College of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China.
College of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China.
Enzyme Microb Technol. 2019 May;124:79-83. doi: 10.1016/j.enzmictec.2019.01.010. Epub 2019 Jan 30.
Pullulanase is a starch-debranching enzyme that is generally employed to efficiently break down starch for the production of high-glucose syrup. Acidic adaptation of pullulanases is of special interest. In this study, we conducted protein engineering to improve the acidic adaptation of Bacillus acidopullulyticus pullulanase (BaPul) and used a hydrogen-bond-based approach to identify promising residues that may change the deprotonation constants (pK) of the catalytic residues. A total of 19 amino acids were selected for mutation according to the crystal structure of BaPul. The pH optimum of the L627R mutant shifted from 5.0 to 4.0, and its relative activity at pH 4.0 was 117% that of the wide-type enzyme. The improved efficacy of the L627R mutant at pH 4.0 was confirmed by kinetic parameters and pK prediction. Moreover, the L627R mutant exhibited increased tolerance against acid-mediated denaturation, and its maximum d-glucose content (97.4%) was obtained after 40 h incubation, which is shorter by 10 h compared with the time required by the wide-type enzyme to produce a comparable amount of the monosaccharide. The L627R mutant may be suitable for industrial application because its shortened reaction time translates to reduced energy consumption.
普鲁兰酶是一种淀粉分支酶，通常用于高效分解淀粉以生产高葡萄糖浆。酸性适应是普鲁兰酶的一个特别关注点。在这项研究中，我们进行了蛋白质工程改造，以提高解淀粉芽孢杆菌普鲁兰酶（BaPul）的酸性适应能力，并采用氢键方法来确定可能改变催化残基去质子化常数（pK）的有前途的残基。根据 BaPul 的晶体结构，总共选择了 19 个氨基酸进行突变。L627R 突变体的最适 pH 从 5.0 转移到 4.0，其在 pH 4.0 时的相对活性是野生型酶的 117%。动力学参数和 pK 预测证实了 L627R 突变体在 pH 4.0 时的效果更好。此外，L627R 突变体对酸介导的变性具有更高的耐受性，其最大 d-葡萄糖含量（97.4%）在 40 小时孵育后获得，比野生型酶生产可比量单糖所需的时间缩短了 10 小时。L627R 突变体可能适合工业应用，因为其缩短的反应时间意味着降低了能源消耗。
