Method for purifying nattokinase
A purification method and technology of nattokinase, applied in the direction of transferase and the like, can solve the problems of difficult operation, cumbersome process of nattokinase, low purity and the like, and achieve the effects of convenient operation, high product purity and simple process
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Embodiment 1
[0017] 1. Fermentation broth pretreatment
[0018] The Bacillus natto fermentation liquid was centrifuged at 4° C. at a speed of 8000 rpm for 10 min with a high-speed refrigerated centrifuge, and the supernatant was collected. The supernatant was filtered with a 0.45 μm filter membrane, and the filtered supernatant was collected and used as the upper column liquid for later use.
[0019] 2. Purification by Ion Exchange Chromatography
[0020] The gel used in ion exchange chromatography is CM Sepharose Fast Flow, and the column specification is 3.5 cm in inner diameter and 20 cm in height; the gel is equilibrated successively with pH 6.8, phosphate buffer containing 70 mM and 6 mM; Concentration (conductivity is adjusted to 1.8mS / cm); the filtered supernatant with adjusted ion concentration is added to the column at a speed of 10mL / min; the column bed is first washed with pH6.8, 6mM phosphate buffer, and then washed with pH6 .8. Linear gradient elution with an eluent containi...
Embodiment 2
[0024] 1. Fermentation broth pretreatment
[0025] Nattokinase was centrifuged at a speed of 12,000 rpm for 5 min at 10° C. in a high-speed refrigerated centrifuge, and the supernatant was collected. The supernatant was filtered with a 0.2 μm filter membrane, and the filtrated liquid was collected and used as the column sample solution for later use.
[0026] 2. Purification by Ion Exchange Chromatography
[0027] The gel used in ion-exchange chromatography is CM Sepharose Fast Flow, and the column specification is 5.5 cm in inner diameter and 30 cm in height; the gel is firstly and then equilibrated with phosphate buffer containing pH 6.8, containing 70 mM and 6 mM; adjust the filtered supernatant The ion concentration (conductivity is adjusted to 2.0mS / cm); the filtered clear solution with adjusted ion concentration is added to the column at a speed of 15mL / min; the column bed is first washed with pH6.8, 6mM phosphate buffer, Then use pH 6.8, containing 6mM sodium dihydrog...
Embodiment 3
[0031] 1. Fermentation broth pretreatment
[0032] The Bacillus natto fermentation liquid was cooled to 15° C. by the interlayer cooling method of the fermenter, centrifuged at a speed of 16000 rpm with a high-speed tube centrifuge, and the supernatant was collected. The supernatant was filtered with a 0.2 μm filter membrane, and the filtrated liquid was collected and used as the column sample solution for later use.
[0033] 2. Purification by Ion Exchange Chromatography
[0034] The gel used in ion-exchange chromatography is CM Sepharose Fast Flow, and the column specification is 16 cm in inner diameter and 38 cm in height; the gel is equilibrated with pH 6.0, containing 70 mM and 6 mM phosphate buffer first and then; adjust the prepared Filter the ionic concentration of the supernatant (adjust the conductivity to 2.2mS / cm), put it into a plastic bucket, add 2L of CM Sepharose FastFlow gel that has been balanced, adjust the pH to 6.0, stir and absorb at a slow speed with a ...
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