Abstract Enzymatic deglycosylation is an important means to study the structure and function of glycoproteins. Traditional enzymatic deglycosylation mostly uses free enzymes, so that the residual deglycosylase becomes a potential source of contamination signals for subsequent glycoprotein analysis (such as mass spectrometry analysis). To explore new utilization methods of deglycosylation is of great value for the analysis of glycoprotein glycan chain structure. In this study, the recombinant endo-β-N-acetylglucosaminidase (EndoEf) of Enterococcus faecaliswas expressed and purified, using sepharose CL 6B activated by epichlorohydrin as the carrier to immobilize, and the catalytic characteristics of immobilized EndoEf were analyzed. The results showed that EndoEf could be efficiently expressed in Escherichia coli BL21 Star (DE3), and 186.5 mg of enzyme protein could be purified per liter of bacterial solution. The optimal conditions for immobilization of EndoEf were 4.0 mg enzyme per gram of carrier in 50 mmol/L phosphate buffer at pH 8.0, 12 h conjugated at 4 ℃. Immobilized enzymes could remove N-glycan chains of ribonuclease B (RNase B) and ovalbumin (Ova), remaining active when stored at 4 ℃ for 60 d. Immobilized EndoEf had higher activity in the range of 30~40 ℃ and pH 6.0~7.0. The tolerance to DL-dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) was significantly improved than that of the free enzyme, which could tolerate denaturation conditions of 12.0 mmol/L DTT and 2.0% SDS, and tolerate 1.0 mol/L NaCl. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that centrifugation completely removed immobilized EndoEf from the reaction system. The establishment of immobilization method and the analysis of catalytic characteristics could provide a basis for the application of immobilized EndoEf in glycoprotein research.
WANG Lu,MU Si-Yu,WANG Yun-Xiao, et al. Immobilization and Enzymatic Characterization Analysis of Endo-β-N-acetylglucosaminidase (EndoEf)[J]. 农业生物技术学报, 2024, 32(3): 655-665.
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