Metagenomic alkaline protease from mangrove sediment

Autor: Natielle Cachoeira Dotivo, Tharcilla Braz Alves Pessoa, Ana Camila Oliveira Freitas, Thalis Ferreira dos Santos, Eric de Lima Silva Marques, João Carlos Teixeira Dias, Luiz C. Salay, Carlos Priminho Pirovani, Carla Cristina Romano, Ana C. S. Gonçalves, Rachel Passos Rezende
Rok vydání: 2017
Předmět:
0106 biological sciences
0301 basic medicine
Bacillus safensis
Geologic Sediments
Hot Temperature
medicine.medical_treatment
Bacillus
01 natural sciences
Applied Microbiology and Biotechnology
Substrate Specificity
03 medical and health sciences
chemistry.chemical_compound
Bacterial Proteins
Tandem Mass Spectrometry
010608 biotechnology
Endopeptidases
Enzyme Stability
medicine
Ammonium
Amino Acid Sequence
Thermostability
Enzyme Assays
chemistry.chemical_classification
Protease
biology
Temperature
General Medicine
Hydrogen-Ion Concentration
biology.organism_classification
Enzyme assay
Enzyme Activation
Molecular Weight
Kinetics
030104 developmental biology
Enzyme
chemistry
Biochemistry
Wetlands
Glutamyl aminopeptidase
biology.protein
Chromatography
Gel
Metagenome
Specific activity
Salts
Metagenomics
Sequence Alignment
Brazil
Biotechnology
Peptide Hydrolases
Zdroj: Journal of basic microbiology. 57(11)
ISSN: 1521-4028
Popis: Functional screening of metagenomic libraries is an important tool for the discovery of new molecules. The metabolic diversity of microorganisms enables survival in harsh environments and is related to the production of enzymes. In this study, we identified a protease-producing clone from a metagenomic library derived from mangrove sediment. The protease was purified by ammonium sulphate precipitation and gel filtration chromatography, with a yield of 77.27% and a specific activity of 8.57 U μg-1 . It had a molecular weight of approximately 70 kDa. MS/MS in ESI-Q-TOF revealed nine peptides similar to a peptidase of Bacillus safensis. The aligned partial sequence showed 47.48% identity and 82.74% similarity to the conserved domains of a glutamyl aminopeptidase from the human gut metagenome and 32.12% total coverage. The protease had an optimal pH of 8.5 and optimal activity at 60°C. At pH 9-12, its activity was greater than 80%. It had moderate thermotolerance and thermostability at temperatures of 40 and 50 °C. The KM and Vmax values were estimated to be 0.92 mg ml-1 , and 13.15 mmol min-1 for azocasein. Substrate specificity analysis showed that PR4A3 was active on gelatin, blood, egg yolk, and milk. These results support the potential use of PR4A3 in biotechnological applications.
Databáze: OpenAIRE
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