Purification, Characterization and Thermodynamic Assessment of an Alkaline Protease by Geotrichum Candidum of Dairy Origin

Document Type: Research Paper

Authors

1 Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan

2 Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan

3 Aliments Bioprocédés Toxicologie Environnement (ABTE), E.A. 4651, Université de Caen Basse-Normandie, Esplanade de la Paix, CAEN Cedex, France

4 Aliments Bioprocédés Toxicologie Environnement (ABTE), E.A. 4651, Université de Caen Basse-Normandie, Esplanade de la Paix, CAEN Cedex, France

Abstract

Background: Alkaline proteases is the important group of enzymes having numerous industrial applications including dairy food formulations.
Objectives: The current study deals with the purification and characterization of an alkaline serine protease produced by Geotrichum candidum QAUGC01, isolated from indigenous fermented milk product, Dahi.
Material and Methods: In total twelve G. candidum strains were screened for their proteolytic activity by using standard protease assay. The protease production from G. candidum QAUGC01 was optimized by varying physio-chemical conditions. The protease was purified by using two-step method: ammonium sulfate precipitation and gel filtration chromatography. Protease was further characterized by studying various parameter like temperature, pH, modulators, metal ions and organic solvent. A thermodynamic study was also carried out to explore the half-life of protease.
Results: The G. candidum grew profusely at 25 °C and at an initial pH of 4.0 for 72 h of incubation producing 26.21 U/mlmaximum extracellular protease. Protease revealed that Vmax and Km was 26.25 U.ml-1.min-1 and 0.05 mg.mL-1, respectively using casein as substrate. The enzyme was stable at a temperature range (25-45 ºC) and pH (8-9). Residual enzyme activity was strongly inhibited in the presence of PMSF (7.5%). The protease could hydrolyze proteinaceous substrates, casein (98%) and BSA (95%). The thermodynamic studies explored that the half-life of the enzyme that was 106.62 min, 38.72 min and 15.71 min at 50, 60 and 70 ºC, respectively.
Conclusions: Purified protease from G. candidum GCQAU01 is an ideal candidate for industrial application.

Keywords

Main Subjects


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