Insight into multicopper oxidase laccase from Myrothecium verrucaria ITCC-8447: a case study using in silico and experimental analysis

Abstract

The oxidation activity of multicopper-oxidases overlaps with different substrates of laccases and bilirubin oxidases, thus in the present study an integrated approach of bioinformatics using homology modeling, docking, and experimental validation was used to confirm the type of multicopper-oxidase in Myrothecium verrucaria ITCC-8447. The result of peptide sequence of M. verrucaria ITCC-8447 enabled to predict the 3 D-structure of multicopper-oxidase. It was overlapped with the structure of laccase and root mean square deviation (RMSD) was 1.53 Å for 533 and, 171 residues. The low binding energy with azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (-5.64) as compared to bilirubin (-4.39) suggested that M. verrucaria ITCC-8447 have laccase-like activity. The experimental analysis confirmed high activity with laccase specific substrates, phenol (18.3 U/L), ampyrone (172.4 U/L) and, ampyrone phenol coupling (50 U/L) as compared to bilirubin oxidase substrate bilirubin (16.6 U/L). In addition, lowest binding energy with ABTS (-5.64), syringaldazine SYZ (-4.83), guaiacol GCL (-4.42), and 2,6-dimethoxyphenol DMP (-4.41) confirmed the presence of laccase. Further, complete remediation of two hazardous model pollutants i.e., phenol and resorcinol (1.5 mM) after 12 h of incubation and low binding energy of −4.32 and, −4.85 respectively confirmed its removal by laccase. The results confirmed the presence of laccase in M. verrucaria ITCC-8447 and its effective bioremediation potential.

Graphical Abstract

Keywords:

• Bilirubin oxidase
• docking
• laccase
• phenol
• resorcinol

Additional information

Funding

The authors acknowledge the support of Dr. Jata Shankar, Genomics Laboratory, Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan, India for the Bioinformatical Studies. The authors are also thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India, (Grant No. BT/304/NE/TBP/2012 and BT/PR7333/PBD/26/373/2012). The authors are also thankful to DST-FIST (Grant No. SR/FST/LSI-676/2016) for the infrastructure facility at the Department of Microbiology, CURAJ. The authors would also thank IIT Bombay, SAIF for HR-LCMS Orbitrap, Agilent Technologies, Centre of Excellence Manesar, Gurugram India for QTOF – LCMS, and Department of Chemistry, CURAJ for HPLC. The authors would also like to acknowledge the financial support provided by CURAJ Ajmer, India.