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Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium.

Santos AF, Valle RS, Pacheco CA, Alvarez VM, Seldin L, Santos AL - Braz. J. Microbiol. (2014)

Bottom Line: This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment.The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way.The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane).

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. ; Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

ABSTRACT
Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

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Related in: MedlinePlus

Time course of hydrolysis of chromogenic substrates from serine proteases secreted by H. blutaparonensis. The chromogenic substrates used are specific to measure the proteolytic activity of elastase (N-Succinyl-Ala-Ala-Ala-p-nitroanilide), trypsin (N-Benzoyl-Phe-Val-Arg-p-nitroanilide) and chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-Phe-p-nitroanilide). The enzymatic activities, expressed in arbitrary units (AU), were determined at 37 °C for 120 min under alkaline conditions of pH (pH 9.0). The values represent the mean ± standard error of three independent experiments performed in triplicate.
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f5-bmj-44-4-1299: Time course of hydrolysis of chromogenic substrates from serine proteases secreted by H. blutaparonensis. The chromogenic substrates used are specific to measure the proteolytic activity of elastase (N-Succinyl-Ala-Ala-Ala-p-nitroanilide), trypsin (N-Benzoyl-Phe-Val-Arg-p-nitroanilide) and chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-Phe-p-nitroanilide). The enzymatic activities, expressed in arbitrary units (AU), were determined at 37 °C for 120 min under alkaline conditions of pH (pH 9.0). The values represent the mean ± standard error of three independent experiments performed in triplicate.

Mentions: The degradation of different serine protease substrates was employed using the supernatant fluid of H. blutaparonensis (Figure 5). To this end, we performed kinetic assays using chromogenic peptide substrates specific to elastase, trypsin and chymotrypsin. The serine proteases secreted by H. blutaparonensis showed elevated degradation rate on the chymotrypsin substrate when compared to trypsin or elastase substrates (Figure 5). As expected, PMSF was able to completely abolish the cleavage of the chromogenic chymotrypsin substrate (data not shown).


Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium.

Santos AF, Valle RS, Pacheco CA, Alvarez VM, Seldin L, Santos AL - Braz. J. Microbiol. (2014)

Time course of hydrolysis of chromogenic substrates from serine proteases secreted by H. blutaparonensis. The chromogenic substrates used are specific to measure the proteolytic activity of elastase (N-Succinyl-Ala-Ala-Ala-p-nitroanilide), trypsin (N-Benzoyl-Phe-Val-Arg-p-nitroanilide) and chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-Phe-p-nitroanilide). The enzymatic activities, expressed in arbitrary units (AU), were determined at 37 °C for 120 min under alkaline conditions of pH (pH 9.0). The values represent the mean ± standard error of three independent experiments performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3958202&req=5

f5-bmj-44-4-1299: Time course of hydrolysis of chromogenic substrates from serine proteases secreted by H. blutaparonensis. The chromogenic substrates used are specific to measure the proteolytic activity of elastase (N-Succinyl-Ala-Ala-Ala-p-nitroanilide), trypsin (N-Benzoyl-Phe-Val-Arg-p-nitroanilide) and chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-Phe-p-nitroanilide). The enzymatic activities, expressed in arbitrary units (AU), were determined at 37 °C for 120 min under alkaline conditions of pH (pH 9.0). The values represent the mean ± standard error of three independent experiments performed in triplicate.
Mentions: The degradation of different serine protease substrates was employed using the supernatant fluid of H. blutaparonensis (Figure 5). To this end, we performed kinetic assays using chromogenic peptide substrates specific to elastase, trypsin and chymotrypsin. The serine proteases secreted by H. blutaparonensis showed elevated degradation rate on the chymotrypsin substrate when compared to trypsin or elastase substrates (Figure 5). As expected, PMSF was able to completely abolish the cleavage of the chromogenic chymotrypsin substrate (data not shown).

Bottom Line: This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment.The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way.The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane).

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. ; Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

ABSTRACT
Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

Show MeSH
Related in: MedlinePlus