Effects Of Glycosylation On The Enzymatic Properties Of ?-Glucosidase Bgl3A Expressed In Pichia Pastoris

Glycosylation is one of the most universal and diverse post-translational modifications.It can affect protein secretion,stability,and immunogenicity.In nature,eukaryotic microbes secrete various glycoside hydrolases to deconstruct plant polysaccharides and lignin.These enzymes are mostly decorated with N/O-glycosylation.It has been reported that glycosylation has effects on enzyme activity and stability.In this study,the?-glucosidase bgl3A derived from Talaromyce leycettanus JCM12802was selected to be produced in three eukaryotic?Talaromyce leycettanus,Pichia pastoris and Humicola insolens?and one prokaryotic?Escherichia coli?expression systems.The enzyme properties were then compared.N/O-deglycosylation mutants were also constructed and expressed in P.pastoris,and the effects of glycosylation sites and glycosylation types were preliminarily analyzed.The main results are:?1?Enzymatic properties of recombinant?-glucosidase Bgl3A produced in four systemsThe?-glucosidase Bgl3A was successfully expressed in three eukaryotic and one prokaryotic expression systems and functionally characterized.SDS-PAGE analysis indicated different levels of N-and O-glycosylation occurred in the eukaryotic expression systems,and the glycosylation degree in H.insolens was lower than that of the other two systems.Their enzyme properties also varied,with the pH stability and cellobiose-catalyzing efficiency following the orders of T.leycettanus>E.coli>H.insolens>P.pastoris and H.insolens(91 s^–1mM^–1)>P.pastoris(89 s^–1mM^–1)>T.leycettanus(27s^–1mM^–1)>E.coli(12 s^–1mM^–1).The results showed that there are different degrees of post-glycosylation modifications in the expressed proteins of different systems.These post-glycosylation modifications have certain effects on the properties of the enzyme proteins.?2?Effect of N-glycosylation on the enzymatic properties of Bgl3AN-glycosylation mutants were constructed by site-directed mutagenesis,and their enzymatic properties were analyzed.In comparison with the wild type,the mutant S228A was low in protein secretion and had trace activity against pNPG,while mutants T44A and S299A showed similar pH and temperature optima,but had improved T_m values and thermostability at 70°C.When using cellobiose as the substrate,T44A and S299A retained almost the same catalytic efficiency.It suggested that N-glycosylation mainly affects the protein secretion and enzyme stability.Of the three N-glycosylation sites,N226 is critical for the proper folding of Bgl3A.?3?Effect of O-glycosylation on the enzymatic properties of Bgl3AO-glycosylation mutants were also constructed by site-directed mutagenesis,and their enzymatic properties were analyzed.Compared with the wild-type,all mutants had similar temperature and pH optima,but broader pH stability ranges?the order of T443A>T436A>T437A>Bgl3A?.Kinetic analysis indicated that the catalytic capabilities of mutant T443A on pNPG and cellobiose were significantly reduced,while the catalytic efficiencies of mutants T436A and T437A on cellobiose were increased.It suggested that O-glycosylation mainly affects the enzyme stability buffers.Of the three O-glycosylation sites,T443 plays a key role in maintaining the enzyme activity,and T436 and T437may have effects on the tertiary structure of Bgl3A and consequently on the catalytic efficiency.In summary,by comparing the enzymatic properties of?-glucosidases Bgl3A expressed in the four systems,effects of glycosylation on the?-glucosidase were preliminarily determined.Constructing glycosylation mutants and comparing the mutant enzymatic properties in expression of Pichia pastoris showed that N-glycosylation mainly affects Bgl3A catalytic efficiency and O-glycosylation mainly affects Bgl3A stability buffers.This provides a theoretical basis for the study of the effect of glycosylation on Bgl3A.