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Journal of Bacteriology, April 2002, p. 1865-1872, Vol. 184, No. 7
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.7.1865-1872.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

Katsushiro Miyamoto,1 Eiji Nukui,1 Hiroyuki Itoh,1 Takaji Sato,1 Takeshi Kobayashi,2 Chiaki Imada,2 Etsuo Watanabe,2 Yoshihiko Inamori,1 and Hiroshi Tsujibo1*

Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094,1 Department of Food Science and Technology, Tokyo University of Fisheries, Minato-ku, Konan 4-5-7, Tokyo 108-8477, Japan2

Received 24 September 2001/ Accepted 4 January 2002

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 ß-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°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.

* Corresponding author. Mailing address: Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Phone: (81-726) 90-1057. Fax: (81-726) 90-1057. E-mail: tsujibo{at}gly.oups.ac.jp.

Journal of Bacteriology, April 2002, p. 1865-1872, Vol. 184, No. 7
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.7.1865-1872.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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