title: In-Vitro Refolding and Characterization of Recombinant Laccase (CotA) From Bacillus pumilus MK001 and Its Potential for Phenolics Degradation.
abstract: Among lignocellulolytic enzymes, laccases are the most versatile, broadly specific, and largely studied enzyme with a wide range of biotechnological potential. Putative laccase (CotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli. In addition to soluble bioactive fraction, inactive inclusion body fraction was also harvested and refolded under optimized conditions resulting in 64 % of refolding efficiency. The enzyme was found to be thermostable exhibiting a half-life of 60 min at 80 °C. UV thermal CD spectra also supported the observation as about 9 % increase in β-sheets was recorded after thermal induction. The 3D CotA structure was constructed through homology modeling and the best selected model was verified through PROCHECK, ERRAT, Verify 3D, and PROSA servers. Final 3D model showed potential binding affinities with ferulic acid, caffeic acid, and vanillin. Results of the docking studies were further validated by HPLC analysis which signified the efficient bioconversion ability of CotA.
authors: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
authors: Sandeep
authors: S
authors: Kumar
authors: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
authors: Kavish Kr
authors: KK
authors: Jain
authors: Department of Biophysics, University of Delhi South Campus, New Delhi, 110021, India.
authors: Shikha
authors: S
authors: Rani
authors: Uttarakhand State Council for Science and Technology [UCOST], Vigyan Dham, Post Office - Jhajra, Dehradun, Uttarakhand, 248007, India.
authors: Kailash N
authors: KN
authors: Bhardwaj
authors: Department of Biophysics, University of Delhi South Campus, New Delhi, 110021, India.
authors: Manisha
authors: M
authors: Goel
authors: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. kuhad85@gmail.com.|Central University of Haryana, Jant-Pali Village, Mahendergarh District, Pali, Haryana, 123029, India. kuhad85@gmail.com.
authors: Ramesh Chander
authors: RC
authors: Kuhad
mesh_terms: D000069979:Bacillus pumilus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D042845:Laccase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010636:Phenols / Q000737:chemistry*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D010636:Phenols; D011994:Recombinant Proteins; D042845:Laccase
keywords: Bacterial laccase; Docking; Modeling; Phenolics degradation; Refolding