A Metalloprotease (MprIII) Involved in the Chitinolytic System of a Marine Bacterium, Alteromonas sp. Strain O-7

Abstract

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50°C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.

Microbial Surface Colonization and Biofilm Development in Marine Environments

“…They can degrade and utilize a broad spectrum of organic substrates, including deep-sea recalcitrant organic matter (245,(250)(251)(252). Alteromonas produces and secretes a variety of extracellular enzymes that contribute to the hydrolysis of biopolymers, including polysaccharides (253)(254)(255)(256)(257), proteins (258,259), nucleic acids (260,261), and lipids (262), the major components of marine POM. Alteromonas and related species respond rapidly to phytoplankton blooms and especially to elevated POC concentrations (109,(263)(264)(265).…”

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

“…Both types of proteases have been cloned and sequenced from many different individual microorganisms, i.e., Bacillus subtilis (56), Enterobacter sakazakii (22), Listeria monocytogenes (4), and Alteromonas sp. (33,34).…”

“…Both types of proteases have been cloned and sequenced from many different individual microorganisms, i.e., Bacillus subtilis (56), Enterobacter sakazakii (22), Listeria monocytogenes (4), and Alteromonas sp. (33,34).In the present study, we sought to isolate proteases by a metagenomic approach. Functional metagenomics comprising the isolation of DNA from environmental samples without prior enrichment of individual microorganisms, the construction of libraries from the recovered DNA, and function-driven screening of the generated libraries has led to the identification and characterization of a variety of novel enzymes (7, 14, 43), i.e., amylases (44, 58), oxidoreductases (21), lipolytic enzymes (9,16,44), monooxygenases (53), and proteins conferring nickel resistance (32).…”

Isolation and identification of protease-producing bacteria from the intertidal zone in Jeju Island, Korea

“…Previous studies have shown the isolation of proteases from several marine bacteria (Alfredsson et al, 1995;Marcello et al, 1996;Irwin et al, 2001;Lee et al, 2002). These bacterial proteases showed algicidal activity or enhanced chitinase activity (Lee et al, 2002;Miyamoto et al, 2002).…”