Abstract：Endo-β-N-acetylglucosaminidase (ENGase) can cleave the N-linked oligosaccharides of glycoprotein and is an essential tool enzyme for protein deglycosylation. This study obtained the genome data of Enterococcus faecalis from the NCBI database and amplified the ENGase gene endoE (endoglycosidase E) by PCR. Then the expression vector pET-28a-endoE was constructed by homologous recombination and transformed into Escherichia coli BL21 star (DE3) competent cells. The recombinant protein was efficiently expressed in E. coli, and the deglycosylation properties of EndoE was systematically analyzed. The results showed that recombinant protein was expressed in soluble form in E. coli, and the yield was 45.6 mg/L after purified by Co2+ affinity chromatography and anion-exchange chromatography. The deglycosylation activity analysis indicated that the recombinant EndoE could hydrolyze the N-linked glycan of both natural and denatured ribonuclease (RNase B), ovalbumin (Ova) and immune globulin G (IgG). Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) proved that recombinant EndoE could remove the N-linked oligosaccharides in RNase B. In addition, in the temperature range of 20~50 ℃ or pH range of 4.0~6.0, EndoE had ideal hydrolytic activity. It also had tolerance up to 100 mmol/L DL-dithiothreitol (DTT), 1 mol/L NaCl, and 2% Triton X-100. Compared to the other reported ENGase from Enterococcus faecalis, EndoE has a broader range of substrates and is a prospective tool enzyme in protein deglycosylation study.
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