RESUMO
This study examined the laxative effects of hot-water extracts of Hovenia dulcis Thunb. (HD), Phyllostachys pubescens Mazel (PM), and a 2:8 mixture of both (HP) in two chronic constipation models. For the loperamide-induced constipation model, animals were divided into an untreated group, negative control group (loperamide 4 mg/kg), positive control group (bisacodyl 4 mg/kg) group, and six treatment groups (HP 100 or 400, HD 50 or 100, and PM 100 or 400 mg/kg). For the lowfiber diet-induced constipation model, animals were divided into an untreated group (normal diet), negative control group (low-fiber diet), positive control group (Agio granule, 620 mg/kg), and the same treatment groups. Fecal number, weight, fecal water content, and intestinal transit ratio were higher in the groups treated with HP, HD, and PM than in the groups treated with loperamide or lowfiber diet. Thickness of colon mucosa and muscle layers were increased in the treated groups. Colon tension increased in the HP groups, and [Ca2+]i measurements using fura-2 as an indicator showed that HP inhibits ATP-mediated Ca2+ influx in IEC-18 cells. These results showed that the HP mixture has laxative activity by increased mucin secretion and inducing contractile activity and relaxation. It may be a useful therapeutic strategy for ameliorating in chronic constipation.
Assuntos
Constipação Intestinal/metabolismo , Laxantes/farmacologia , Extratos Vegetais/farmacologia , Poaceae/química , Rhamnaceae/química , Animais , Colo/efeitos dos fármacos , Constipação Intestinal/induzido quimicamente , Dieta , Fibras na Dieta , Modelos Animais de Doenças , Loperamida/efeitos adversos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Bacillus is an antagonistic bacteria that shows high effectiveness against different phytopathogenic fungi and produces various lytic enzymes, such as chitosanase, chitinase, protease, and gluconase. The aim of this study is to determine Bacillus spp. for lytic enzyme production and to evaluate the antifungal effects of the selected strains for biocontrol of mycotoxigenic and phytopathogenic fungi. A total of 92 endospore-forming bacterial isolates from the 24 fig orchard soil samples were screened for chitosanase production, and six best chitosanolytic isolates were selected to determine chitinase, protease, and N-acetyl-ß-hexosaminidase activity and molecularly identified. The antagonistic activities of six Bacillus strains against Aspergillus niger EGE-K-213, Aspergillus foetidus EGE-K-211, Aspergillus ochraceus EGE-K-217, and Fusarium solani KCTC 6328 were evaluated. Fungal spore germination inhibition and biomass inhibition activities were also measured against A. niger EGE-K-213. The results demonstrated that Bacillus mojavensis EGE-B-5.2i and Bacillus thuringiensis EGE-B-14.1i were more efficient antifungal agents against A. niger EGE-K-213. B. mojavensis EGE-B-5.2i has shown maximum inhibition of the biomass (30.4%), and B. thuringiensis EGE-B-14.1i has shown maximum inhibition of spore germination (33.1%) at 12 h. This is the first study reporting the potential of antagonist Bacillus strains as biocontrol agents against mycotoxigenic fungi of fig orchads.
Assuntos
Aspergillus/efeitos dos fármacos , Bacillus/metabolismo , Toxinas Bacterianas/farmacologia , Ficus/microbiologia , Fusarium/efeitos dos fármacos , Micotoxinas/biossíntese , Controle Biológico de Vetores , Aspergillus/classificação , Aspergillus/metabolismo , Bacillus/classificação , Bacillus/crescimento & desenvolvimento , Quitosana/metabolismo , Enzimas/metabolismo , Fusarium/metabolismo , Microbiologia do Solo , Especificidade da Espécie , Esporos BacterianosRESUMO
Chitin and chitosan oligosaccharides (COS) have been traditionally obtained by chemical digestion with strong acids. In light of the difficulties associated with these traditional production processes, environmentally compatible and reproducible production alternatives are desirable. Unlike chemical digestion, biodegradation of chitin and chitosan by enzymes or microorganisms does not require the use of toxic chemicals or excessive amounts of wastewater. Enzyme preparations with chitinase, chitosanase, and lysozymeare primarily used to hydrolyze chitin and chitosan. Commercial preparations of cellulase, protease, lipase, and pepsin provide another opportunity for oligosaccharide production. In addition to their hydrolytic activities, the transglycosylation activity of chitinolytic enzymes might be exploited for the synthesis of desired chitin oligomers and their derivatives. Chitin deacetylase is also potentially useful for the preparation of oligosaccharides. Recently, direct production of oligosaccharides from chitin and crab shells by a combination of mechanochemical grinding and enzymatic hydrolysis has been reported. Together with these, other emerging technologies such as direct degradation of chitin from crustacean shells and microbial cell walls, enzymatic synthesis of COS from small building blocks, and protein engineering technology for chitin-related enzymes have been discussed as the most significant challenge for industrial application.
Assuntos
Quitina/química , Oligossacarídeos/química , Animais , Quitosana/química , Crustáceos/metabolismo , Humanos , Hidrólise , Engenharia de Proteínas/métodosRESUMO
Application of poly-gamma-glutamic acid (γ-PGA), an unusual macromolecular anionic polypeptide, is limited due to the high cost associated with its low productivity. Screening bacterial strains to find a more efficient producer is one approach to overcome this limitation. Strain MJ80 was isolated as a γ-PGA producer among 1,500 bacterial colonies obtained from soil samples. It was identified as Bacillus subtilis, based on the biochemical and morphological properties and 16S rDNA gene sequencing. It produced γ-PGA from both glutamic acid and soybean powder, identifying it as a facultative glutamic acid-metabolizing bacterium. After optimization of its culture conditions, B. subtilis MJ80 showed γ-PGA productivity of 75.5 and 68.7 g/l in 3 and 300 l jar fermenters for 3 days cultivation, respectively, the highest productivity reported to date, suggesting MJ80 to be a promising strain for γ-PGA production.
Assuntos
Bacillus subtilis/metabolismo , Ácido Poliglutâmico/análogos & derivados , Bacillus subtilis/genética , Meios de Cultura/química , Meios de Cultura/metabolismo , Ácido Glutâmico , Microbiologia Industrial , Ácido Poliglutâmico/análise , Ácido Poliglutâmico/metabolismo , Cloreto de Sódio , Amido/metabolismo , Ureia/metabolismoRESUMO
Bacillus cereus MP-310 was incubated on various culture media substrates as LB, colloidal chitin, chitosan powder, and chitosan beads to investigate the concurrent expression patterns of chitinase and chitosanase isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase activity increased rapidly with a maximum level after 6 days of incubation in CM-chitin medium. Major bands of chitinase isozymes were strongly expressed on SDS-PAGE in LB medium (four bands) and in colloidal chitin medium (five bands) after 6 days after incubation, and in chitosan powder medium (one band) and in chitosan beads medium (five bands) after 12 days after incubation. A major band of chitosanase isozymes was strongly expressed on SDS-PAGE in chitosan powder medium (one band) and in chitosan beads medium (one band) after 12 days of incubation.
Assuntos
Bacillus cereus/enzimologia , Quitinases/biossíntese , Expressão Gênica , Glicosídeo Hidrolases/biossíntese , Bacillus cereus/crescimento & desenvolvimento , Quitina/metabolismo , Quitosana/metabolismo , Meios de Cultura/química , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacosRESUMO
A novel chitosan-cinnamon bead carrier was prepared in this study. Chitosan was mixed with cinnamon powder (CP) and cinnamon extract (CE) to obtain chitosan-cinnamon powder (CCP) beads and chitosan-cinnamon extracted (CCE) beads, respectively. The potential antifungal and nematicidal activities of CCP and CCE were investigated against Rhizoctonia solani and Meloidogyne incognita in vitro. Relative antifungal activity of the CCP (5% CP) bead-treated R. solani was 30.9 and 23.9% after 1 and 2 day incubations, respectively. Relative antifungal activity of the CCE (0.5% CE) bead-treated R. solani was 4.3, 3.0 and 4.2% after 1, 2 and 3 days of incubation. Inhibition of hatch by CCP beads with CP of 5% was 78.8%. Inhibition of hatch by CCE beads with CE of 0.5% was 82.0%. J2 mortality following the CCP (5% CP) and CCE (0.5% CE) bead treatments was 85.0 and 95.8%, respectively against M. incognita after 48 h incubations.
Assuntos
Quitosana/química , Cinnamomum zeylanicum/química , Extratos Vegetais/farmacologia , Rhizoctonia/efeitos dos fármacos , Tylenchoidea/efeitos dos fármacos , Animais , Antifúngicos/farmacologia , Antinematódeos/farmacologia , Extratos Vegetais/química , Pós/química , Pós/farmacologiaRESUMO
An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl2, 2 g Tween 80/l, and 0.02 mM pyridoxal 5'-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.
Assuntos
Levilactobacillus brevis/metabolismo , Ácido gama-Aminobutírico/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Microbiologia de Alimentos , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Maltose/metabolismo , Nitrogênio/metabolismo , Ácido gama-Aminobutírico/análiseRESUMO
In this study, the 3,4-dihydroxybenzoic acid (3,4-DHBA) from Terminalia nigrovenulosa bark (TNB) was purified and its in vitro nematicidal activity was investigated against Meloidogyne incognita. The purification of 3,4-DHBA used a silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the 3,4-DHBA was conducted using (1)H nuclear magnetic resonance (NMR), (13)C NMR, and liquid chromatography time-of-flight mass spectrometry. Nematicidal activity bioassays revealed that 3,4-DHBA treatment resulted in 33.3, 47.5, 72.5 and 94.2% J2 mortality at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively after 12 h incubation. J2 mortality was increased significantly (P < 0.0001) with increasing incubation time in the range of 54.2-94.2% from 3 to 9 h after incubation with 3,4-DHBA (1.0 mg/ml), but with no significant difference observed where the incubation time was increased from 9 to 12 h. The 3,4-DHBA treatment resulted in 33.3, 65.0, 76.7 and 85.0% hatch inhibition at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively, 3 days after incubation. Changes in the shape of the eggs were determined after incubation for 1 day with a 3,4-DHBA concentration of 1.0 mg/ml.
Assuntos
Anti-Helmínticos/farmacologia , Hidroxibenzoatos/farmacologia , Terminalia/química , Tylenchoidea/efeitos dos fármacos , Animais , Anti-Helmínticos/isolamento & purificação , Bioensaio , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Hidroxibenzoatos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Casca de Planta/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise de Sobrevida , Tylenchoidea/fisiologia , Zigoto/efeitos dos fármacosRESUMO
In this study, a bacterium Serratia marcescens PRC-5 that displayed strong chitinolytic activity on 0.5% colloidal chitin-containing agar medium was isolated from soil. The chitinase activity increased rapidly with a maximum level (6.14 U/mL) on 4 days of incubation with swollen chitin (pH 5.0). Three active bands of chitinase isozymes were observed (53, 44, and 34 kDa) on SDS-PAGE gel and there pI values ranged from pI 5.4 to 5.8 on 2D gels. The chitinase from the PRC-5 strain was also able to produce GlcNAc monomers on TLC plates. The chitinase of PRC-5 inhibited the mycelial growth of Rhizoctonia solani KACC40111, which indicates that it could be used as a biocontrol agent for phytophathogens. The chitinase isozyme N1, which had a molecular weight of 62 kDa, was transferred from a native and SDS-PAGE gel onto an immunoblot and was probed using an anti-PrGV-chitinase.
Assuntos
Quitinases/análise , Quitinases/biossíntese , Immunoblotting/métodos , Imunoglobulina G/imunologia , Serratia marcescens/metabolismo , Sequência de Aminoácidos , Animais , Quitinases/química , Quitinases/imunologia , Dados de Sequência Molecular , Ratos , Rhizoctonia/crescimento & desenvolvimento , Serratia marcescens/fisiologia , Microbiologia do SoloRESUMO
The antifungal activities of methanolic extracts from Terminalia nigrovenulosa bark (TNB) was investigated for effects on the initial growth of mycelia against Fusarium solani. The ethyl acetate fraction separated from TNB demonstrated the highest antifungal activity against F. solani. The antifungal compound was isolated from TNB using silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the antifungal compound was conducted using (1)H NMR, (13)C NMR, and liquid chromatography-tandem mass spectrometry. The purified antifungal compound was gallic acid (GA) or 3,4,5-trihydroxy benzoic acid. Purified-GA possesses the high antifungal activity against F. solani, and that antifungal activity was dosage-dependent. The hyphae became collapsed and shrunken after 24 h incubation with GA (500 ppm). In pot experiments, the application of TNB crude extract was found to be effective in controlling the cucumber Fusarium root rot disease by enhancing activities of chitinase, peroxidase thereby promoting the growth of plants. The applied TNB extract significantly suppressed root rot disease compared to control. It resulted in 33, 75 and 81% disease suppression with 100, 500 and 1000 ppm of TNB crude extract, respectively. The study effectively demonstrated biological activities of the TNB extract, therefore suggesting the application of TNB for the control of soil-borne diseases of cucumber plants.
Assuntos
Antifúngicos/farmacologia , Fusarium/efeitos dos fármacos , Ácido Gálico/farmacologia , Terminalia/química , Antifúngicos/isolamento & purificação , Cromatografia , Cucumis sativus/microbiologia , Relação Dose-Resposta a Droga , Fusarium/crescimento & desenvolvimento , Ácido Gálico/isolamento & purificação , Espectroscopia de Ressonância Magnética , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Casca de Planta/química , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Espectrometria de Massas em TandemRESUMO
To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.
Assuntos
Quitinases/genética , Meios de Cultura/farmacologia , Paenibacillus/efeitos dos fármacos , Paenibacillus/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Íons/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Metais/farmacologia , Técnicas Microbiológicas/métodos , Paenibacillus/genética , Paenibacillus/crescimento & desenvolvimentoRESUMO
The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.
Assuntos
Glycine max/imunologia , Glycine max/microbiologia , Doenças das Plantas/microbiologia , Pseudomonas/fisiologia , Rhizoctonia/fisiologia , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/microbiologia , Glycine max/enzimologia , Glycine max/genéticaRESUMO
Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2-4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1-10 mM Co(2+) and strongly inhibited by 10 mM Cu(2+). It required at least two GlcNAc residues for catalysis. When (GlcNAc)(6) was used as substrate, K(m) and V(max) were determined to be 1.1 mM and 54.6 µmol min(-1) respectively.
Assuntos
Amidoidrolases/isolamento & purificação , Amidoidrolases/metabolismo , Mortierella/metabolismo , Amidoidrolases/biossíntese , Amidoidrolases/química , Cátions Bivalentes/farmacologia , Glicosilação , Concentração de Íons de Hidrogênio , Peso Molecular , TemperaturaRESUMO
Chitin deacetylases, occurring in marine bacteria, several fungi and a few insects, catalyze the deacetylation of chitin, a structural biopolymer found in countless forms of marine life, fungal cell and spore walls as well as insect cuticle and peritrophic matrices. The deacetylases recognize a sequence of four GlcNAc units in the substrate, one of which undergoes deacetylation: the resulting chitosan has a more regular deacetylation pattern than a chitosan treated with hot NaOH. Nevertheless plain chitin is a poor substrate, but glycolated, reprecipitated or depolymerized chitins are good ones. The marine Vibrio sp. colonize the chitin particles and decompose the chitin thanks to the concerted action of chitinases and deacetylases, otherwise they could not tolerate chitosan, a recognized antibacterial biopolymer. In fact, chitosan is used to prevent infections in fishes and crustaceans. Considering that chitin deacetylases play very important roles in the biological attack and defense systems, they may find applications for the biological control of fungal plant pathogens or insect pests in agriculture and for the biocontrol of opportunistic fungal human pathogens.
Assuntos
Amidoidrolases , Proteínas de Bactérias , Proteínas Fúngicas , Proteínas de Insetos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Amidoidrolases/genética , Amidoidrolases/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Quitina/química , Quitina/metabolismo , Quitina/farmacologia , Quitosana/química , Quitosana/metabolismo , Quitosana/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Fungos/enzimologia , Fungos/patogenicidade , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Insetos/enzimologia , Controle Biológico de Vetores/métodos , Vibrionaceae/enzimologiaRESUMO
The purified endochitosanase (Mw 41 kDa) from bacterium Bacillus cereus D-11 hydrolyzed chitooligomers (GlcN)5-7 into chitobiose, chitotriose, and chitotetraose as the final products. The minimal size of the oligosaccharides for enzymatic hydrolysis was a pentamer. To further investigate the cleavage pattern of this enzyme, chitooligosaccharide alcohols were prepared as substrates and the end products of hydrolysis were analyzed by TLC and HPLC. The chitosanase split (GlcN)4GlcNOH into (GlcN)3+ (GlcN)1GlcNOH, and (GlcN)5GlcNOH into (GlcN)4+ (GlcN)1GlcNOH and (GlcN)3+(GlcN)2GlcNOH. The heptamer (GlcN)6GlcNOH was split into (GlcN)5 [thereafter hydrolyzed again into (GlcN)3+(GlcN)2]+(GlcN)1GlcNOH, (GlcN)4+(GlcN)2GlcNOH, and (GlcN)3+(GlcN)3GlcNOH, whereas (GlcN)1-3GlcNOH was not hydrolyzed. The monomers GlcN and GlcNOH were never detected from the enzyme reaction. These results suggest that D-11 chitosanase recognizes three glucosamine residues in the minus position and simultaneously two residues in the plus position from the cleavage point.
Assuntos
Álcoois/metabolismo , Bacillus cereus/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Glucosamina/metabolismo , HidróliseRESUMO
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60 degrees C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag(+) and Hg(2+) while Chi46 by Hg(2+) and Pb(2+) at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co(2+). On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)( n ), n = 2-6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.
Assuntos
Quitinases/isolamento & purificação , Hypocreales/classificação , Hypocreales/enzimologia , Óvulo/microbiologia , Tylenchoidea/microbiologia , Sequência de Aminoácidos , Animais , Biotecnologia , Quitinases/química , Quitinases/metabolismo , Interações Hospedeiro-Parasita , Hypocreales/genética , Hypocreales/isolamento & purificação , Dados de Sequência Molecular , Tylenchoidea/crescimento & desenvolvimentoRESUMO
Among more than a hundred colonies of fungi isolated from soil samples, DY-52 has been screened as an extracellular chitin deacetylase (CDA) producer. The isolate was further identified as Mortierella sp., based on the morphological properties and the nucleotide sequence of its 18S rRNA gene. The fungus exhibited maximal growth in yeast peptone glucose (YPD) liquid medium containing 2% of glucose at pH 5.0 and 28 degrees C with 150 rpm. The CDA activity of DY-52 was maximal (20 U/mg) on the 3rd day of culture in the same medium. The CDA was inducible by addition of glucose and chitin. The enzyme contained two isoforms of molecular mass 50 kDa and 59 kDa. This enzyme showed a maximal activity at pH 5.5 and 60 degrees C. In addition, it had a pH stability range of 4.5-8.0 and a temperature stability range of 4-40 degrees C. The enzyme was enhanced in the presence of Co2+ and Ca2+. Among various substrates tested, WSCT-50 (water-soluble chitin, degree of deacetylation 50%), glycol chitin, and crab chitosan (DD 71-88%) were deacetylated. Moreover, the CDA can handle N-acetylglucosamine oligomers (GlcNAc)2-7.
Assuntos
Amidoidrolases/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/metabolismo , Mortierella/enzimologia , Mortierella/isolamento & purificação , Microbiologia do Solo , Amidoidrolases/química , Meios de Cultura/química , DNA Fúngico/genética , DNA Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Proteínas Fúngicas/química , Mortierella/química , Mortierella/genética , Transporte Proteico , RNA Ribossômico 18S/genética , Especificidade por Substrato , TemperaturaRESUMO
Two chitinolytic fungal strains, Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9, were isolated from soil samples of Korea and Vietnam, respectively. DY-59 and VS-9 crude chitinases secreted by these fungi in the 0.5% swollen chitin culture medium had an optimal pH of 4 and the optimal temperatures of 40 degrees C and 60 degrees C, respectively. Enzymatic hydrolysis products from crab swollen chitin were N-acetyl-beta-D-glucosamine (GlcNAc) by DY-59 chitinase, and GlcNAc and N, N'-diacetylchitobiose (GlcNAc)2 by VS-9 chitinases. The chitinases degraded the cell wall of Fusarium solani hyphae to produce oligosaccharides, among which GlcNAc, (GlcNAc)2, and pentamer (GlcNAc)5 were identified by high-pressure liquid chromatography. DY-59 and VS-9 chitinases inhibited F. solani microconidial germination by more than 70% and 60% at final protein concentrations of 5 and 27 microg mL(-1), respectively, at 30 degrees C for 20 h treatment.
Assuntos
Antifúngicos/farmacologia , Quitinases/farmacologia , Fusarium/efeitos dos fármacos , Rhizopus/enzimologia , Trichoderma/enzimologia , Acetilglucosamina/metabolismo , Antifúngicos/metabolismo , Parede Celular/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Dissacarídeos/metabolismo , Concentração de Íons de Hidrogênio , Coreia (Geográfico) , Oligossacarídeos/metabolismo , Rhizopus/isolamento & purificação , Microbiologia do Solo , Temperatura , Trichoderma/isolamento & purificação , VietnãRESUMO
Chitinases are enzymes that hydrolyze internal beta-1,4-N-acetyl-D-glucosamine linkages of chitin. Since the backbone of Nod factors is a chitin oligomer, we investigated whether chitinases produced by soil bacteria Paenibacillus illinoisensis KJA-424 and Bacillus thuringiensis subsp. Pakistani HD 395 are able to degrade Nod factor produced by Bradyrhizobium japonicum, a phenomenon that could disrupt B. japonicum-soybean signaling and nodule establishment when chitinases are present. Purified Nod factor [LCO Nod Bj-V (C(18:1), MeFuc)] was isolated from Bradyrhizobium japonicum and incubated with crude chitinases isolated from KJA-424 and HD395, with or without acetate buffer. After 15 h of incubation, Nod factor in the resulting solution was quantified by HPLC. Degradation was greatest following treatment with KJA-424 (91.9%) and HD395 (86.5%) chitinases in acetate buffer. Treatments that included acetate buffer had higher levels of degradation than those without. For all treatments degradation was greater than 77%.
Assuntos
Quitinases/metabolismo , Bactérias Gram-Positivas/enzimologia , Lipopolissacarídeos/metabolismo , Acetatos/metabolismo , Soluções Tampão , Quitinases/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Lipopolissacarídeos/isolamento & purificaçãoRESUMO
An extract of the leaves of Yucca smalliana Fern. (Agavaceae) showed potential antimicrobial activity. Employing a bioassay linked fractionation method, one of the active principles, namely yuccalan, was isolated as a new steroidal saponin. The structure of the new steroidal saponin was elucidated as 3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(3beta, 5alpha, 6alpha, 25S)-spirostan-3,6,27-triol (1) using various spectroscopic techniques, including IR, MS, 1D and 2D 1H-NMR, and 13C-NMR. The purified yuccalan showed antifungal activities against both Rhizoctonia solani and Fusarium oxysporum.