Li Pi-Wu, Ma Jun, Wei Xiao-Feng, Zhang Zi-Yang, Wang Rui-Ming, Xiao Jing, Wang Jun-Qing
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, 250353, Shandong, Republic of China.
Key Laboratory of Shandong Microbial Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250353, Shandong, Republic of China.
AMB Express. 2022 Oct 9;12(1):130. doi: 10.1186/s13568-022-01472-0.
Alkaline pectate lyase has developmental prospects in the textile, pulp, paper, and food industries. In this study, we selected BacPelA, the pectin lyase with the highest expression activity from Bacillus clausii, modified and expressed in Escherichia coli BL21(DE3). Through fragment replacement, the catalytic activity of the enzyme was significantly improved. The optimum pH and temperature of the modified pectin lyase (PGLA-rep4) were 11.0 and 70 °C, respectively. It also exhibited a superior ability to cleave methylated pectin. The enzyme activity of PGLA-rep4, measured at 235 nm with 0.2% apple pectin as the substrate, was 554.0 U/mL, and the specific enzyme activity after purification using a nickel column was 822.9 U/mg. After approximately 20 ns of molecular dynamics simulation, the structure of the pectin lyase PGLA-rep4 tended to be stable. The root mean square fluctuation (RMSF) values at the key catalytically active site, LYS168, were higher than those of the wildtype PGLA. In addition, PGLA-rep4 was relatively stable in the presence of metal ions. PGLA-rep4 has good enzymatic properties and activities and maintains a high pH and temperature. This study provides a successful strategy for enhancing the catalytic activity of PGLA-rep4, making it the ultimate candidate for degumming and various uses in the pulp, paper, and textile industries.
碱性果胶裂解酶在纺织、制浆、造纸和食品工业中具有发展前景。在本研究中，我们选择了来自克劳氏芽孢杆菌的表达活性最高的果胶裂解酶BacPelA，在大肠杆菌BL21(DE3)中进行改造和表达。通过片段替换，该酶的催化活性得到显著提高。改造后的果胶裂解酶(PGLA-rep4)的最适pH和温度分别为11.0和70℃。它还表现出优异的裂解甲基化果胶的能力。以0.2%苹果果胶为底物，在235nm处测得的PGLA-rep4的酶活性为554.0U/mL，用镍柱纯化后的比酶活为822.9U/mg。经过约20ns的分子动力学模拟，果胶裂解酶PGLA-rep4的结构趋于稳定。关键催化活性位点LYS168处的均方根波动(RMSF)值高于野生型PGLA。此外，PGLA-rep4在金属离子存在下相对稳定。PGLA-rep4具有良好的酶学性质和活性，能在高pH和高温下保持稳定。本研究为提高PGLA-rep4的催化活性提供了成功策略，使其成为制浆、造纸和纺织工业中脱胶及各种用途的最终候选酶。
