Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system.
Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.
Miyamoto
Katsushiro
K
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.
Nukui
Eiji
E
Itoh
Hiroyuki
H
Sato
Takaji
T
Kobayashi
Takeshi
T
Imada
Chiaki
C
Watanabe
Etsuo
E
Inamori
Yoshihiko
Y
Tsujibo
Hiroshi
H
D020591:Alteromonas / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D002686:Chitin; D002688:Chitinases; D010450:Endopeptidases