Cloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp NCIM 5124)

TitleCloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp NCIM 5124)
Publication TypeJournal Article
Year of Publication2015
AuthorsRohamare, S, Gaikwad, SM, Jones, D, Bhavnani, V, Pal, J, Sharma, R, Chatterjee, P
JournalProcess Biochemistry
Volume50
Issue3
Pagination378-387
Date PublishedMAR
ISSN1359-5113
KeywordsActinomycetes, Cloning and expression, Kinetic stability, Protease, Thermal simulation
Abstract

A serine protease (N. protease), from Nocardiopsis sp., was cloned and expressed in Escherichia coli and investigated for its potential kinetic stability. Protein expression using two vectors, pET-22b (+) and pET-39b (+) was compared based on proper folding and soluble expression of the protein. pET-39b (+) was found to be a better vector for soluble expression of this protease containing disulfide bonds. In silico studies were also carried out for N. protease. Homology modeling suggested N. protease to be a member of PA clan of proteases. The phylogenetic analysis showed relatedness of N. protease to kinetically stable proteases. Molecular docking studies performed exhibited interaction of a peptide substrate with catalytic pocket of the enzyme. High temperature MD simulations were performed on N. protease to study its unfolding behavior and comparisons were made with alpha LP. A novel approach to study `cooperativity' of protein unfolding was undertaken, wherein `P' value analysis based on phi and psi values of the protein was performed. Data showed sharper P value transition for alpha LP when compared to N. protease thus indicating relatively less kinetic stability of N. protease. Present study holds significance as the non-streptomycete actinomycetes group is least explored and ensures industrially important enzymes with exceptional stabilities. (C) 2015 Elsevier Ltd. All rights reserved.

DOI10.1016/j.procbio.2014.12.025
Type of Journal (Indian or Foreign)

Foreign

Impact Factor (IF)2.529
Divison category: 
Biochemical Sciences
Physical and Materials Chemistry