ABSTRACT
A thermostable keratinase designated as KBALT was purified from Bacillus altitudinis RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in Escherichia coli. Recombinant KBALT (rKBALT) when purified using a single step Ni-NTA His affinity chromatography achieved a yield of 38.20% and a 76.4-fold purification. Comparison of the deduced amino acid sequence of rKBALT with known proteases of Bacillus species and inhibitory effect of PMSF suggest that rKBALT was a subtilisin-like serine protease. Both native and rKBALT exhibited higher activity at 85 °C and pH 8.0 in the presence of Mg2+, Mn2+, Zn2+, Ba2+ and Fe3+ metal ions. Interestingly, 70% of their activity was retained at temperatures ranging from 35 to > 95 °C. The keratinolytic activity of both nKBALT and rKBALT was enhanced in the presence of reducing agents. They exhibited broad substrate specificity towards various protein substrates. KBALT was determined for its kinetic properties by calculating its Km (0.61 mg/ml) and Vmax (1673 U/mg/min) values. These results suggest KBALT as a robust and promising contender for enzymatic processing of keratinous wastes in waste processing plants.
ABSTRACT
BACKGROUND: 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. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. RESULTS: Wild 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. CONCLUSIONS: The 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.
