title: Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system.
abstract: 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.
authors.affiliation: Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.
authors.forename: Katsushiro
authors.initials: K
authors.lastname: Miyamoto
authors.forename: Eiji
authors.initials: E
authors.lastname: Nukui
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Itoh
authors.forename: Takaji
authors.initials: T
authors.lastname: Sato
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Chiaki
authors.initials: C
authors.lastname: Imada
authors.forename: Etsuo
authors.initials: E
authors.lastname: Watanabe
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Inamori
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsujibo
mesh_terms: 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
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D002686:Chitin; D002688:Chitinases; D010450:Endopeptidases