The Effect Of The N-Glycosylation From Windmill Palm Tree Peroxides (WPTP) On Its Function Using Computer-aided Design And Site-directed Mutagenesis

Palm tree peroxidase is highly stable,and it has more and more application in food industry.Academic research on peroxidase is gradually increasing,but there is little research about the windmill palm tree peroxidase(WPTP).In this paper,the effect of two-thirteenth glycosylation sites of WPTP was studied.Firstly,the optimized wild-type WPTP gene was inserted into Pichia pastor is GS115 and was induced expression.Next wild-type WPTP site-directed mutagenesis was done at N60 and N144 sites,which Asn was changed into Gln,then induced expression again in Pichia pastoris;secondly,three kinds of purified proteins were identified by MALDI-TOF MS,then the enzyme activity and substrate specificity were analyzed and compared,in order to determine the relationship between the two glycosylation sites and the functional properties of the enzyme;finally,homology modeling method was used to obtain the three-dimensional structure model of the above three enzymes,then molecular docking simulations were carried out between the enzymes and small molecules,which could study the effects of the two glycosylation sites on WPTP.The results obtained are as follows:1.Three recombinant strains were obtained successfully:GS115/pPICZaA-WT-WPTPY,GS115/pPICZaA-N60Q-WPTPY,GS115/pPICZaA-N144Q-WPTPY,which could be induced expression of wild-type and two mutant enzyme,then the enzymes were identified by SDS-PAGE and MALDI-TOF MS,and the result was accurate.2.Using TMB as the substrate,the activity of N60Q-WPTP was measured 50%less than WT-WPTP,and the activity of N144Q decreased nearly 80%comparing with WT-WPTP,which indicated that N60 and N144 sites played important roles in WPTP activity.N60 and N144 sites of wild-type WPTP were linked with sugar chains,and the enzyme activity was decreased seriously without those sugar chains.3.The three-dimensional structure model of WPTP molecule was obtained by homology modeling method for the first time.The model showed that both N60 and N144 sites are located near the Heme active pocket,and the latter is nearer.4.The analysis of experimental results and simulation results showed:The sugar chain attached to the N144 site near the active site played an active role in the binding of the substrate and the enzyme.Although the substrate didn't need to be bonded directly to the hydrophobic heme center,the substrate molecules still needed to approach to the amino acid residues which around the heme activity pocket to transfer electron,and the sugar chains like protractile tentacles of enzyme molecules could help the substrate molecules approach to the active pocket and complete electron transfer faster.This is a major advance in the study of the structure and functional relationship of the peroxidase in the palm tree,which provides an inspiration for the study of the role of glycosylation sites in the future.