Design of mutants of GH11 xylanase from Bacillus pumilus for enhanced stability by amino acid substitutions in the N-terminal region: an in silico analysis

Abstract

GH11 xylanases are versatile small-molecular-weight single-polypeptide chain monofunctional enzymes. This family of glycoside hydrolases has important applications in food, feed and chemical industries. We designed mutants for improved thermal stability with substitutions in the first six residues of the N-terminal region and evaluated the stability in silico. The first six residues RTITNN of native xylanase have been mutated accordingly to introduce β structure, increase hydrophobic clusters and enhance conformational rigidity in the molecule. To design stable mutants, the approach consisted of constructing root mean square fluctuation (RMSF) plots of both mesophilic and thermophilic xylanases to check the localized backbone displacement maxima, identify the hydrophobic interaction cluster in and around the peaks of interest, construct mutants by substituting appropriate residues based on beta propensity, hydrophobicity, side chain occupancy and conformational rigidity. This resulted in the decreased number of possible substitutions from 19 to 6 residues. Introduction of conformational rigidity by substitution of asparagine residues at 5th and 6th residue position with proline and valine enhanced the stability. Deletion of N-terminal region increased the stability probably by reducing entropic factors. The structure and stability of GH11 xylanase and resultant mutants were analyzed by root mean square deviation, RMSF, radius of gyration and solvent accessible surface area analysis. The stability of the mutants followed the order N-del > Y1P5 >Y1V5 > ATRLM. The contribution of N-terminal end to overall stability of the molecule is significant because of the proximity of the C-terminal end to the N-terminal end which reinforces long-range interactions.

Communicated by Ramaswamy H. Sarma

Keywords:

• MD simulations
• GH11 xylanase
• hydrophobic interaction clusters
• protein unfolding
• root mean square fluctuation

Acknowledgements

Authors thank Mr. Krishna Bhat Kadappu, Managing Director and staff of KAYPEEYES Biotech private limited, R&D center, Mysuru, India, for their support and guidance while carrying out the research. Authors also thank Dr. Pradeep H., Department of Studies in Biotechnology, University of Mysore, Mysuru, India, for fruitful discussions.

Disclosure statement

All the authors have read the manuscript and have no conflict of interest.

Author contributions

AGA and KRK conceived the project, designed the experiments and critically evaluated and edited the manuscript, SKB performed the experiments and wrote the manuscript, KP assisted in the interpretation of the results.

Additional information

Funding

Authors gratefully acknowledge the financial support from KAYPEEYES Biotech private limited, Mysuru, India.