Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern
Bo Pang, Jing He, Weijiao Zhang, Hao Huang, Yang Wang, Miao Wang, Guocheng Du, Zhen Kang
Abstract
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 Pichia pastoris under a methanol-induced promoter P AOX1 . By optimizing the composition of the C-terminal domain and fusing protein tags, we constructed a fusion mutant AP 2 -△491C with the extracellular hyaluronidase activity of 258.1 U·L −1 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 6 NA 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.