Xia Bingbing, Ma Dai, Ye Zifan, Yang Jingwen, Zhang Hongbin, Hu Xueqin
School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, Anhui, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Sep 25;40(9):3072-3082. doi: 10.13345/j.cjb.230896.
Dextranase is an enzyme that specifically hydrolyzes the α-1, 6 glucoside bond. In order to improve the activity of dextranase from KQ11, site-directed mutagenesis was used to modify the amino acids involved in the "tunnel-like binding site". A saturating mutation at position 507 was carried out on this basis. The mutant enzymes A356G, S357W, W507Y, and W507F with improved enzyme activities and catalytic efficiency were successfully obtained. Compared with wild type (WT), W507Y showed the specific activity increasing by 3.00 times, the value increasing by 3.62 times, the value decreasing by 54%, and the catalytic efficiency (/) increasing by 8.98 times. The three-dimensional structure analysis showed that the increase of the number of hydrogen bonds and the distance between "tunnel-like binding sites" were important factors affecting enzyme activity. Compared with WT, W507Y had a shortened distance from the residues on the other side of the "tunnel-like binding site", which made it easier to generate hydrogen binding forces. Accordingly, the substrate hydrolysis and product efflux were accelerated, which dramatically increased the enzyme activity and catalytic efficiency.
葡聚糖酶是一种特异性水解α-1,6糖苷键的酶。为了提高来自KQ11的葡聚糖酶的活性，采用定点诱变来修饰“隧道样结合位点”中涉及的氨基酸。在此基础上对第507位进行饱和突变。成功获得了酶活性和催化效率提高的突变酶A356G、S357W、W507Y和W507F。与野生型（WT）相比，W507Y的比活性提高了3.00倍，Km值提高了3.62倍，Kcat值降低了54%，催化效率（Kcat/Km）提高了8.98倍。三维结构分析表明，氢键数量的增加和“隧道样结合位点”之间的距离是影响酶活性的重要因素。与WT相比，W507Y与“隧道样结合位点”另一侧的残基距离缩短，这使得更容易产生氢键结合力。因此，底物水解和产物流出加速，从而显著提高了酶活性和催化效率。
