College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, 315100, China.
College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
World J Microbiol Biotechnol. 2020 Feb 14;36(2):33. doi: 10.1007/s11274-020-2809-4.
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 (k and V). 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.
食草动物瘤胃中的微生物群是挖掘糖苷水解酶用于木质纤维素生物质生物炼制的重要目标。我们之前从中国本土高繁殖品种湖羊的瘤胃液 fosmid 文库中分离出一种活性很高但热稳定性差的木聚糖酶（LXY）。在这项研究中，我们使用了一种称为 SpyTag/SpyCatcher 分子环化的通用酶工程策略，通过异肽键介导的连接来提高 LXY 的稳定性。线性和环化的 LXY（分别为 L-和 C-LXY）具有相似的最适 pH 和温度、pH 稳定性和动力学常数（k 和 V）模式。然而，C-LXY 比 L-LXY 具有更好的热稳定性、离子稳定性以及对聚集和冻融处理的恢复能力，而不影响其催化效率。圆二色性、内源和 8-苯胺-1-萘磺酸结合荧光分析表明，环化酶比线性酶更能保持其二级和三级结构。综上所述，这些结果促进了环化酶在饲料、食品、纸浆和生物能源等行业的潜在应用。
