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

Rohamare, Sonali, Gaikwad, Sushama, Jones, Dafydd ORCID: https://orcid.org/0000-0001-7709-3995, Bhavnani, Varsha, Pal, Jayanta, Sharma, Ranu and Chatterjee, Prathit 2015. Cloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp. NCIM 5124). Process Biochemistry 50 (3) , pp. 378-387. 10.1016/j.procbio.2014.12.025

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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 αLP. A novel approach to study ‘cooperativity’ of protein unfolding was undertaken, wherein ‘P’ value analysis based on φ and ψ values of the protein was performed. Data showed sharper P value transition for αLP 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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	Elsevier
ISSN:	1359-5113
Date of Acceptance:	22 December 2014
Last Modified:	09 Nov 2022 10:23
URI:	https://orca.cardiff.ac.uk/id/eprint/139305

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