Protease-producing psychrotrophic bacteria isolated from Antarctica

Vázquez, Susana; Merino, L. N. Rios; Cormack, Walter P. Mac; Fraile, Elda R.

doi:10.1007/bf00241051

Cited by 37 publications

(24 citation statements)

References 28 publications

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“…Proteolytic activity assay was carried out by the method of Vázquez et al [11]. One protease unit is defined as the amount of enzyme causing a rise in absorbance at 340 nm of 0.003 per min, at 55 • C and pH 6.0.…”

Section: Enzyme and Protein Assaysmentioning

confidence: 99%

Purification of lumbrokinase from Eisenia fetida using aqueous two-phase systems and anion-exchange chromatography

Iannucci

Camperi

Cascone

2008

Separation and Purification Technology

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Section: Enzyme and Protein Assaysmentioning

confidence: 99%

Purification of lumbrokinase from Eisenia fetida using aqueous two-phase systems and anion-exchange chromatography

Iannucci

Camperi

Cascone

2008

Separation and Purification Technology

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“…Savinase was assayed at 408C for 30 min using two different concentrations (0.6 or 1.5% (w/v)) of azocasein as substrate in 0.2 M Tris/H 2 SO 4 , pH 8.5 as previously described (Vazquez et al, 1995) with minor modifications. Protease activity units were defined as the total amount of azocasein hydrolyzed (mg) per min per mg savinase and was determined by completely hydrolyzing a known amount of azocasein.…”

Section: Protease Assaymentioning

confidence: 99%

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

Siddiqui

Parkin

Curmi

et al. 2009

Biotech & Bioengineering

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The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15 degrees C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K(i) values of modified savinases sixfold higher than the unmodified savinase. Modeling of small-angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold-active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme-formulation.

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“…To study the protease activities of the strains producing clearing zones on skim milk plates, a fluorimetric assay was used (28) that was far more sensitive than the azocasein method (7,30) and was suitable for continuous assays. Protease activity was detected for all the strains tested.…”

Section: Resultsmentioning

confidence: 99%

Use of a Packed-Column Bioreactor for Isolation of Diverse Protease-Producing Bacteria from Antarctic Soil

Wéry

Gerike

Sharman

et al. 2003

Appl Environ Microbiol

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Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica. The column was maintained at 10°C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45°C. 16S ribosomal DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial divisions: ␣-, ␤-, and ␥-Proteobacteria; the Flexibacter-Cytophaga-Bacteroides group; and high G؉C grampositive bacteria and low G؉C gram-positive bacteria. Some strains represent candidates for new species of the genera Chryseobacterium and Massilia. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.In the laboratory, microorganisms are generally cultivated as cell suspensions in liquid media. However, their natural environments are often more complex, and most microorganisms are members of mixed populations, growing frequently in a nearly static situation (e.g., embedded in soil or as biofilms) resulting from the passage of an aqueous stream of dissolved gases and nutrients over them. A wide variety of surfaces are available in natural environments for attachment and colonization by microorganisms, and these attached bacteria are often more active than free cells (29).Developing new strategies to recover the microbial population that is not accessible by using classical enrichment techniques has become a real issue for microbiologists. Molecular studies of microbial diversity have shown that these "not yet cultivated" microorganisms can correspond to 99% of the total microbial population (2). Furthermore, access to this reservoir of genetic and metabolic diversity is of interest in areas such as food production, medicine, bioremediation of waste materials, and agriculture. In particular, there is an important biotechnological interest in the isolation of extremophilic microorganisms that are adapted to growth under extreme conditions of temperature, pH, salinity, pressure, and/or in the presence of radioactivity or high concentrations of metal. These extremophiles present a commercial potential for various industries and products, including agricultural applications, chemical synthesis, laundry detergents, and pharmaceuticals (22).In the present study, microrganisms were enriched by using a reactor designed to mimic natural ecosystems. This reactor contained a column filled with packing to allow microbial colonization and was fed by culture medium in a slow upflow mode. It was used in an attempt to isolate microorganisms growing at low temperatures and producing cold-active enzymes. Psychrophiles ar...

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Protease-producing psychrotrophic bacteria isolated from Antarctica

Cited by 37 publications

References 28 publications

Purification of lumbrokinase from Eisenia fetida using aqueous two-phase systems and anion-exchange chromatography

Purification of lumbrokinase from Eisenia fetida using aqueous two-phase systems and anion-exchange chromatography

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

Use of a Packed-Column Bioreactor for Isolation of Diverse Protease-Producing Bacteria from Antarctic Soil

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