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
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.