RESUMEN
The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0-9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL-1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL-1 and Vmax of 5.75 mmol min-1. Kcat and catalytic efficiency were measured at 1.91 s-1 and 191 mL mg-1 s-1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent.
Asunto(s)
Antifúngicos , Bacillus , Quitina , Quitinasas , Quitinasas/metabolismo , Quitinasas/aislamiento & purificación , Quitinasas/química , Quitinasas/farmacología , Quitina/química , Quitina/metabolismo , Quitina/farmacología , Antifúngicos/farmacología , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Bacillus/enzimología , Fusarium/enzimología , Fusarium/efectos de los fármacos , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
To promote the bioconversion of marine chitin waste into value-added products, we expressed a novel pH-stable Micromonospora aurantiaca-derived chitinase, MaChi1, in Escherichia coli and subsequently purified, characterized, and evaluated it for its chitin-converting capacity. Our results indicated that MaChi1 is of the glycoside hydrolase (GH) family 18 with a molecular weight of approximately 57 kDa, consisting of a GH18 catalytic domain and a cellulose-binding domain. We recorded its optimal activity at pH 5.0 and 55 °C. It exhibited excellent stability in a wide pH range of 3.0-10.0. Mg2+ (5 mM), and dithiothreitol (10 mM) significantly promoted MaChi1 activity. MaChi1 exhibited broad substrate specificity and hydrolyzed chitin, chitosan, cellulose, soluble starch, and N-acetyl chitooligosaccharides with polymerization degrees ranging from three to six. Moreover, MaChi1 exhibited an endo-type cleavage pattern, and it could efficiently convert colloidal chitin into N-acetyl-D-glucosamine (GlcNAc) and (GlcNAc)2 with yields of 227.2 and 505.9 mg/g chitin, respectively. Its high chitin-degrading capacity and exceptional pH tolerance makes it a promising tool with potential applications in chitin waste treatment and bioactive oligosaccharide production.
Asunto(s)
Quitina , Quitinasas , Micromonospora , Quitinasas/metabolismo , Quitinasas/química , Quitinasas/aislamiento & purificación , Quitinasas/genética , Quitina/análogos & derivados , Quitina/metabolismo , Quitina/química , Concentración de Iones de Hidrógeno , Especificidad por Sustrato , Micromonospora/enzimología , Micromonospora/genética , Hidrólisis , Escherichia coli/genética , Quitosano/química , Estabilidad de EnzimasRESUMEN
The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm-3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.
Asunto(s)
Quitinasas , Nicotiana , Fosfatos , Polietilenglicoles , Proteínas Recombinantes , Quitinasas/química , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Nicotiana/enzimología , Fosfatos/química , Proteínas Recombinantes/aislamiento & purificación , Polietilenglicoles/química , Trichoderma/enzimología , Sales (Química)/química , Sales (Química)/farmacología , Agua/químicaRESUMEN
Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul-1 h-1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol-1, 100.59 kJ mol-1, 102.64 kJ mol-1 and 5.95 J mol-1 K-1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2-4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2-4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.
Asunto(s)
Quitina , Quitinasas , Estabilidad de Enzimas , Oligosacáridos , Temperatura , Quitinasas/química , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Concentración de Iones de Hidrógeno , Quitina/química , Oligosacáridos/química , Quitosano/química , Especificidad por Sustrato , CinéticaRESUMEN
Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401T, with an ability to degrade crystalline chitin into (GlcNAc)2 (N,N'-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45-50 °C and pH optima of 7.0-7.5. They are all stable at 40 °C and in the pH range of 5.0-11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)2, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)2.
Asunto(s)
Proteínas Bacterianas/química , Quitina/química , Quitinasas/química , Disacáridos/química , Pseudoalteromonas/enzimología , Proteínas Bacterianas/aislamiento & purificación , Quitinasas/aislamiento & purificación , Genoma Bacteriano , Pseudoalteromonas/genética , Cloruro de Sodio/químicaRESUMEN
The investigation for novel unique extremozymes is a valuable business for which the marine environment has been overlooked. The marine fungus Clonostachys rosea IG119 was tested for growth and chitinolytic enzyme production at different combinations of salinity and pH using response surface methodology. RSM modelling predicted best growth in-between pH 3.0 and 9.0 and at salinity of 0-40‱, and maximum enzyme activity (411.137 IU/L) at pH 6.4 and salinity 0‱; however, quite high production (>390 IU/L) was still predicted at pH 4.5-8.5. The highest growth and activity were obtained, respectively, at pH 4.0 and 8.0, in absence of salt. The crude enzyme was tested at different salinities (0-120‱) and pHs (2.0-13.0). The best activity was achieved at pH 4.0, but it was still high (in-between 3.0 and 12.0) at pH 2.0 and 13.0. Salinity did not affect the activity in all tested conditions. Overall, C. rosea IG119 was able to grow and produce chitinolytic enzymes under polyextremophilic conditions, and its crude enzyme solution showed more evident polyextremophilic features. The promising chitinolytic activity of IG119 and the peculiar characteristics of its chitinolytic enzymes could be suitable for several biotechnological applications (i.e., degradation of salty chitin-rich materials and biocontrol of spoiling organisms, possibly solving some relevant environmental issues).
Asunto(s)
Quitinasas/metabolismo , Hypocreales/enzimología , Hypocreales/metabolismo , Biotecnología , Quitina/química , Quitinasas/aislamiento & purificación , Extremófilos/aislamiento & purificación , Extremófilos/metabolismo , Fermentación , SalinidadRESUMEN
Mungbean root rot caused by Rhizoctonia bataticola (Taub.) Butler is the most devastating disease inflicting yield loss up to 60%. The use of beneficial antagonist, viz., Streptomyces with diverse antifungal activity and prolific secondary metabolites production, is the ecofriendly and environmentally acceptable alternative to the existing chemical control methods. In this investigation we have identified the promising isolate of Streptomyces sp. which potentially reduced the mungbean root rot. A total of nine mungbean rhizospheric actinobacterial isolates were evaluated for their antagonistic potential against root rot pathogen and growth promoting trait of mungbean. The actinobacterial isolate GgS 48 was shown to be effective in reducing the mycelial growth of R. bataticola by 65.3% in dual culture technique and enhancing the growth of mugbean under in vitro condition. Morphological, biochemical and molecular characterization confirmed the isolate GgS 48 as Streptomyces rameus. The actinobacteria S. rameus GgS 48 exerted antifungal action against R. bataticola by hyphal coiling, which was confirmed under scanning electron microscopy (SEM), and promoted the growth through the production of IAA. It showed positive for the production of siderophore and hydrolytic enzymes, viz., chitinase and protease. The chitinase produced by the GgS 48 was purified and its molecular weight was determined as 40 kDa and it had great potential in reducing the mycelial growth of R. bataticola. The talc-based formulation of S. rameus GgS 48 was found to be promising in suppressing the root rot severity and enhancing the growth and yield attributes of mungbean both under glass house and field conditions.
Asunto(s)
Antibiosis/fisiología , Ascomicetos/patogenicidad , Streptomyces , Vigna/microbiología , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Microscopía Electrónica de Rastreo , Péptido Hidrolasas/metabolismo , Filogenia , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Rizosfera , Sideróforos/metabolismo , Streptomyces/genética , Streptomyces/aislamiento & purificación , Vigna/crecimiento & desarrolloRESUMEN
A novel chitinase (P1724) was discovered from a Qinghai-Tibetan plateau microbial metagenome. P1724 contains two GH18 family catalytic domains and is phylogenetically distant from any of the chitinases studied. P1724 and its truncated versions, P1724(∆cGH18) and P1724(∆nGH18), were produced in Escherichia coli and characterized. Using colloidal chitin as substrate, the three recombinant proteins showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive, as they remained active at 4 °C; their chitinase activities were decreased with the presence of Cu2+ and EDTA, but increased with Ba2+ and Ca2+; they all showed both chitobiosidase and endochitinase activities. Compared to P1724(∆nGH18), P1724 and P1724(∆cGH18) shared more similarities in temperature and pH stabilities, NaCl tolerance, and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. kcat/Km value of P1724 was significantly higher than the sum values of P1724(∆cGH18) and P1724(∆nGH18), which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin. This study has not only broadened the understanding of unknown chitinases in nature but also discussed the strategy of adding additional catalytic domains in enzyme engineering.
Asunto(s)
Quitina/genética , Quitinasas/genética , Metagenoma/genética , Dominio Catalítico/genética , Quitina/química , Quitinasas/química , Quitinasas/aislamiento & purificación , Hidrólisis , Filogenia , Microbiología del Suelo , Tibet , HumedalesRESUMEN
The chitinase-producing bacteria Paenibacillus sp. was isolated from soil samples. The chitinase was purified successively by ammonia sulfate fractional precipitation followed by chromatography on DEAE 52-cellulose column and then on Sephadex G-75 column. The chitinase has a molecular weight of ca. 30 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Its optimum pH is 4.5, and its optimum temperature is 50 °C with colloidal chitin as a substrate. The enzyme is stable below 45 °C and in pH ranges between 4.5 and 5.5. It is activated by glucosamine, glucose, N-acetylglucosamine, and metal ions including Ca2+ , Fe2+ , Fe3+ , and Ni2+ . It is inhibited by SDS, H2 O2 , ascorbic acid, Cu2+ , Mg2+ , Ba2+ , Sn2+ , Cr3+ , and K+ . With colloidal chitin as substrate, the Km and the Vmax of the chitinase are 4.28 mg/mL and 14.29 µg/(Min·mL), respectively, whereas the end products of the enzymatic hydrolysis are 14.33% monomer and 85.67% dimer of N-acetylglucosamine. The viscosity of carboxymethyl chitin decreased rapidly at the initial stages when subjected to chitinase hydrolysis, which indicates that the chitinase acts in an endosplitting pattern.
Asunto(s)
Proteínas Bacterianas , Quitinasas , Paenibacillus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Quitinasas/química , Quitinasas/aislamiento & purificación , Estabilidad de EnzimasRESUMEN
The insect group II chitinase (ChtII, also known as Cht10) is a unique chitinase with multiple catalytic and chitin-binding domains. It has been proven genetically to be an essential chitinase for molting. However, ChtII's role in chitin degradation during insect development remains poorly understood. Obtaining this knowledge is the key to fully understanding the chitin degradation system in insects. Here, we investigated the role of OfChtII during the molting of Ostrinia furnacalis, a model lepidopteran pest insect. OfChtII was expressed earlier than OfChtI (OfCht5) and OfChi-h, at both the gene and protein levels during larva-pupa molting as evidenced by quantitative polymerase chain reaction and western blot analyses. A truncated OfChtII, OfChtII-B4C1, was recombinantly expressed in Pichia pastoris cells and purified to homogeneity. The recombinant OfChtII-B4C1 loosened compacted chitin particles and produced holes in the cuticle surface as evidenced by scanning electron microscopy. It synergized with OfChtI and OfChi-h when hydrolyzing insoluble α-chitin. These findings suggested an important role for ChtII during insect molting and also provided a strategy for the coordinated degradation of cuticular chitin during insect molting by ChtII, ChtI and Chi-h.
Asunto(s)
Quitinasas , Muda , Mariposas Nocturnas , Animales , Sitios de Unión , Quitina/metabolismo , Quitinasas/química , Quitinasas/genética , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Genes de Insecto , Proteínas de Insectos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Mariposas Nocturnas/genética , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/metabolismo , Conformación Proteica , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomycetales/genética , Especificidad por SustratoRESUMEN
Chitinases play crucial roles in enzymatic conversion of chitin and biocontrol of phytopathogenic fungi. Herein, a chitinase of glycoside hydrolase (GH) family 19, SaChiB, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21(DE3). The purified SaChiB displayed maximal activities at 45 °C and pH 8.0, and showed good stability up to 55 °C and in the range of pH 4.0-11.0, respectively. It exhibited substrate specificity towards chitin and chitooligosaccharides (degree of polymerization 3-6) with the endo-cleavage manner. In combination with the N-acetyl hexosaminidase SaHEX, it yielded a conversion rate of 95.2% from chitin powder to N-acetyl-D-glucosamine in 8 h and a product purity of >98.5%. Furthermore, the enzyme strongly inhibited the growth of tested pathogenic fungi. These results indicated that SaChiB has the great potential for applications in the conversion of raw chitinous waste in industries as well as the biocontrol of fungal diseases in agriculture.
Asunto(s)
Fenómenos Químicos , Quitina/química , Quitinasas/química , Streptomyces/enzimología , Secuencia de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacología , Biodegradación Ambiental , Catálisis , Quitinasas/genética , Quitinasas/aislamiento & purificación , Clonación Molecular , Activación Enzimática , Expresión Génica , Hidrólisis , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes , Análisis de Secuencia de ADN , Streptomyces/genética , Especificidad por SustratoRESUMEN
This paper reports the isolation and identification of an acido-thermostable chitinase (ChiA-Ba43) which was purified, from the culture liquid of Bacillus altitudinis strain KA15, and characterized. Purification of ChiA-Ba43 produced a 69.6-fold increase in the specific activity (120,000 U/mg) of the chitinase, with a yield of 51% using colloidal chitin as substrate. ChiA-Ba43 was found to be a monomeric protein with a molecular mass of 43,190.05 Da as determined by MALDI-TOF/MS. N-terminal sequence of the first 27 amino-acids (aa) of ChiA-Ba43 displayed homology to chitinases from other Bacillus species. Interestingly, ChiA-Ba43 exhibited optimum pH and temperature of 4-5.5 and 85 °C, respectively. Thin-layer chromatography (TLC) showed that the final hydrolyzed products of the enzyme from chitin-oligosaccharides and colloidal chitin are a mixture of (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, which indicates that ChiA-Ba43 possesses an endo-acting function. More interestingly, compared to ChiA-Mt45, ChiA-Hh59, Chitodextrinase®, N-acetyl-ß-glucosaminidase®, and ChiA-65, ChiA-Ba43 demonstrated a high level of catalytic efficiency and outstanding tolerance towards various organic solvents. The chiA-Ba43 gene (1332 bp) encoding ChiA-Ba43 (409 aa) was cloned, sequenced, and expressed in Escherichia coli strain HB101. The biochemical properties of the recombinant chitinase (rChiA-Ba43) were equivalent to those of the natively expressed enzyme. These properties make ChiA-Ba43 an ideal candidate for industrial bioconversion of chitinous waste.
Asunto(s)
Bacillus/enzimología , Quitinasas/metabolismo , Residuos Industriales , Temperatura , Residuos , Secuencia de Aminoácidos , Bacillus/genética , Bacillus/crecimiento & desarrollo , Biocatálisis , Quitinasas/química , Quitinasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Alineación de SecuenciaRESUMEN
Chitinase from the leaves of Simarouba glauca, a plant used in traditional anti-inflammatory therapy is purified and characterized. Peptide mass finger print analysis revealed the protein as an endo-chitinase which was further confirmed using chitin-agar assay. The enzyme exhibited significant anti-fungal efficacy against phyto-pathogens such as Macrophomina phaseolina, Fusarium oxysporum and Sclerotium rolfsii. Chitinolysis was also examined against insoluble chitin using SEM. Using X-ray diffraction data up to 1.66 Å, the structure was determined by Molecular Replacement using crystal structure of GH19 Chitinase-like protein from Hevea brasiliensis. During structure refinement, an extra domain could be traced and identified as hevein domain. To our knowledge, this is the first report of any chitinase with intact hevein domain. The GH19 chitinase and hevein domains though connected by a lengthy loop, are restricted to be close by disulfide bridges. These bridges connecting each domain with the loop may be important for proper chitin feeding into the active site. By considering reports on hevein and chitinase domains as well as the traditional use of the plant, this report of an intact hevein-chitinase protein and their relative orientation may add further insights for the usefulness of this protein.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Péptidos Catiónicos Antimicrobianos/química , Quitinasas/química , Quitinasas/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Lectinas de Plantas/química , Simarouba/enzimología , Secuencia de Aminoácidos , Antiinflamatorios/aislamiento & purificación , Antifúngicos/química , Antifúngicos/farmacología , Dominio Catalítico , Quitinasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Hidrólisis , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Extractos Vegetales/aislamiento & purificación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Análisis EspectralRESUMEN
Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket-Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition-arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.
Asunto(s)
Achromobacter denitrificans/enzimología , Achromobacter denitrificans/aislamiento & purificación , Quitina/metabolismo , Quitinasas/biosíntesis , Crustáceos/microbiología , Eliminación de Residuos , Aminoácidos/análisis , Animales , Quitina/química , Quitinasas/aislamiento & purificación , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Filogenia , TemperaturaRESUMEN
Biomineralization, especially shell formation, is a sophisticated process regulated by various matrix proteins. Pinctada fucata chitinase-like protein 1 (Pf-Clp1), which belongs to the GH18 family, was discovered by our group using in-depth proteomic analysis. However, its function is still unclear. In this study, we first obtained the full-length cDNA sequence of Pf-Clp1 by RACE. Real-time polymerase chain reaction results revealed that Pf-Clp1 was highly expressed in the important biomineralization tissues, the mantle edge and the mantle pallial. We expressed and purified recombinant protein rPf-Clp1 in vitro to investigate the function of Pf-Clp1 on CaCO3 crystallization. Scanning electron microscopy imaging and Raman spectroscopy revealed that rPf-Clp1 was able to affect the morphologies of calcite crystal in vitro. Shell notching experiments suggested that Pf-Clp1 might function as a negative regulator during shell formation in vivo. Knockdown of Pf-Clp1 by RNAi led to the overgrowth of aragonite tablets, further confirming its potential negative regulation on biomineralization, especially in the nacreous layer. Our work revealed the potential function of molluscan Clp in shell biomineralization for the first time and unveiled some new understandings toward the molecular mechanism of shell formation.
Asunto(s)
Exoesqueleto/metabolismo , Quitinasas , Clonación Molecular , Regulación de la Expresión Génica , Pinctada , Animales , Quitinasas/biosíntesis , Quitinasas/química , Quitinasas/genética , Quitinasas/aislamiento & purificación , Pinctada/enzimología , Pinctada/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
A recombinant strain producing a complex of extracellular enzymes including chitinase from Myceliophtora thermophila was created based on the fungus Penicillium verruculosum. The activity of the enzyme preparations obtained from the cultural fluid of the producer strain was 0.55, 0.53, and 0.66 U/mg protein with chitin and chitosans with the molecular weight of 200 and 1000 kDa, respectively. The temperature optimum for the recombinant chitinase was 52-65°C; the pH optimum was 4.5-6.2, which corresponded to the published data for this class of the enzymes. The content of heterologous chitinase in the obtained enzyme preparations was 47% of total protein content in the cultural fluid. Enzyme preparations produced by the recombinant P. verruculosum XT403 strain and containing heterologous chitinase were able to degrade the mycelium of micromycetes, including phytopathogenic ones, and were very efficient in the bioconversion of microbiological industry waste.
Asunto(s)
Pared Celular/metabolismo , Quitina/metabolismo , Quitinasas/metabolismo , Proteínas Recombinantes/metabolismo , Sordariales/enzimología , Quitinasas/genética , Quitinasas/aislamiento & purificación , Hidrolasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sordariales/genética , Sordariales/metabolismoRESUMEN
Subtilases are a large family of serine proteases that occur throughout biology. A small subset contain protease-associated (PA) domains that are structurally separate from but encoded within the active site. In bacteria, subtilase PA domains function to recruit specific protein substrates. Here we demonstrate that a protease secreted by the fungal corn pathogen Stenocarpella maydis, which truncates corn ChitA chitinase, is a PA domain subtilase. Protease was purified from S. maydis cultures and tryptic peptides were analyzed by LC-MS/MS. Ions were mapped to two predicted PA domain subtilases. Yeast strains were engineered to express each protease. One failed to produce recombinant protein while the other secreted protease that truncated ChitA. This protease, that we named kilbournase, was purified and characterized. It cleaved multiple peptide bonds in the amino-terminal chitin binding domain of ChitA while leaving the catalytic domain intact. Kilbournase was more active on the ChitA-B73 alloform compared to ChitA-LH82 and did not cleave AtChitIV3, a homolog from Arabidopsis thaliana, indicating a high level of specificity. Truncation of corn ChitA by kilbournase resembles truncation of human C5a by Streptococcus pyogenes ScpA, arguing that PA domain proteases in bacteria and fungi may commonly target specific host proteins.
Asunto(s)
Ascomicetos/genética , Péptido Hidrolasas/genética , Subtilisinas/genética , Zea mays/genética , Arabidopsis/genética , Ascomicetos/patogenicidad , Dominio Catalítico/genética , Quitinasas/genética , Quitinasas/aislamiento & purificación , Cromatografía Liquida , Péptido Hidrolasas/aislamiento & purificación , Espectrometría de Masas en Tándem , Zea mays/microbiologíaRESUMEN
Xenorhabdus nematophila strain ATCC 19061 is an insect pathogen that produces various protein toxins which intoxicate and kill its larval host. In the present study, we have described the cloning, expression and characterization of a 76-kDa chitinase protein of X. nematophila. A 1.9 kb DNA sequence encoding the chitinase gene was PCR amplified and cloned. Further, the chitinase protein was expressed in Escherichia coli and purified by using affinity chromatography. Two highly conserved domains were identified GH18 and ChiA. The purified chitinase protein showed chitobiosidase activity, ß-N-acetylglucosaminidase and endochitinase activity, when enzyme activity was measured using respective substrates. The purified chitinase protein was found to be orally toxic to the larvae of a major crop pest, Helicoverpa armigera when fed to the larvae mixed with artificial diet. It also had adverse effect on the growth and development of the surviving larvae. Surviving larvae showed 9-fold reduction in weight, as a result the transformation of larvae into pupae was adversely affected. Our results demonstrated that the chitinase protein of X. nematophila has insecticidal property and can prove to be a potent candidate for pest control in plants.
Asunto(s)
Quitinasas/química , Quitinasas/farmacología , Insecticidas/química , Insecticidas/farmacología , Xenorhabdus/enzimología , Fenómenos Químicos , Quitinasas/genética , Quitinasas/aislamiento & purificación , Dicroismo Circular , Clonación Molecular , Relación Dosis-Respuesta a Droga , Expresión Génica , Modelos Biológicos , Conformación Proteica , Proteínas Recombinantes , Análisis de Secuencia de ADN , Análisis Espectral , Xenorhabdus/genéticaRESUMEN
Antifreeze proteins restrict the growth of ice crystals during recrystallization and therefore find application in the protection of food products from damage upon freezing. Hippophae rhamnoides (seabuckthorn) is a freeze tolerant Himalayan shrub exhibiting antifreeze properties. Here, ~39 kDa class IV chitinase (HrCHI4) was purified from seabuckthorn seeds using chitin-affinity chromatography that showed antifreeze property by ice recrystallization inhibition. The application of HrCHI4 in cryopreservation of green beans was analyzed to verify its antifreeze potential. HrCHI4 pretreatment reduced the drip loss and electrolytic leakage in frozen beans, revealing that it preserved the membrane integrity upon cryopreservation. The texture analysis and SEM further validated structural maintenance. The volatile component analysis using GC-MS was performed to evaluate the quality of frozen beans. HrCHI4 contributed positively towards the retention of volatile components after freeze-thaw. In conclusion, a class IV chitinase HrCHI4 was purified from seabuckthorn seeds and its cryoprotective function was reported.
Asunto(s)
Quitinasas/química , Criopreservación/métodos , Conservación de Alimentos/métodos , Verduras , Proteínas Anticongelantes/química , Proteínas Anticongelantes/aislamiento & purificación , Proteínas Anticongelantes/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Crioprotectores , Hippophae/enzimología , PhaseolusRESUMEN
A chitinase gene from Serratia marcescens was cloned and expressed in Escherichia coli BL21(DE3) and the properties of recombinant chitinase rCHI-2 were characterized. The optimum catalytic pH of rCHI-2 was 6.0. It was stable in the pH range of 6.0-9.0 and could maintain more than 90% of its relative enzyme activity after incubation at 37 °C for 1 h. The optimum catalytic temperature of the enzyme was 55 °C and 85% of enzyme activity was remained after incubation at 45 °C for 1 h. The activation energy of the thermal inactivation of the enzyme was 10.9 kJ/mol and the Michaelis-Menten constant was 3.2 g/L. The purified rCHI-2 was found to be highly stable at 45 °C with half-life (t1/2) of 289 min and thermodynamic parameters ΔH*, ΔG* and ΔS* revealed high affinity of rCHI-2 for chitin. Hg2+ was found to be able to inhibit the enzyme activity reversibly, while IC50 and inhibition constant of Hg2+ on the enzyme were 34.8 µmol/L and 44.6 µmol/L, respectively. Moreover, rCHI-2 could specifically hydrolyze colloidal chitin into GlcNAc2 as the major product.