Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides  

作　　者：Lizhi Liao Hao Huang Yang Wang Guocheng Du Zhen Kang 

机构地区：[1]The Science Center for Future Foods,Jiangnan University,Wuxi 214122,China [2]The Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China [3]The Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China

出　　处：《Engineering Microbiology》2023年第4期11-19,共9页工程微生物学（英文）

基　　金：supported by the National Natural Science Foundation of China(32000058);the Jiangsu Province Natural Science Fund for Distinguished Young Scholars(BK20200025);the National Key Research and Development Program of China(2021YFC2103100).

摘　　要：Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

关 键 词：HYALURONAN Leech hyaluronidase Yeast display BIOCATALYSIS Enzyme immobilization 

分 类 号：Q55[生物学—生物化学]