Hu Weixiong, Liu Xiaoyun, Li Yufeng, Liu Daling, Kuang Zhihe, Qian Chuiwen, Yao Dongsheng
Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China.
Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China; Department of Bioengineering, Jinan University, Guangzhou City, Guangdong Province 510632, China.
Enzyme Microb Technol. 2017 Feb;97:82-89. doi: 10.1016/j.enzmictec.2016.11.005. Epub 2016 Nov 14.
β-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant β-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation. The mutant enzymes were expressed in Pichia pastoris SMD1168, and their thermal stability, pH stability, trypsin resistance and pepsin resistance were determined. Two mutant MAN47 enzymes, g-123 and g-347, were glycosylated as expected when expressed in yeast, and their thermal stability, pH stability, and protease resistance were significantly improved compared to the wild-type enzyme. An enzyme with multiple stability characterizations has broad prospects in practical applications, and the rational design N-glycosylation strategy may have applications in simultaneously improving several properties of other biotechnological targets.
β-甘露聚糖酶已广泛应用于食品和饲料加工等行业，因此一直是生物技术开发的目标酶。在本研究中，我们试图通过合理设计的N-糖基化来提高来自蜜环菌的重组β-甘露聚糖酶MAN47的稳定性和蛋白酶抗性。基于同源建模、分子对接、二级结构分析和糖基化可行性分析，将增强的芳香序列引入特定的MAN47环区域以促进N-糖基化。突变酶在毕赤酵母SMD1168中表达，并测定其热稳定性、pH稳定性、胰蛋白酶抗性和胃蛋白酶抗性。两种突变型MAN47酶g-123和g-347在酵母中表达时按预期进行了糖基化，与野生型酶相比，它们的热稳定性、pH稳定性和蛋白酶抗性得到了显著提高。具有多种稳定性特征的酶在实际应用中具有广阔前景，合理设计的N-糖基化策略可能在同时改善其他生物技术目标的多种特性方面具有应用价值。
