RESUMO
A high phytase-producing strain of Aspergillus niger N-3 was identified by screening 104 microbial strains. The gene for A. niger N-3 was cloned and expressed in Pichia pastoris. The coding region without the introns and putative signal sequence was comprised of 1347 nucleotides. It encoded a polypeptide of 448 amino acids, exhibiting high amino acid sequence homologies (94.87%) with the typical phytase of A. niger NRRL 3135. The molecular mass of the recombinant phytase as determined by SDS-PAGE was 60-70 kDa, with maximum activity at approximately 55 degrees C (after incubation at 10 min). The phytase retained about 45% of its enzymatic activity under heat treatment at 90 degrees C for 5 min. It showed a greater affinity for sodium phytate than for p-nitrophenyl phosphate. Dual optima pH (2.0 and 5.5) was gained. The activity at pH 2.0 was about 30% higher than at pH 5.5, which was more suitable to the circumstance of the stomachs of monogastric animals. The extent of glycosylation influenced the characterization of phytase. The deglycosylated phytase showed pH optima at 3.5 and 5.5, and the molecular mass had dropped to 50 kDa.
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Aspergillus niger/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microbiologia Industrial , 6-Fitase/química , Sequência de Aminoácidos , Animais , Aspergillus niger/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Proteínas Fúngicas/química , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Íntrons , Dados de Sequência Molecular , Peso Molecular , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Ácido Fítico/metabolismo , Pichia/genética , Sinais Direcionadores de Proteínas , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.
