Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2.
A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N'-diacetylchitobiose were 1.06 mM and 44.4 micromol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N'-diacetylchitobiose, indicating that ChiA is a chitobiosidase.
Lee
H S
HS
Department of Biotechnology, College of Engineering and Bioproducts Research Center, Yonsei University, Seoul, Korea.
Han
D S
DS
Choi
S J
SJ
Choi
S W
SW
Kim
D S
DS
Bai
D H
DH
Yu
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D002686:Chitin; D002688:Chitinases; C094590:chitinase ChiA