Abstract：Endo-N-acetyl-β-glucosaminidase (Endo/ENGase, EC3.2.1.96) is a kind of glycohydrolase which recognizes and cleaves the β-1,4-glycosidic bonds of N,N′-acetylglucosamine in core structure of N-linked glycans. It is widely used in analyzing N-glycosylation of protein. In order to characterize the enzymatic properties of endo-β-N-acetylglucosaminidase from Enterococcus faecalis, the endo-N-acetyl-β-glucosaminidase gene endoEf of Enterococcus faecalis was cloned by PCR and the prokaryotic expression vector pET-28a-endoEf was constructed using seamless cloning technology. The catalytic properties and pivotal catalytic residues of EndoEf were analyzed on the basis of recombinant expression and purification. The results showed that EndoEf was efficiently expressed in Escherichia coli BL21 Star (DE3) in soluble form, and that the target protein was successfully purified from bacteria solution by one step Co2+ affinity chromatography with a yield of 202.1 mg per liter. The specific activity of EndoEf was measured as 1.0×104 U/mg using ribonuclease B (RNaseB) as substrate. EndoEf belonged to the glycoside hydrolase family 18 (GH18) which contained conservative motifs DXDXE. In addition, both natural or denatured RNaseB and ovalbumin (Ova) were able to be hydrolyzed by EndoEf. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) further confirmed the hydrolysis on N-linked sugar chains in RNaseB. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of EndoEf was 30.2 kD. Enzymatic properties analysis showed that the optimum temperature and pH range of EndoEf were 40 ℃ and 5.0~7.0, and EndoEf was relatively more stable at 40~50 ℃ and pH 7.0~9.0. EndoEf had salt-tolerance up to 1 mol/L NaCl, and remained full activity under either condition of 100 mmol/L DTT, 2% SDS and Triton X-100. Three mutants D125N, D127N and E129Q of EndoEf were constructed by site-directed mutagenesis method. Activity analysis showed that E129Q was almost inactive, and D127N lost most of its activity, while D125N had no significant change in activity. It could be concluded that the glutamic acid at position 129 was essential for catalytic reactions. The establishment of recombinant expression system and analysis of enzyme properties may lay a foundation for the practical application of EndoEf on glycoprotein study.
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