RESUMO
The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel ß-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Penicillium/enzimologia , Penicillium/genética , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Polissacarídeo-Liases/química , Polissacarídeo-Liases/isolamento & purificação , Conformação Proteica , Proteólise , Alinhamento de Sequência , TemperaturaRESUMO
The CT1 mutant of Penicillium occitanis hyperproduces extracellular pectinases constitutively since it secretes pectinases even on glucose-containing medium. We show here that all other hydrolytic enzymes remain at low activities in CT1, confirming the specificity of the regulatory mutation towards pectinases. We isolated, by RT-PCR and through the construction of a cDNA library, three fragments coding for: a pectin lyase (pnl1), a polygalacturonase (pga1) and a pectate lyase (pal1). These fragments were used as probes in Northern blots analysis of the wild type strain CL100 and the CT1 mutant of P. occitanis grown in three culture conditions. The CT1 mutant showed a very high amount of pnl1, pga1 and pal1 mRNA either in pectin, glucose or glycerol grown cells while in the wild type CL100 strain, all transcripts were undetectable even on pectin. These results suggest that the CT1 mutation affects a trans-regulatory transcriptional factor regulating pectinase expression.
Assuntos
Penicillium/enzimologia , Penicillium/genética , Poligalacturonase/genética , Poligalacturonase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biotecnologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Penicillium/crescimento & desenvolvimento , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 degrees C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.
