A1 Journal article (refereed)
Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase (2020)
Sharma, V., Kumar, R., Sharma, V. K., Yadav, A. K., Tiirola, M., & Sharma, P. K. (2020). Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase. Molecular Biology Reports, 47(1), 533-544. https://doi.org/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
Publication open access: Not open
Publication channel open access:
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
JUFO rating: 1
