RESUMEN

Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548, was cloned into the Streptomyces/Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in S. lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 and 16.1 mg/mL, respectively. The Vmax values toward Avicel and filter paper were 0.432 and 0.084 µM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co(2+), Cu(2+), Ni(2+) and Mn(2+) (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-ß-cellobiosidase that hydrolyzes cellulose into cellobiose.

Asunto(s)

Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Streptomyces coelicolor/enzimología , Streptomyces coelicolor/genética , Cationes Bivalentes/metabolismo , Celobiosa/metabolismo , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/química , Clonación Molecular , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Expresión Génica , Vectores Genéticos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Peso Molecular , Papel , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura