AUTHOR=Pang Bo , He Jing , Zhang Weijiao , Huang Hao , Wang Yang , Wang Miao , Du Guocheng , Kang Zhen 

TITLE=Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 10 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.885888

DOI=10.3389/fbioe.2022.885888

ISSN=2296-4185

ABSTRACT=Hyaluronidases are a group of glycosidases catalyzing the degradation of hyaluronic acid (HA). Because the advantages of effectively hydrolyzing the HA-rich matrix and low immunogenicity, human hyaluronidase PH20 (hPH20) is widely used in medicine field. Here, we realized the active expression of recombinant hPH20 by Pichia pastoris under a methanol-induced promoter PAOX1. Through optimizing the composition of C-terminal domain and fusing protein tags, we constructed a fusion mutant AP2-△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 HA6NA as the smallest substrate. The result will provide important theoretical guidance for directed evolution of the enzyme to prepare multi-functional o-HAs with specific molecular weights.