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农业生物技术学报  2019, Vol. 27 Issue (6): 1118-1125    DOI: 10.3969/j.issn.1674-7968.2019.06.018
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Recombinant Expression and Catalytic Properties of EndoE from Enterococcus faecalis
HAN Xiao-Wei1, HUANG Yun-Na1,2, NIU Yi-Nan1, LI Xue-Jun1, XU Quan-Le1,*, CHEN Peng1,*
1 College of Life Sciences, Northwest A&F University, Yangling 712100, China;;
2 Conghua Middle School, Guangzhou 510900, China
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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.
Key wordsEndo-β-N-acetylglucosaminidase (ENGase)      N-glycosylation      Recombination expression      Enzymatic property     
Received: 19 December 2018     
ZTFLH:  S182  
  Q936  
Corresponding Authors: pengchen@nwsuaf.edu.cn   
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HAN Xiao-Wei
HUANG Yun-Na
NIU Yi-Nan
LI Xue-Jun
XU Quan-Le
CHEN Peng
Cite this article:   
HAN Xiao-Wei,HUANG Yun-Na,NIU Yi-Nan, et al. Recombinant Expression and Catalytic Properties of EndoE from Enterococcus faecalis[J]. 农业生物技术学报, 2019, 27(6): 1118-1125.
URL:  
http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2019.06.018     OR     http://journal05.magtech.org.cn/Jwk_ny/EN/Y2019/V27/I6/1118
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