Pang Bo, He Jing, Zhang Weijiao, Huang Hao, Wang Yang, Wang Miao, Du Guocheng, Kang Zhen
School of Food Science and Technology, Jiangnan University, Wuxi, China.
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Front Bioeng Biotechnol. 2022 May 13;10:885888. doi: 10.3389/fbioe.2022.885888. eCollection 2022.
Hyaluronidases are a group of glycosidases catalyzing the degradation of hyaluronic acid (HA). Because of the advantages of effectively hydrolyzing the HA-rich matrix and low immunogenicity, human hyaluronidase PH20 (hPH20) is widely used in the medical field. Here, we realized the active expression of recombinant hPH20 by under a methanol-induced promoter P. By optimizing the composition of the C-terminal domain and fusing protein tags, we constructed a fusion mutant AP-△491C with the extracellular hyaluronidase activity of 258.1 U·L in a 3-L bioreactor, the highest expression level of recombinant hPH20 produced by microbes. Furthermore, we found recombinant hPH20 hydrolyzed the β-1,4 glycosidic bonds sequentially from the reducing end of o-HAs, with HA as the smallest substrate. The result will provide important theoretical guidance for the directed evolution of the enzyme to prepare multifunctional o-HAs with specific molecular weights.
透明质酸酶是一类催化透明质酸（HA）降解的糖苷酶。由于能有效水解富含HA的基质且免疫原性低，人透明质酸酶PH20（hPH20）在医学领域得到广泛应用。在此，我们通过甲醇诱导型启动子P实现了重组hPH20的活性表达。通过优化C末端结构域的组成并融合蛋白标签，我们在3-L生物反应器中构建了具有258.1 U·L胞外透明质酸酶活性的融合突变体AP-△491C，这是微生物产生的重组hPH20的最高表达水平。此外，我们发现重组hPH20从o-HAs的还原端依次水解β-1,4糖苷键，其中HA是最小的底物。该结果将为酶的定向进化以制备具有特定分子量的多功能o-HAs提供重要的理论指导。
