Desheng introduced the purification method of neuraminidase
Release time:
2020-12-30
Neuraminidase is the common name for Acetylneuraminyl Hydrolase (sialidase). Its pH stability is 4.0-9.5, specific activity: ≥400U/mg, the enzyme can catalyze the hydrolysis of glycoprotein and oligosaccharides α2-3, α2-6 and α2-8 bond N-acetylneuraminic acid residues. Basically, Desheng Technology will introduce the purification method of neuraminidase for you below. This purification method includes the following steps.
(1) Ultrafiltration concentration: the fermentation broth h 淸 1~20U of the urea-producing joint rod post was selected for membrane ultrafiltration, and the concentrated solution after ultrafiltration was diluted to 0.5~10L with buffer A;
(2) Ammonium sulfate precipitation: slowly add 226~516g money sulfate, stir for 20~4()m1ndW for 1~5h, centrifuge at 5000~15000g for 10~30m1n, remove the supernatant, dissolve the precipitation to 100~1000ml with buffer B, stir After full dialysis overnight;
(3) DEAE Sepharose FF column chromatography: 1) After the RAE Sepharose FF column is equilibrated with buffer B, sample the dialyzed sample for h, equilibrate with buffer B, elute with buffer C, collect and wash Take off the sample
⑷Phenyl Sepharose FF column chromatography: Add (NH 4) 2 S0 4 to the collected elution sample to make the final concentration reach 0.6~().8M, then Phenyl Sepharose FF column Equilibrate with buffer 丨), load the eluted sample added with / (NH 4) 2 S0 4, equilibrate with buffer 丨), wash with buffer E, and elute with buffer F;
The eluted sample obtained in step (4) was subjected to ultrafiltration and concentration, and then exchanged with buffer B, and then lyophilized. The above operations were carried out at 0~5°C; the above buffer A, B, C, 1 ), E, F are:
Buffer A: 10 ~100mM Tr1s-HCL, 100 ~500mM NaCL, pH6. 5 ~8.5;
Buffer B: 10 〜100mM Tr1 s-HCl, pH6. 5 〜8.5;
Buffer C: 10 〜100mM Tr1s-HCL, 10 〜1000mM NaCl, PH6. 5 〜8.5;
Buffer D: 10 〜l00mM Tr1s_HCl, 0.7 〜0. 9M(NH4) 2 SO4, PH6. 5 〜8.5;
Buffer E: 10 〜l00mM Tr1s_HCl, 0.4 〜0. 6M(NH4 )2 SO4, PH6. 5 〜8.5;
Buffer F: 10~100mM Tr1s-HCl, 0.2~0.4M(NH4)2 SO4, PH6.5~8.5;
The above is the purification method of Acetylneuraminyl Hydrolase introduced by Desheng, which includes ultrafiltration concentration, ammonium sulfate precipitation, DEAE sepharose FF column chromatography, phenyl sepharose FF column chromatography, and liquid exchange and lyophilization. And other steps. The above operations are all carried out at 0~5°C. The neuraminidase purified by this method has high purity and high substrate specific binding ability. The neuraminidase obtained by this method is used to detect the content of human serum sialic acid with higher accuracy.
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