Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris (2024)

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A novel member of the thermolysin family, cloning and biochemical characterization of metalloprotease from Staphylococcus pseudintermedius

Adam Dubin, Artur Sabat

Acta biochimica Polonica, 2008

Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression o...

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Purification and Characterization of a Thermostable Neutrophilic Metalloprotease from Pseudomonas Sp. DR89

ahmad asoodeh

Iranian Journal of Biotechnology, 2012

A novel neutrophilic metalloprotease was isolated from Pseudomonas sp. DR89 isolate which was identified in a mineral spring in Iran. The enzyme was purified from the isolate to 21-folds in a three-step procedure involving ammonium sulfate precipitation, Q-Sepharose ionic exchange and Sephadex G-100 gel filtration chromatography. Resuts showed that the enzyme was active at high temperatures and in a wide-range pH of 5-11 with the optimum of 8.0. The zymogram and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of one protease with a molecular weight of 74 kDa. The enzyme activity was decreased by Zn2+, Mn2+, H 2 O 2 and cetyl trimethylammonium bromide (CTAB), whereas its activity was increased by Ca2+, Mg2+, Cu2+ and dimethyl sulfoxide (DMSO). Na + , phenylmethyl sulfonylfluoride (PMSF), β-mercaptoethanol, sodium dodecyl sulfate (SDS), and Triton X-100 did not show a considerable effect on its activity. Casein was a better substrate than bovin...

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In silico characterization and structural modeling of bacterial metalloprotease of family M4

MD. Nazmul Haq Rony

Journal of Genetic Engineering & Biotechnology, 2021

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Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

Danilo Morales

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Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5

Fatma Matpan Bekler, Ömer Acer, Kemal Guven

Abstract Background: Due to the importance of microbial proteases in biotechnological applications, a number of microorganisms are being explored. The production, purification and characterisation of extracellular metallo-proteases by producing Bacillus sp. KG5 was studied. Material and Methods: Bacterial strain KG5 was isolated from Kös (Bingöl) hot spring. The strain KG5 was identified by morphological, physiological, biochemical and 16S rRNA gene sequencing. The effects of various parameters on protease production, such as time, temperature, pH, carbon and nitrogen sources and CaCl2 were studied. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel permeation chromatography. Molecular weight was calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymographic analysis. The effects of some metal ions, chelators and inhibitors on enzyme activity were determined. Results: The optimum temperature, pH and incubation period for protease production were 40- 45°C, 7.0 and 24 h, respectively. It was determined that the best nitrogen sources were yeast extract and urea, while the best carbon sources were lactose and galactose. However, glucose as a source of carbon was found to inhibit the production of the enzyme. The maximum enzyme production was increased in the presence of CaCl2. The molecular weight of purified enzyme was found to be approximately 48 kDa. It was found that the enzyme was fully stable in the presence of 2 mM CaCl2 at 50°C after 120 min. Purified protease was significantly activated by Ca2+ and Mg2+, while it was greatly inhibited by Cu2+, Zn2+, Hg2+ and SDS as well as by the metal ion chelators ethylenediaminetetraacetic (EDTA) and 1,10-phenanthroline. Phenylmethylsulfonyl fluoride (PMSF) had a little effect on the enzyme. Conclusions: Our findings suggest the potential of this isolate for protease production and that this enzyme may be suitable for biotechnological applications. Keywords: Bacillus sp. KG5, biotechnology, protease production and characterisation.

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Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic <em>Pseudomonas</em> isolate

Danilo Morales

AIMS Bioengineering, 2017

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Involvement of Propeptides in Formation of Catalytically Active Metalloproteinase from Thermoactinomyces sp

Ilya Demidyuk

2011

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Temperature-dependent activation of Bacillus brevis metalloprotease expressed in mesophilic Bacillus subtilis

Alex Strongin

FEMS Microbiology Letters, 2000

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Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase TA metalloenzyme endowed with dual substrate specificity

Nikolay Yankovsky

FEBS letters, 1991

A gene coding for an extracellular Zn-carboxypeptidase of Thermoactinomyces vulgaris has been cloned and sequenced (EMBL X56901). This enzyme named carboxypeptidase T reveals simultaneously both types of substrate specificity ...

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Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris (2024)
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