Isolation and Characterization of the Genes Encoding Two Metalloproteases (MprI and MprII) from a Marine Bacterium,<i>Alteromonas</i>sp. Strain O-7

Miyamoto, Katsushiro; Tsujibo, Hiroshi; Nukui, Eiji; Itoh, Hiroyuki; Kaidzu, Yoshihiko; Inamori, Yoshihiko

doi:10.1271/bbb.66.416

Cited by 18 publications

(12 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Waschkowitz

Rockstroh

Daniel

2009

Appl Environ Microbiol

View full text Add to dashboard Cite

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per g of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.Proteolytic enzymes catalyze the hydrolytic cleavage of peptide bonds. These enzymes are present in all living organisms and are essential for cell growth and differentiation. Microorganisms produce a variety of intracellular and/or extracellular proteases. Intracellular microbial proteases are highly specific and are involved in several cellular and metabolic processes such as activation of inactive precursors, maintenance of the cellular protein pool, and sporulation. Extracellular proteases degrade proteins in cell-free environments. The resulting hydrolytic products (small peptides and amino acids) can be transported into the cells and utilized as carbon or nitrogen sources (13, 39). Especially, extracellular proteases are of industrial importance and are used as cleaning agents and food and feed additives.Proteases can be divided into various groups with respect to the functional group present at the active site and the pH profile of the activity. Microbial alkaline proteases, which are defined to be active in a neutral to alkaline pH range (13), possess either a serine center (serine proteases) or are of metal-type (metalloproteases). Extracellular...

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Waschkowitz

Rockstroh

Daniel

2009

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…strain O-7 secretes four serine proteases and three metalloproteases (15). The genes for three of the serine proteases (AprI, AprII, and AprIV) and two of the metalloproteases (MprI and MprII) have been cloned and sequenced, and the relationship between the structure and function of these enzymes has been investigated (15,16,28,32). Our final goal was to investigate the physiological role and gene regulation of each protease of the strain.…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that MprI, which is a metalloprotease produced by Alteromonas sp. strain O-7, is one of the typical enzymes in the thermolysin family (15). MprIII showed a high activity towards gelatin among the substrates tested, whereas MprI showed a high activity towards casein (Table 2).…”

Section: Discussionmentioning

confidence: 99%

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

Miyamoto

Nukui

Hirose

et al. 2002

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Although some serine proteases and metalloproteases from terrestrial bacteria are reported to be elastases, there are very few studies on marine elastase-producing bacteria and elastases (13,14). For this reason, the mechanism of marine elastin degradation remains unclear.…”

mentioning

confidence: 99%

Elastolytic Mechanism of a Novel M23 Metalloprotease Pseudoalterin from Deep-sea Pseudoalteromonas sp. CF6-2

Zhao

Chen

Xie

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:The mechanism of marine elastin degradation is unclear. Results: A novel M23 metalloprotease pseudoalterin from a marine bacterium degraded elastin by cleaving both the glycyl bonds and the peptide bonds involved in cross-linking. Conclusion: Pseudoalterin adopts a novel elastolytic mechanism different from other M23 metalloproteases. Significance: The results shed light on the mechanism of marine elastin degradation.

show abstract

Isolation and Characterization of the Genes Encoding Two Metalloproteases (MprI and MprII) from a Marine Bacterium,Alteromonassp. Strain O-7

Cited by 18 publications

References 28 publications

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

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

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

Elastolytic Mechanism of a Novel M23 Metalloprotease Pseudoalterin from Deep-sea Pseudoalteromonas sp. CF6-2

Contact Info

Product

Resources

About