Abstract
BackgroundThe aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates.ResultsWild type and mutated genes were cloned and overexpressed in BL21. Optimum pH and temperature of rBAxn exhibited pH 9.0 and 50 °C respectively and it was stable up to 215 h. Along with the physical properties of rBAxn, kinetic parameters (Km 19.34 ± 0.72 mg/ml; kcat 6449.12 ± 155.37 min− 1 and kcat/Km 333.83 ± 6.78 ml min− 1 mg− 1) were also compared with engineered enzymes. Out of five mutations, W63A, Y128A and W144A lost almost 90% activity and Y124A and W187A retained almost 40–45% xylanase activity.ConclusionsThe site-specific single mutation, led to alteration in substrate specificity from xylan to CMC while in case of double mutant the substrate specificity was altered from xylan to CMC, FP and avicel, indicating the role of aromatic residues on substrate binding, catalytic process and overall catalytic efficiency.
Highlights
The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis
The present study aims to identify the role of key aromatic amino acid residues present in the substrate binding pocket
Based on the reported structure of the enzyme in a substrate bound state (Fig. 3(a)) in the Protein Data Bank (PDB) (ID: 2B46), using PyMoL [19] we identified aromatic amino acids for point mutations which could lead to a change in substrate binding properties of the enzyme
Summary
The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis These residues are highly conserved and play an important role in substrate binding through steric hindrance. Lignocellulose is the principal and most plentiful component of the renewable biomass produced by photosynthesis and is synthesized at an estimated rate of some 200 billion tons per year [1] These biopolymers are the major stored carbon source in the nature and are composed of cellulose (40%), hemicellulose (20–30%) and lignin (20–30%) [2]. Cellulase enzymes access the glucose polymer cellulose while xylanases act on xylose polymer xylan and both enzymes share a common mechanism of action [5] This is the reason why many xylanases are found to be bifunctional in nature. The difference lies in the architecture of the substrate binding site consist of many aromatic amino acid residues and the orientation of amino acids in the binding
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