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

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.

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.

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 ß 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.