Study On The Separation, Purification, Stability And Activity Improvement Of ?-glucosidase Based On Elastin-like Protein

In this study,a recombinant beta-glucosidase?Glu-ELP-His?labled with elastin-like Polypeptide?ELP?-?VPGVG?₅₀ tag and His-tag?6xHis?was expressed and purified by inverse transition cycling?ITC?depend on thermal phase denaturation of ELP tag.Then we evaluated the purification efficiency by comparing with the traditional affinity chromatography method.The effects of ELP labeling on the kinetic parameters,stability and enzyme activity of Glu were studied,and the effects of immobilization on the kinetic parameters,stability and enzyme activity of Glu and GEH were also studied.The results are as follows:Firstly,the recombinant?GEH?with ELP tag and His-tag was whole-gene synthesized.The recombinant expression vector was confirmed by PCR and double enzyme digestion,and the correct recombinant expression vector was verified by sequencing.Secondly,the fusion protein GEH was purified by ITC using thermal denaturation of ELP tag.The optimum salt ion and concentration of ITC for purification of GEH was confirmed to be 0.5 M?NH₄?₂SO₄.Compared with commercial purification material non-magnetic nickel column High Affinity Ni-NTA Resin,the specific activity of purified GEH by ITC was?75.50 U/mg?,which was slightly higher than that of non-magnetic nickel column?68.22.U/mg?.The recovery rate of total purification activity of purified GEH by ITC was?76.09%?,which was slightly lower than that of non-magnetic nickel column?77.35%?.the efficiency of repeated purification of GEH by ITC showed that the specific activity of CEH in the second round was?76.22 U/mg?,which was slightly higher than a round of the ITC?75.50 U/mg?,and the recovery rate of the total activity in the second round was?64.82%?,which was slightly lower than that in the first round of ITC?76.09%?.Thirdly,ELP labeling did not affect the kinetic parameters and enzyme activity of Glu,but significantly improved the stability of the enzyme.The affinity of GEH to p-NPG was?Km=5.27±0.5 mM?,which was higher than that of Glu?Km=5.73±0.4mM?.The catalytic efficiency of p-NPG was?Kcat/Km=14.79±0.5 S-1mM-1?higher than that of Glu?Kcat/Km=12.10±0.7 S-1mM-1?.The residual activity of GEH was 80%and 50%of the original activity after incubated at 60??70?for 30 minutes,while the residual activity of Glu was almost undetectable after 30 minutes at 60??70?.The residual enzyme activity of GEH and Glu at 25?for 28 days were 70%and 30%respectively,which indicated that the thermal stability and storage stability of GEH were higher than that of Glu.The ELP label did not change the secondary structure of the Glu enzyme,but the results showed that the stability of the secondary structure of GEH was higher than that of Glu under the treatment of protein denaturant reagent?guanidine hydrochloride and urea?.Finally,it showed that the immobilization of the enzyme could improve the stability and tolerance of the enzyme.In this paper,Glu and GEH were immobilized on magnetic nanoparticles Fe₃O₄/PMG/IDA-Ni²⁺and magnetic nickel column HisPurTMNi-NTA Magnetic Beads with histidine label,and the kinetic parameters,stability and enzyme activity of immobilized Glu and Glu?GEH?with ELP label were compared.The bounds of Glu and GEH on the magnetic nanoparticles Fe3O4/PMG/IDA-Ni2 were 90 mg/g and 170 mg/g,respectively,while those of Glu and GEH bound on the HisPur Ni-NTA Magnetic Beads were 150 and 220mg/g,respectively.The Michaelis constant of the immobilized enzyme was slightly higher than that of the free enzyme,but the catalytic efficiency was maintained.In terms of stability,the effect of free GEH was similar to that of immobilized Glu enzyme;The immobilization of GEH did not significantly improve the stability of free GEH.