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

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Abstract
Alteromonassp. 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 inEscherichia coli. DNA sequencing analysis revealed that the open reading frame ofaprIVencoded 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 α-chitin and β-chitin. Western and Northern analyses demonstrated thataprIVwas induced in the presence ofN-acetylglucosamine,N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity fromAlteromonassp. 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.