RESUMO
AIMS: Isolation and characterization of the glucose oxidase (GOX)-encoding gene from a Penicillium variabile strain (P16) having a high level of GOX activity and comparison of its expression with that of another strain of P. variabile (NRRL 1048) characterized by low GOX activity. METHODS AND RESULTS: The gene, isolated by PCR consisted of 1818 bp encoding 605 amino acid residues. Gene expression was analysed by Northern blotting and compared with that of P. variabile NRRL 1048. The higher GOX activity of strain P16 appeared likely because of de novo mRNA synthesis. Southern blotting analyses of the genomic DNA showed that the hybridization pattern of the two strains differed for the size of hybridizing fragment detected by the probe and slightly for their signal intensity. CONCLUSIONS: The GOX-encoding gene of P. variabile P16 was isolated and characterized to identify the molecular bases of its high level of expression and in view of improving enzyme production by developing a process based on heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: GOX-encoding genes can be subjected to high difference in their expression levels. The P16 strain of P. variable producing large amount of GOX as well as its encoding gene might be exploited for industrial applications.
Assuntos
Glucose Oxidase/biossíntese , Glucose Oxidase/genética , Penicillium/enzimologia , Penicillium/genética , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , Cromossomos Fúngicos/genética , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Expressão Gênica , Genes Fúngicos , Genoma Fúngico , Glucose Oxidase/metabolismo , Dados de Sequência Molecular , Polimorfismo Genético , RNA Mensageiro/análise , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de TempoRESUMO
The effects of medium composition on the production of beta-glucosidase (amygdalase and linamarase) by Penicillium aurantiogriseum P35 were studied and the medium optimized as follows (g/l of deionized water): pectin, 10.0; (NH4)2SO4, 8.0; KH2PO4, 8.0; Na2HPO4, 2.8; MgSO4.7H2O, 0.5; yeast extract, 4.0; initial pH 6.0. When grown in a bench fermenter on this medium, the fungus produced 50.5 mU of amygdalase and 9.4 mU of linamarase per ml of culture broth. Two beta-glucosidases (PGI and PGII), each having amygdalase and linamarase activities, were recovered from the culture broth and purified; their relative molecular weights, as native enzymes, were estimated to be about 247,000 and 147,000, respectively. Both enzymes showed the same optimum pH (6.0) but different optimum temperatures (55 and 60 degrees C for PGI and PGII, respectively). Thermostability (10 min at 60 degrees C) and half-life of enzyme activity (7 hours at 60 degrees C) of PGII were higher than those of PGI (10 min at 50 degrees C and 2 hours at 55 degrees C, respectively). A wide range of cyanogenic glycosides (such as tetraphyllin B, epivolkenin, gynocardin, passibiflorin, prunasin, taxiphyllin, amygdalin, lucumin, sambunigrin, dhurrin, linamarin and cardiospermin sulfate) were hydrolyzed by both enzymes.
Assuntos
Penicillium/enzimologia , beta-Glucosidase/biossíntese , Reatores Biológicos , Configuração de Carboidratos , Sequência de Carboidratos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Especificidade por Substrato , Temperatura , beta-Glucosidase/isolamento & purificação , beta-Glucosidase/metabolismoRESUMO
Mucor circinelloides LU M40 and Penicillium aurantiogriseum P 35, characterized by extracellular beta-glucosidase activity on cyanogenic glycosides, hydrolyse amygdalin by a two-step reaction mechanism being the first step of hydrolysis, from amygdalin to prunasin, very rapid (15 min) and the second one, from prunasin to mandelonitrile, much slower (120 min).
