| There are many ways to modify glycosylation of proteins,of which o-glycosylation is an important one.In order to study the effect of O-glycosylation on protein,the analysis of sugar chain structure is essential.In order to analyze the sugar chain,the first step is to remove the sugar chain from the protein.At present,the method of dissolving the sugar chain from the protein is hydrazine solution ?-elimination,biodissociation and enzymatic hydrolysis.The first three methods have obvious limitations,and the enzymatic hydrolysis can avoid these shortcomings,so the study of N-acetylglucosidase(endo-?-N-acetylgalactosaminidase)is of great significance.At present,there are few studies on endo-?-N-acetylgaiactosaminidase,and there are few applications for the commercialization of this enzyme.Therefore,explore of high substrate specificity recombinant endo-?-N-acetylgalactosaminidase which can be expressed in e.coli is of great significance.The analysis of the sugar chain structure and its biological functions in O-glycosylation modification research is keen interest.In view of the current problems existing in the research of endo-?-N-acetylgalactosaminidase,this experiment using biological engineering technology from the human gut microbes Tyzzerella nexilis to discover new endo-?-N-acetylgalactosaminidase,its activity and biochemistry test.1.In this study,three encoding endo-?-N-acetylgalactosaminidase genes were found in Tyzzerella nexilis(DSM 1787).Molecular cloning of these three genes was carried out,and the recombinant expression vector was constructed,and the recombinant expression vector was induced into E.coli BL21(DE3).A relatively large amount of recombinant endo-?-N-acetylgalactosaminidase can be obtained in a short period of time under certain culture and induction conditions.The recombinant endo-?-N-acetylgalactosaminidase was isolated and purified by Ni-NTA,and the results of SDS-PAGE showed that the recombinant endo-?-N-acetylgalactosaminidase with relatively high purity could be obtained.2.After induced expression,the enzyme crude extract obtained from the strain E.coli BL21(DE3)was based on pNP-core 1,and the enzyme activity was preliminarily detected according to the color reaction.Enzyme by nickel(Ni-NTA)affinity column,purified using different O-glycan structure to the substrate(pNP-core 1,pNP-core 2,pNP-alpha-Ga1NAc,pNP-beta-Ga1NAc,pNP-alpha-galactose and pNP-beta-galactose)determination of its enzyme specificity,Tn0153 and Tn2105 have activity to the pNP-core 1,but for Tn1787 not.Tn2105 was only specific to pNP-core 1,while Tn0153 was active to pNP-core 1,and it was also active to pNP-a1pha-GalNAc,but with low activity.3.With improved bradford kit to measure the restructuring of the purified enzyme in the enzyme protein content,and then each use quantitative restructuring of endo-?-N-acetylgalactosaminidase and pNP-core 1 to determine biochemical characteristics inherent in enzyme.According to the experimental results,Tn0153 and Tn2105 have the highest activity in acid environment,and the optimal pH is pH4.0.Tn0153 and Tn2105 optimum reaction temperature 50?.Cu2+completely inhibited the activity of Tn0153 and Tn2105,and the activity of Tn0153 was completely inhibited with the addition of Zn2+.Each addition had a certain effect on Tn0153 and Tn2105 activity.It was found that SDS and imidazole were the most obvious inhibitors to the activity of these two enzymes.It is important to note that the final concentration of 200mM imidazole completely inhibits the activity of Tn0153.To sum up,the successful cloning of the two N-acetylglucosidase genes of Tyzzerella nexilis and using the recombinant expression system of escherichia coli successfully obtained the enzyme with high substrate specificity.This study provides an important tool enzyme for the analysis of O-glycosylated protein glycosylation chains,and lays an important foundation for the study of the effect of sugar chain on the function of O-glycosylation modified protein. |
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