Family 19 chitinases from Streptomyces thermoviolaceus OPC-520: molecular cloning and characterization.
Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70 degrees C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.
Tsujibo
H
H
Osaka University of Pharmaceutical Sciences, Takatsuki, Japan. tsujibo@oysun01.oups.ac.jp
Okamoto
T
T
Hatano
N
N
Miyamoto
K
K
Watanabe
T
T
Mitsutomi
M
M
Inamori
Y
Y
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D000935:Antifungal Agents; D004269:DNA, Bacterial; D002688:Chitinases