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 with 1% chitin, three activity bands corresponding to proteins of 40 kDa (Chi40), 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 estimated to be 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its isoelectric point was 3.8. The optimum pH and temperature of Chi30 were 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 nucleotides sequenced. The open reading frame of chi30 encoded a protein consisting 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 existence 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 induction and glucose repression.
Tsujibo
H
H
Osaka University of Pharmaceutical Sciences, Japan. tsujibo@oysun01.oups.ac.jp
Hatano
N
N
Endo
H
H
Miyamoto
K
K
Inamori
Y
Y
D001483:Base Sequence; D002688:Chitinases / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D004271:DNA, Fungal; D002688:Chitinases