AUTHOR=Li Piwu , Wei Xiaofeng , Wang Yun , Liu Hui , Xu Yanpeng , Zhang Ziyang , Li Junlin , Wang Jianbin , Guo Chuanzhuang , Sui Songsen , Wang Junqing , Wang Ruiming 

TITLE=Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1242123

DOI=10.3389/fbioe.2023.1242123

ISSN=2296-4185

ABSTRACT=<p><bold>Background:</bold> Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance.</p><p><bold>Methods:</bold> The alkaline pectate lyase BspPel from <italic>Bacillus</italic> RN.1 was heterologously expressed in <italic>Escherichia coli</italic> BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored.</p><p><bold>Results:</bold> Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0–11.0). The specific enzyme activity after purification was 139.4 U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the <italic>V</italic><sub>max</sub> and <italic>K</italic><sub><italic>m</italic></sub> were 29 μmol/min. mL and 0.46 mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved.</p><p><bold>Conclusion:</bold> The modified BspPel-th has considerable potential for industrial applications with high pH processes.</p>