Engineering non-conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34 for improved thermal stability: an in-silico evaluation

Abstract

GH11 xylanases are commercially important enzymes for degradation of xylan fibers. We have identified the presence of nine non-conserved and five conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34. We have designed two sets of mutants viz., (1) substitution mutants in which non-conserved charged amino acid residues have been replaced with appropriate hydrophobic residues based on side chain occupancy and hydrophobicity and (2) deletion mutants where non-conserved charged residues have been deleted. The stability of the mutants has been evaluated in-silico by analyzing the contributions of non-covalent interactions like hydrophobic interaction clusters and salt bridges. The stability of the resultant mutants was evaluated using parameters such as radius of gyration, solvent accessible surface area, root mean square deviation, root mean square fluctuations and protein unfolding measurements using molecular dynamic simulations. The deletion of certain charged residues resulted in mutants having lowered radius of gyration and decreased surface areas. However, RMSD and RMSF measurements indicated lowered stability in comparison to substitution mutants. Of the substitution mutants, the SBM 3 was the most stable mutant as indicated by Rg, SASA, RMSF and simulated protein unfolding measurements. The major contributing factors for improved stability could be strengthening of hydrophobic interactions in the GH11 xylanase from B. pumilus. These in-silico stability measurements of salt bridge mutants may lead to better design of GH11 xylanases for commercial applications.

Communicated by Ramaswamy H. Sarma

Keywords:

• MD simulations
• GH 11 xylanase
• conserved and non-conserved salt bridges
• protein unfolding

Acknowledgment

Authors gratefully acknowledge the financial support and microbial cultures from KAYPEEYES Biotech private limited, Mysuru, India. Authors thanks Mr. Krishna Bhat Kadappu, Managing Director and staff of KAYPEEYES Biotech private limited, 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.

Authors Contribution

KRK and AGA 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.