RESUMO
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V (max) of 2.03 x 10(-9) s (-1) and a K (m) of 206 microM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70 degrees C and the enzyme had a half life at 60 degrees C of 20.8 h.
Assuntos
Bacillus/enzimologia , Bacillus/genética , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Purina-Núcleosídeo Fosforilase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
This paper describes a high yielding coupled enzymatic reaction using Bacillus halodurans purine nucleoside phosphorylase (PNP) and E. coli uridine phosphorylase (UP) for synthesis of 5-methyluridine (5-MU) by transglycosylation. Key parameters such as reaction temperature, pH, reactant loading, reactor configuration and enzyme loading were investigated. A guanosine conversion of 95% and a 5-MU yield of 85% were achieved at 1l scale, with a productivity of 10 g l⻹ h⻹.