Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil

Two psychrophilic bacterial samples were isolated from King George Island soil, in Antarctica. The phylogenetic analysis based on the 16S rRNA (rrs) gene led to the correlation with the closest related isolates as Sporosarcina aquimarina (99%) and Algoriphagus antarcticus (99%), with query coverage of 99% and 98%, respectively. The spent culture media from both isolates displayed proteolytic activities detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis containing gelatin as protein substrate. Under the employed conditions, S. aquimarina showed a 55 kDa protease with the best activity detected at pH 7.0 and at 27°C. A. antarcticus also showed a single extracellular protease, however its molecular mass was around 90kDa and its best activity was detected at pH 9.0 and at 37°C. The proteases from both isolates were inhibited by 1,10-phenanthroline and EDTA, two metalloprotease inhibitors. This is the first record of protease detection in both species, and our results may contribute to broaden the basic knowledge of proteases from the Antarctica environment and may help prospecting future biotechnological applications of these enzymes.

Algoriphagus antarcticus; Antarctica; Sporosarcina aquimarina; Protease

Authorship

Anderson F. Santos

Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, 21941-902 Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrasilRio de Janeiro, RJ, BrasilDepartamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, 21941-902 Rio de Janeiro, RJ, Brasil

Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, 21941-909 Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrasilRio de Janeiro, RJ, BrasilPrograma de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, 21941-909 Rio de Janeiro, RJ, Brasil

Fabiano Pires

Hugo E. Jesus

André L.S. Santos

Raquel Peixoto

Alexandre S. Rosado

Claudia M. D'Avila-Levy

Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil, 4365, 21040-360 Rio de Janeiro, RJ, BrasilFundação Oswaldo CruzBrasilRio de Janeiro, RJ, BrasilInstituto Oswaldo Cruz, Fundação Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil, 4365, 21040-360 Rio de Janeiro, RJ, Brasil

Marta H. Branquinha

Correspondence to: Marta Helena Branquinha E-mail: mbranquinha@micro.ufrj.br

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Figures

Figures (5)

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Figure 1 -
Location of collection of bacterial samples: (A) The Antarctic continent, with the Antarctic Peninsula highlighted. (B) Antarctic Peninsula and archipelago of the South Shetland Islands. (C) The Admiralty Bay highlighted in King George Island. The circle indicates the location of the Comandante Ferraz Antarctic Station (EACF), from Brazil. Adapted from Simões et al. (2004).

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Figure 2 -
Phylogenetic tree of 16S rRNA gene sequences (around 1500 bp) showing the relationship between two Antarctic bacterial isolates (Samples 1 and 2) and related species identified using BLASTn searches. The tree was constructed based on a neighborjoining method. Bootstrap analyses were performed with 10,000 repetitions, and results are represented at the branch points. The GenBank accession number of each species is cited in parentheses (S. aquimarina JX45902 and A. antarcticus JX45901). Type species are referred to as (T).

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Figure 3 -
Gelatin-SDS-PAGE showing the detection of extracellular proteases by Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at distinct pH values and optimal temperature conditions, as described in Materials and Methods. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the optimal pH conditions, is shown on the right.

Thumbnail

Figure 4 -
Gelatin-SDS-PAGE showing the detection of extracellular proteases in Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at distinct temperature values and optimal pH conditions, as described in Materials and Methods. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the optimal temperature conditions, is shown on the right.

Thumbnail

Figure 5 -
Gelatin-SDS-PAGE showing the detection of extracellular proteases in Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at pH and temperature conditions that better resolved the proteolytic profile, as described in Materials and Methods, in the absence (Control) or in the presence of different proteolytic inhibitors: 10 mM PMSF, 100 µM TLCK, 10 mM 1,10-phenanthroline (PHEN) and 10 mM EDTA. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the control, is shown on the right.

Figure 1 - Location of collection of bacterial samples: (A) The Antarctic continent, with the Antarctic Peninsula highlighted. (B) Antarctic Peninsula and archipelago of the South Shetland Islands. (C) The Admiralty Bay highlighted in King George Island. The circle indicates the location of the Comandante Ferraz Antarctic Station (EACF), from Brazil. Adapted from Simões et al. (2004).

Figure 2 - Phylogenetic tree of 16S rRNA gene sequences (around 1500 bp) showing the relationship between two Antarctic bacterial isolates (Samples 1 and 2) and related species identified using BLASTn searches. The tree was constructed based on a neighborjoining method. Bootstrap analyses were performed with 10,000 repetitions, and results are represented at the branch points. The GenBank accession number of each species is cited in parentheses (S. aquimarina JX45902 and A. antarcticus JX45901). Type species are referred to as (T).

Figure 3 - Gelatin-SDS-PAGE showing the detection of extracellular proteases by Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at distinct pH values and optimal temperature conditions, as described in Materials and Methods. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the optimal pH conditions, is shown on the right.

Figure 4 - Gelatin-SDS-PAGE showing the detection of extracellular proteases in Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at distinct temperature values and optimal pH conditions, as described in Materials and Methods. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the optimal temperature conditions, is shown on the right.

Figure 5 - Gelatin-SDS-PAGE showing the detection of extracellular proteases in Sporosarcina aquimarina (a) and Algoriphagus antarcticus (b) at pH and temperature conditions that better resolved the proteolytic profile, as described in Materials and Methods, in the absence (Control) or in the presence of different proteolytic inhibitors: 10 mM PMSF, 100 µM TLCK, 10 mM 1,10-phenanthroline (PHEN) and 10 mM EDTA. Molecular masses of the proteases, expressed in kiloDaltons, are represented on the left. The densitometric analysis of the proteolytic halos, expressed as the percentage of the remaining activity with respect to the control, is shown on the right.

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Detection of proteases from Sporosarcina aquimarina and Algoriphagus antarcticus isolated from Antarctic soil

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[1] Correspondence to: Marta Helena Branquinha E-mail: mbranquinha@micro.ufrj.br