Purification and characterization of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 and cloning of the encoding gene

When Streptomyces thermoviolaceus OPC-520 was grown in a minimal medium wit h 1% chitin, three activity bands corresponding to proteins of 40 kDa (Chi4 0), 30 kDa (Chi30), and 25 kDa (Chi25) were detected. Among them, Chi30 was purified from the culture filtrate of the strain. The molecular mass was e stimated to be 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrop horesis and its isoelectric point was 3.8. The optimum pH and temperature o f Chi30 mere 4.0 and 60 degrees C, respectively. Chi30 was stable at pH 6-8 up to 60 degrees C. The gene encoding Chi30 (chi30) was cloned and its nuc leotides sequenced. The open reading frame of chi30 encoded a protein consi sting of 347 amino acids with a calculated molecular weight of 35,621. The mature Chi30 consisted of only a catalytic domain and showed a significant similarity with ChiA from S. coelicolor and ChiA from S. lividans. The exis tence of a 12-bp direct repeat sequence in the promoter region of chi30 was detected, which have Been suggested to be involved in both chitin inductio n and glucose repression.