The Heterologous Expression And Application Of Endo-β-N-acetyglucosaminidase H From Streptomyces Avermitilis In Bacillus Subtilis

β-N-acetyl-glucosaminidase H(Endo H) is one of the important glucosidases. It cleaves the β-1,4-glycosidic bond of the N-acetylglucosamine core of oligosacchardes and leaves one N-acetylchitobiose attached to the asparagine residue of the glycoprotein. Endo H usually does not affect the biological function of the target protein, and is widely used in the research of glycomics.In this study, the gene for Endo H from Streptomyces avermitilis was amplified and cloned into the plasmid p MA0911.1. The recombined plasmid was further transformed into Bacillus subtilis WB600. Bacillus subtilis WB600 containing recombined plasmid was cultured at 37°C for 48 h. The fermentation broth was purified by hydrophobic chromatography and ionic exchange, and relatively 145 μg purified Endo H was obtained in 100 m L broth.In order to study the enzyme acticity and enzymatic properties, the optimum temperature, the optimum p H and the thermal stability of recombinant Endo H, and the effect of metal ions on the enzymatic activities were explored, using RNase B as the substrate. Our data showed that the enzyme acticity of the recombine Endo H was 90000 U×mg-1,the optimum temperature temperature was 40°C, the optimum reaction temperature was between 35°C-40°C,and recombinant Endo H keep high enzyme activity between p H 5.0-10. Recombinant Endo H showed good thermal stability from 35°C to 60°C, and in the condition containing 5 mmol×L-1various metal ions.In this study, to investigate the deglycosylation function of the recombinant Endo H on different glycoprotein, chicken ovalbumin standard glycoprotein(OVA), human breast cancer serum and proteins from human epidermal carcinoma A431 were used as substrate. We validated high deglycosylation efficiency of heterologous expressed Endo H, and we profiled the N-glycan structure of these three samples. Among thirteen identified N-glycans in OVA, eight structures were complex type N-glycans, and the others were high-mannose type Nglycans. In human breast cancer serum, eight N-glycans were identified, six strutures were highmannose type N-glycans, the others were complex type N-glycans. In A431 cells, five Nglycans were identified, and all strutures were high-mannose type N-glycans.