P. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel University, Sardar Patel Maidan, Bakrol-Vadtal Road, PO Box 39, Vallabh Vidyanagar, Gujarat, 388 120, India.
P. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology (CHARUSAT), Changa, Anand, Gujarat, India.
BMC Biotechnol. 2018 Feb 13;18(1):9. doi: 10.1186/s12896-018-0420-7.
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.
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 (K 19.34 ± 0.72 mg/ml; k 6449.12 ± 155.37 min and k/K 333.83 ± 6.78 ml min mg) 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.
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.
通过同源建模和序列分析研究了来自解淀粉芽孢杆菌的木聚糖酶酶的底物结合口袋的芳香残基 W187、Y124、W144、Y128 和 W63 在底物结合中的作用。这些残基高度保守，通过空间位阻在底物结合中起重要作用。用丙氨酸取代这些残基可以使酶容纳非特异性底物。
野生型和突变型基因在 BL21 中克隆和过表达。rBAxn 的最适 pH 和温度分别为 9.0 和 50°C，并且稳定至 215 h。除了 rBAxn 的物理性质外，还比较了动力学参数（K 19.34±0.72 mg/ml；k 6449.12±155.37 min 和 k/K 333.83±6.78 ml min mg）与工程酶。在这五个突变中，W63A、Y128A 和 W144A 失去了近 90%的活性，而 Y124A 和 W187A 保留了近 40-45%的木聚糖酶活性。
定点单突变导致从木聚糖到 CMC 的底物特异性改变，而在双突变体中，底物特异性从木聚糖改变到 CMC、FP 和 Avicel，表明芳香残基在底物结合、催化过程和整体催化效率中的作用。
