Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (km and Vmax). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze–thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.
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This work was supported by the Zhejiang Provincial Natural Science Foundation (No. LQ19C050001), Xinmiao Talents Program (2018R420012), Ningbo Public Service Platform for High-Value Utilization of Marine Biological Resources (NBHY-2017-P2), Zhejiang Provincial Top Key Discipline (ZS2018003), and the Zhejiang Wanli University Research & Development Funding.
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Zhou, Y., Cao, J., Sun, X. et al. Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization. World J Microbiol Biotechnol 36, 33 (2020). https://doi.org/10.1007/s11274-020-2809-4
- Rumen microbe
- Molecular cyclization