Screening and characterization of the protease CP1 produced by the moderately halophilic bacterium Pseudoalteromonas sp. strain CP76.
A total of 26 proteolytic moderate halophiles were isolated and characterized. Most isolates were members of the genus Salinivibrio (16 strains), while others were identified as Bacillus (4 strains), Salinicoccus (2 strains), or members of the gamma-Proteobacteria (4 strains). Strain CP76 was selected as the best producer of an extracellular protease, designated CP1, and was used for further studies. Sequence analysis of the 16S rRNA gene in addition to phenotypic tests led to the placement of this organism in the genus Pseudoalteromonas. Maximal protease production was detected at the end of the exponential growth phase. This CP1 protease was purified and biochemically characterized, showing optimal activity at 55 degrees C, pH 8.5, and high tolerance to a wide range of NaCl concentrations (0 to 4 M NaCl). The most interesting features of this enzyme are its moderate thermoactivity, its activity at a range of pH values (6-10), and, especially, its salt tolerance (optimal activity at 7.5% total salts). The purified protease has a molecular mass of 38 kDa, and the N-terminal amino acid sequence determined showed similarity to metalloproteases previously described. The protease activity was strongly inhibited by EDTA, PMSF, and Pefabloc. No significant inhibition was detected with E-64, bestatin, chymostatin, or leupeptin. These results suggest that Pseudoalteromonas sp. strain CP76 produces an extracellular metalloprotease moderately thermotolerant and stable at high salt concentrations.
Sánchez-Porro
Cristina
C
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Seville, Spain.
Mellado
Encarnación
E
Bertoldo
Costanzo
C
Antranikian
Garo
G
Ventosa
Antonio
A
D004275:DNA, Ribosomal / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D010641:Phenotype; D017434:Protein Structure, Tertiary; D031161:Pseudoalteromonas / Q000201:enzymology*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*; D012492:Salts / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D004275:DNA, Ribosomal; D007477:Ions; D012336:RNA, Ribosomal, 16S; D012492:Salts; D012965:Sodium Chloride; D010450:Endopeptidases