期刊
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
卷 122, 期 2, 页码 131-139出版社
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2015.12.021
关键词
Alternaria solani; Homology modeling; Protease production; Protease purification; Matrix-assisted laser desorption and ionization time of flight/mass spectrometry; Metalloprotease
资金
- University Research Studentship, UGC-India, Bharathidasan University, Tamilnadu, India
- KU Brain Pool of Konkuk University, Seoul, Republic of Korea
The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A. solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0-10.0 and a temperature range of 45-50 degrees C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na+, K+) had little effect. Metal ions such as MgSO4, CaCL2, KCL at 10 mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A. solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5 lcDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-beta-sheets, and 9-alpha-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His(57), His(130), His(169), and Cys(123)). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.
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