the glycosynthase-like activity of six GH18 chitinases from bacterial and archaeal origin by mutating the catalytic assisting residue in the substrate-assisted mechanism of this enzyme family. The alanine mutants at the assisting residue have a significant, but not essential, effect on the hydrolase activity. We studied the ability of the alanine mutants at the assisting residue to catalyze the polymerization of an oxazoline derivative as donor substrate, selecting the oxazoline of pentaacetylchitopentaose (DP5ox) with the aim of obtaining larger oligomers/polymers that, being insoluble, might be resistant to further reactions by the hydrolytically compromised mutant enzymes. For all the enzymes, insoluble polymeric material was obtained, with DP10 as major component, but other COS with different DPs were also obtained, limiting the practical application to produce oligomers/polymers with a defined DP. The balance between the residual hydrolase activity of the mutant enzymes and the solubility/precipitation kinetics still lead to hydrolysis and/or transglycosylation reactions on the newly formed products. From the selected enzymes, the Thermococcus kodakaraensis ChiA D1022A mutant gave the best results, with the formation of insoluble polymers in 45% yield (w/w) and containing about 55% of the target DP10 product.


Assuntos
Quitina/análogos & derivados , Quitinases/genética , Quitinases/metabolismo , Biocatálise , Configuração de Carboidratos , Quitina/biossíntese , Quitina/química , Mutação , Polimerização
12.
Microb Pathog ; 131: 128-134, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30959097

RESUMO

Quorum sensing (QS) is the cell density dependent communication network which coordinates the production of pathogenic determinants in majority of pathogenic bacteria. Pseudomonas aeruginosa causes hospital-acquired infections by virtue of its well-defined QS network. As the QS regulatory network in P. aeruginosa regulates the virulence determinants and antibiotic resistance, attenuating the QS system seems to be influential in developing next-generation anti-infective agents. In the current study, the QS attenuation potential of a flavonoid, mosloflavone was investigated against P. aeruginosa virulence and biofilm formation. Mosloflavone inhibited the pyocyanin production, LasB elastase and chitinase by 59.52 ±â€¯2.74, 35.90 ±â€¯4.34 and 61.18 ±â€¯5.52% respectively. The QS regulated biofilm formation and development was also reduced when supplemented with sub-MIC of mosloflavone. The gene expression studies of mosloflavone using RT-PCR depicted its ability to down-regulate the expression levels of QS regulated virulence genes such as lasI (60.64%), lasR (91.70%), rhlI (57.30%), chiC (90.20%), rhlA (47.87%), rhlR (21.55%), lasB (37.80%), phzM (42.40%), toxA (61.00%), aprA (58.4%), exoS (78.01%), algD (46.60%) and pelA (50.45%). The down-regulation of QS virulence phenotypes by mosloflavone could be attributed to its binding affinity with the QS regulatory proteins, LasR and RhlR by competitively inhibiting the binding of natural autoinducers as evidenced from simulation studies. Mosloflavone also exhibited promising potential in controlling bacterial infection in Caenorhabditis elegans model system, in vivo. The anti-biofilm and anti-QS potential of mosloflavone in the current study illustrated the candidature of mosloflavone as a promising biocide.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Quitinases/metabolismo , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Piocianina/metabolismo , Transativadores/genética , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética
13.
Enzyme Microb Technol ; 126: 50-61, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000164

RESUMO

The biocontrol activity of some soil strains of Chromobacterium sp. against pathogenic fungi has been attributed to secreted chitinases. The aim of this work was to characterize biochemically a recombinant chitinase (CvChi47) from C. violaceum ATCC 12472 and to investigate its effects on phytopathogenic fungi. CvChi47 is a modular enzyme with 450 amino acid residues, containing a type I signal peptide at the N-terminal region, followed by one catalytic domain belonging to family 18 of the glycoside hydrolases, and two type-3 chitin-binding domains at the C-terminal end. The recombinant enzyme was expressed in Escherichia coli as a His-tagged protein and purified to homogeneity. The native signal peptide of CvChi47 was used to direct its secretion into the culture medium, from where the recombinant product was purified by affinity chromatography on chitin and immobilized metal. The purified protein showed an apparent molecular mass of 46 kDa, as estimated by denaturing polyacrylamide gel electrophoresis, indicating the removal of the signal peptide. CvChi47 was a thermostable protein, retaining approximately 53.7% of its activity when heated at 100 °C for 1 h. The optimum hydrolytic activity was observed at 60 °C and pH 5. The recombinant chitinase inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth. Furthermore, atomic force microscopy experiments revealed a pronounced morphological alteration of the cell surface of conidia incubated with CvChi47 in comparison to untreated cells. Taken together, these results show the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fusarium species.


Assuntos
Antifúngicos/farmacologia , Quitinases/metabolismo , Chromobacterium/enzimologia , Fusarium/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Quitinases/química , Quitinases/genética , Clonagem Molecular , Estabilidade Enzimática , Fusarium/efeitos dos fármacos , Doenças das Plantas/microbiologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Temperatura Ambiente
14.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871033

RESUMO

An unstudied ß-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The ß-N-acetylhexosaminidase was able to hydrolyze ß1,2-linked, ß1,3-linked, ß1,4-linked, and ß1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the ß-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.


Assuntos
Actinomycetales/metabolismo , Dissacarídeos/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Aminoácidos/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Escherichia coli/metabolismo , Oximas/metabolismo , Fenilcarbamatos/metabolismo , Microbiologia do Solo
15.
Appl Microbiol Biotechnol ; 103(8): 3289-3295, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30850873

RESUMO

Chitinases are hydrolases that catalyze the cleavage of the ß-1,4-O-glycosidic linkages in chitin, a polysaccharide abundantly found in nature. Although numerous chitinolytic enzymes have been studied in detail, relatively little is known about chitinases capable of broad specificity. Broad-specificity chitinases are a sort of novel chitinases possessing two or three different catalytic activities among exochitinase, endochitinase, and N-acetylglucosaminidase. In the light of the difference of module composition and catalytic mechanism, the broad-specificity chitinases included two broad categories, broad-specificity chitinases with a single catalytic domain or multi-catalytic domains. This broad-specificity chitinases have great potential in chitin conversion. In this review, we summarize all reported cases of broad-specificity chitinases and provide an overview of the recent findings on their origin, characterization, catalytic mechanism, and potential application. Moreover, in-depth study into these chitinases could contribute to our understanding of other broad-specificity enzymes which may have some benefits on progress of biotechnology.


Assuntos
Quitina/metabolismo , Quitinases/metabolismo , Acetilglucosaminidase/metabolismo , Biotecnologia , Catálise , Domínio Catalítico , Quitinases/química , Hexosaminidases/química , Hexosaminidases/metabolismo , Especificidade por Substrato
16.
J Agric Food Chem ; 67(13): 3575-3582, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865442

RESUMO

Insect chitinases play an indispensable role in shedding old cuticle during molting. Targeting chitinase inhibition is a promising pest control strategy. Of ChtI, a chitinase from the destructive insect pest Ostrinia furnacalis (Asian corn borer), has been suggested as a potential target for designing green pesticides. A 4,5,6,7-tetrahydrobenzo[ b]thiophene-3-carboxylate scaffold was previously obtained, and further derivatization generated the lead compound 1 as Of ChtI inhibitor. Here, based on the predicted binding mode of compound 1, the pocket-based lead optimization strategy was applied. A series of analogues was synthesized, and their inhibitory activities against Of ChtI were evaluated. Compound 8 with 6- tert-pentyl showed preferential inhibitory activity with a Ki value of 0.71 µM. Their structure-activity relationships suggested that the compound with larger steric hindrance at the 6-nonpolar group was essential for inhibitory activity due to its stronger interactions with surrounding amino acids. This work provides a strategy for designing potential chitinase inhibitors.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas de Insetos/antagonistas & inibidores , Inseticidas/química , Mariposas/enzimologia , Animais , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Cinética , Mariposas/química , Mariposas/efeitos dos fármacos , Domínios Proteicos
17.
Braz J Microbiol ; 50(2): 481-494, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30877665

RESUMO

Fusarium crown and root rot (FCRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL), is a soilborne tomato disease of increased importance worldwide. In this study, Withania somnifera was used as a potential source of biological control and growth-promoting agents. Seven fungal isolates naturally associated with W. somnifera were able to colonize tomato seedlings. They were applied as conidial suspensions or a cell-free culture filtrate. All isolates enhanced treated tomato growth parameters by 21.5-90.3% over FORL-free control and by 27.6-93.5% over pathogen-inoculated control. All tested isolates significantly decreased by 28.5-86.4% disease severity over FORL-inoculated control. The highest disease suppression, by 86.4-92.8% over control and by 81.3-88.8% over hymexazol-treated control, was achieved by the I6 isolate. FORL radial growth was suppressed by 58.5-82.3% versus control when dual cultured with tested isolates and by 61.8-83.2% using their cell-free culture filtrates. The most active agent was identified as Fusarium sp. I6 (MG835371), which displayed chitinolytic, proteolytic, and amylase activities. This has been the first report on the potential use of fungi naturally associated with W. somnifera for FCRR suppression and for tomato growth promotion. Further investigations are required in regard to mechanisms of action involved in disease suppression and plant growth promotion.


Assuntos
Antifúngicos/metabolismo , Agentes de Controle Biológico/metabolismo , Endófitos/metabolismo , Fusarium/crescimento & desenvolvimento , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/microbiologia , Doenças das Plantas/prevenção & controle , Withania/microbiologia , Amilases/metabolismo , Quitinases/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Caules de Planta/microbiologia , Proteólise
18.
J Microbiol ; 57(5): 372-380, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30806979

RESUMO

Chitin is the most abundant biopolymer in marine environments. To facilitate its utilization, our laboratory screened marine-derived fungal strains for chitinolytic activity. One chitinolytic strain isolated from seawater, designated YS2-2, was identified as Acremonium species based on morphological and phylogenetic analyses. Acremonium species are cosmopolitan fungi commonly isolated from both terrestrial and marine environments, but their chitinolytic activity is largely unknown. The extracellular crude enzyme of YS2-2 exhibited optimum chitinolytic activity at pH 6.0-7.6, 23-45°C, and 1.5% (w/v) NaCl. Degenerate PCR revealed the partial cDNA sequence of a putative chitinase gene, chiA, in YS2-2. The expression of chiA was dramatically induced in response to 1% (w/v) colloidal chitin compared to levels under starvation, chitin powder, and glucose conditions. Moreover, the chiA transcript levels were positively correlated with chitinolytic activities under various colloidal chitin concentrations, suggesting that ChiA mediates chitinolytic activity in this strain. Our results provide a basis for additional studies of marinederived chitinolytic fungi aimed at improving industrial applications.


Assuntos
Acremonium/genética , Acremonium/metabolismo , Quitina/metabolismo , Quitinases/genética , Quitinases/metabolismo , Acremonium/classificação , Acremonium/isolamento & purificação , Filogenia , Água do Mar/microbiologia
19.
Mol Biol Rep ; 46(2): 2243-2257, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30759297

RESUMO

MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803 bp with an open reading frame of 1152 bp (361 aa), signal sequence of 63 bp (21 aa), 5' and 3' UTR of 144 bp and 507 bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7 h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.


Assuntos
Búfalos/metabolismo , Proteína 1 Semelhante à Quitinase-3/metabolismo , Proteína 1 Semelhante à Quitinase-3/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Búfalos/genética , Búfalos/fisiologia , Células COS , Cercopithecus aethiops , Proteína 1 Semelhante à Quitinase-3/genética , Quitinases/genética , Quitinases/metabolismo , Clonagem Molecular/métodos , DNA Complementar/genética , Escherichia coli/genética , Feminino , Glicoproteínas/genética , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Fases de Leitura Aberta , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética
20.
Enzyme Microb Technol ; 124: 54-62, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30797479

RESUMO

Enzymatic production of chitooligosaccharides has high value in medicine and other fields. However, low chitinase activity and yield of chitooligosaccharides limit the production and application. Herein, we used a series of molecular biology strategies to increase the expression of chitinase in Bacillus subtilis WB600. Upon addition of the signal peptide NprB, Chisb was successfully secreted to the outside of the cell and extracellular expression level reached 35.54 U/mL. Furthermore, optimizing Ribosome Binding Sites (RBSs) with spacer sequences, and combining molecular docking technology with site-directed mutagenesis, the expression level and the specific activity of Chisb was further increased to 51.67 U/mL and 249.62 U/mg, respectively. When colloidal chitin was used as the substrate, the chitooligosaccharides detected by ion chromatography were (GlcNAc)1-5, and the total yield of chitooligosaccharides was 14.4%. Our results indicate that strategies for increasing Chisb expression contribute to the study and application of chitinase and the production of chitooligosaccharides.


Assuntos
Bacillus subtilis/enzimologia , Quitina/análogos & derivados , Quitinases/genética , Quitinases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sítios de Ligação , Quitina/metabolismo , Quitinases/isolamento & purificação , Clonagem Molecular , DNA Espaçador Ribossômico/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura Ambiente
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