A1 Journal article (refereed)
Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase (2020)


Sharma, Vinay; Kumar, Rajender; Sharma, Vishal Kumar; Yadav, Ashok kumar; Tiirola, Marja; Sharma, Pushpender Kumar (2020). Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase. Molecular Biology Reports, 47 (1), 533-544. DOI: 10.1007/s11033-019-05159-x


JYU authors or editors


Publication details

All authors or editors: Sharma, Vinay; Kumar, Rajender; Sharma, Vishal Kumar; Yadav, Ashok kumar; Tiirola, Marja; Sharma, Pushpender Kumar

Journal or series: Molecular Biology Reports

ISSN: 0301-4851

eISSN: 1573-4978

Publication year: 2020

Volume: 47

Issue number: 1

Pages range: 533-544

Publisher: Springer Netherlands

Publication country: Netherlands

Publication language: English

DOI: https://doi.org/10.1007/s11033-019-05159-x

Open Access: Publication channel is not openly available


Abstract

In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2 mg of BRPD with specific activity of 6.25 U mg−1 from 600 ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.


Keywords: bioremediation; enzymes; oxidoreductases; harmful substances; pesticides; aromatic hydrocarbons; molecular dynamics

Free keywords: metagenomics; dioxygenase; aromatic hydrocarbons; pesticides; pollutant; molecular modelling


Contributing organizations


Ministry reporting: Yes

Reporting Year: 2020

Preliminary JUFO rating: 1


Last updated on 2020-09-07 at 23:12