Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. strain O-7.
The chitinase B (ChiB) secreted by Alteromonas sp. strain O-7 was purified, and the corresponding gene (chiB) was cloned and sequenced. The open reading frame of the chiB gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da. ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18. The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains. Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in Escherichia coli. Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin. The optimum pH and temperature of ChiB were 6.0 and 30 degrees C, respectively. The enzyme showed relatively high catalysis, even at low temperatures close to 0 degrees C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain. The kca)/Km value for the ChiB reaction at 10 degrees C was about 4.7 times higher than that of ChiC. These results suggest that ChiB is a cold-adapted enzyme. The RNA transcript of chiB was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease. Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.
Orikoshi
Hideyuki
H
Department of Microbiology, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan.
Baba
Nao
N
Nakayama
Shigenari
S
Kashu
Hiroshi
H
Miyamoto
Katsushiro
K
Yasuda
Masahide
M
Inamori
Yoshihiko
Y
Tsujibo
Hiroshi
H
D000222:Adaptation, Physiological; D020591:Alteromonas / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012623:Seawater / Q000382:microbiology; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D002688:Chitinases