Effects Of N-glycosylation On The Biochemical Properties Of Recombinant BEK_L Expressed In Pichia Pastoris

Glycosylation is one of the most complicated and ubiquitous post-translational modifications that may affect the function and structure of enzyme.Enterokinase,a type II serine protease found in the duodenum,is a frequently-used tool enzyme.To study the effects of N-glycosylation on the biochemical properties of bEK_L,the enzyme was deglycosylated via site-directed mutagenesis.Firstly,the recombinant bEK_L was treated with Endo H,and the molecular mass of recombinant bEK_L showed a band shift from 43 kDa to 34 kDa on SDS-PAGE,which means the bEK_L expressed in Pichia pastoris is a glycoprotein.The analysis of amino acid sequences indicated that the bovine enterokinase light chain(bEK_L)contains three putative N-glycosylation sites at N64,N103 and N165,which were located on the surface of the protein in random coil.Then,we constructed a series of glycosylation-deficient mutants,including single-site N-glycan-deficient mutants(N64Q,N103Q,N165Q)and double-site mutants(MD1,MD2,MD3),as well as the triple-site mutant(MT).Through fermentation,dialysis,affinity chromatography and desalting,the purified glycosylated and deglycosylated bEK_L were obtained.The biochemical properties of wild type with deglycosylated bEK_L were compared,and the results showed that the deglycosylation did not affect the protein secretion level in P.pastoris,but it is important to the enzymatic activity.The N64Q increase the affinity of enzyme and substrate,thus increased the specific activity and improved its catalytic efficiency.Moreover,the glycosylated bEK_L is more thermostable than the wilt type,and the removal of N-linked glycan chains will lead to decreased thermostability,commensurate with the number of glycan chains available.Structural analysis of glycosylated and deglycosylated bEK_L revealed that the removal of N-glycosylation did not have pronounced changes on the secondary structure but there was a significant difference in the tertiary structure.In conclusion,this study demonstrated that the effects of glycosylation at different degrees and sites in bEK_L were diverse.Moreover,this work will provide theoretical support for designing enzymes on the basis of N-glycosylation to meet the demands of the biochemical industry.