RESUMO
A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.
Assuntos
Antifúngicos/química , Quitina/análogos & derivados , Chromobacterium/genética , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos/genética , Quitina/biossíntese , Quitina/química , Quitina/genética , Quitosana/química , Chromobacterium/enzimologia , Escherichia coli/genética , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , OligossacarídeosRESUMO
LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40⯰C and pHâ¯6.5. Under these conditions, the highest activity was against tributyrin (1650â¯Uâ¯mg-1), but it also had high activity against olive oil (862â¯Uâ¯mg-1). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24â¯h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24â¯h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5â¯h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)1phenylethyl acetate for the kinetic resolution of (R,S)1phenyl1ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.
Assuntos
Ácidos Graxos/análise , Biblioteca Gênica , Lipase/química , Lipase/metabolismo , Metagenoma , Microbiologia do Solo , Solo/química , Sequência de Aminoácidos , Chromobacterium/enzimologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , Solventes/farmacologia , Temperatura , Triglicerídeos/metabolismoRESUMO
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 , TemperaturaRESUMO
Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics.
Assuntos
Aminopeptidases/farmacologia , Antivirais/farmacologia , Chromobacterium/enzimologia , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Proteínas do Envelope Viral/metabolismo , Proteínas de Bactérias/farmacologia , Dengue/virologia , Vírus da Dengue/fisiologia , Sistema Digestório/virologia , Proteólise , Vírion/efeitos dos fármacos , Ligação Viral/efeitos dos fármacosRESUMO
The named "green chemistry" has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a "green" technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions.
Assuntos
Enzimas/química , Química Verde , Lipase/química , Ultrassom , Álcoois , Biocombustíveis , Burkholderia cepacia/enzimologia , Catálise , Chromobacterium/enzimologia , Dicroísmo Circular , Enzimas Imobilizadas , Ésteres , Ácidos Graxos não Esterificados/química , Proteínas Fúngicas , Glicerol , Hidrólise , Microscopia Eletrônica de Varredura , Polímeros/química , Solventes/químicaRESUMO
Phytases are a group of enzymes that catalyze phytic acid hydrolysis with release of phosphorus (P). The ability of Chromobacterium sp. to produce phytase was detected in 115 out of 118 candidate bacteria isolated from different Brazilian biomas. This is the first report revealing the genus Chromobacterium as phytase producer.
Assuntos
Sequência de Bases , Biomassa , Chromobacterium/enzimologia , Chromobacterium/isolamento & purificação , Microbiologia Ambiental , Reativadores Enzimáticos , Eutrofização , Monoéster Fosfórico Hidrolases , Peptídeo Hidrolases/análise , Catálise , Ativação Enzimática , Variação Genética , Hidrólise , Métodos , Métodos , Ecossistema TropicalRESUMO
Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).