Method for concentrating hirudin in fermentation broth
A technology of hirudin and fermentation broth, applied in the field of biomedicine, can solve the problems of large consumption of ultrafiltration membrane, high concentration cost, low protein recovery rate and the like, and achieve the effects of cost reduction, short concentration time and simple operation.
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Embodiment 1
[0026] 100L tank high-density fermentation, centrifuged to obtain hirudin fermentation broth volume 35L, secreted hirudin expression: 1.2mg / ml, protein activity: 16800IU / ml.
[0027] Pretreatment of reversed-phase chromatographic packing: take 2500 g of reversed-phase chromatographic packing, particle size 20 μm, pore size Put it in a 15000m1 container, add 7500ml of 95% ethanol and shake well, put it in an ultrasonic cleaner and vibrate for 15min, put it into a Buchner funnel and filter it to remove ethanol, rinse it with 10000ml of distilled water and 10000ml of distilled water with a pH of 2.8, drain it and store it at 4°C for later use.
[0028] Take 2100 g of the pretreated reversed-phase chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth, and stir it magnetically for 20 min. After standing still for 1.5 hours, the reversed-phase chromatography packing completely settled, and the supernatant was removed by siphoning, and the precipi...
Embodiment 2
[0033] High-density fermentation in 100L tank, centrifuged to obtain hirudin fermentation broth volume 38L, secreted hirudin expression: 1.18mg / ml, activity: 16100IU / ml.
[0034] Pretreatment of reversed-phase chromatographic packing: take 2500 g of reversed-phase chromatographic packing, particle size 20 μm, pore size Put it in a 15000m1 container, add 7500ml of 93% ethanol, shake well, put it in an ultrasonic cleaner and vibrate for 20min, add it into a Buchner funnel and filter to remove ethanol, rinse with 10000ml of distilled water and 10000ml of distilled water with a pH of 3.2, and then drain and store at 5°C for later use.
[0035]Take 2300g of the pretreated reversed-phase chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth, and stir it magnetically for 15 minutes. After standing still for 2 hours, the reversed-phase chromatography packing completely settled, and the supernatant was removed by siphoning, and the precipitate was s...
Embodiment 3
[0040] 100L tank high-density fermentation, centrifuged to obtain hirudin fermentation broth volume 40L, secreted hirudin expression: 1.08mg / ml, protein activity: 15800IU / ml.
[0041] Pretreatment of reversed-phase chromatographic packing: take 2500 g of reversed-phase chromatographic packing, particle size 20 μm, pore size Put it in a 15000m1 container, add 7500ml of 96% ethanol to shake well, put it in an ultrasonic cleaner and vibrate for 20min, put it into a Buchner funnel and filter to remove ethanol, rinse with 10000ml of distilled water and 10000ml of distilled water with a pH of 3.0, and then drain and store at 6°C for later use.
[0042] Take 2400g of the pretreated reversed-phase chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth, and stir it magnetically for 20 minutes. After standing still for 2 hours, the reversed-phase chromatography packing completely settled, and the supernatant was removed by siphoning, and the precipita...
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