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[目的]优化枯草杆菌重组水蛭素的分离纯化工艺。[方法]通过系列预处理和初步层析和精细纯化试验。发酵液固液分离和预处理:大批的发酵液采用连续流离心,取上清液,滴加50%三氯乙酸,当pH值达到2.2～2.5时停止滴加,静置1 h,离心,取上清液,用饱和NaOH调pH值回中性。然后利用SYNDER-UF202型超滤装置进行超滤浓缩脱盐,先用孔径为0.1μm的滤膜芯进行微滤除大固粒,再用截留分子量为1 000 Da的超滤膜芯进行超滤浓缩,加蒸馏水重复超滤浓缩脱盐。然后在等电点pH 4.0左右进行6倍体积的乙醇沉淀浓缩。离子交换初步层析:最佳pH值8.0,以Tris-HCl缓冲体系为佳;强阴离子交换选用Q Sepharose F.F.介质;系统电导率6 ms/cm,水蛭素的最大上样量以每毫升介质240 ATU为佳,流速1 ml/min;优化工艺在强阴离子交换HiPrep 16/10Q上放大。Sephacryl S-100凝胶过滤柱纯化水蛭素:在一定流速范围内,流速对纯化效果影响不大,上样量可在10ml左右。[结果]优化的分离纯化路线为:大量发酵液→离心→三氯乙酸处理(91%回收率)→再离心→超滤浓缩脱盐(80%回收率)→乙醇沉淀浓缩(82%回收率)→强阴离子交换(90%回收率)→硫酸氨沉淀浓缩(90%回收率)→Sepharcyl S-100凝胶过滤(回收率93%)→纯品(总回收率=91%×80%×82%×90%×90%×93%=45%,纯度95.1%)。[结论]可为进一步工业化分离纯化水蛭素研究提供借鉴。
[Objective] The research aimed to optimize the separation and purification of recombinant hirudin by Bacillus subtilis. [Method] Through a series of pretreatment and preliminary chromatography and fine purification experiments. Fermentation broth solid-liquid separation and pretreatment: a large number of fermentation broth by continuous flow centrifugation, the supernatant was added dropwise 50% trichloroacetic acid, when the pH reached 2.2 ~ 2.5 stop dropping, standing 1 h, centrifuged, Take the supernatant, with saturated NaOH pH value back to neutral. Then use the SYNDER-UF202-type ultrafiltration device for ultrafiltration concentration desalination, first with a pore size of 0.1μm filter core microfiltration large solids removed, and then cut off the molecular weight of 1 000 Da ultrafiltration membrane core ultrafiltration concentration , Add distilled water repeated ultrafiltration concentration desalination. Then 6 times the volume of ethanol precipitation concentration at the isoelectric point of about pH 4.0. Ion exchange preliminary chromatography: the best pH 8.0, Tris-HCl buffer system is better; strong anion exchange using Q Sepharose FF medium; system conductivity 6 ms / cm, the maximum load of hirudin per ml of medium 240 ATU is preferred at a flow rate of 1 ml / min; the optimization process is amplified on a strongly anion-exchanged HiPrep 16 / 10Q. Purification of hirudin by Sephacryl S-100 gel filtration column: The flow rate has little effect on the purification effect within a certain flow rate, and the sample loading can be about 10ml. [Result] The optimized separation and purification routes were as follows: a large amount of fermentation broth → centrifugation → trichloroacetic acid treatment (91% recovery) → re-centrifugation → ultrafiltration concentration desalination (80% recovery) → ethanol precipitation concentration (82% recovery) → Strong Anion Exchange (90% recovery) → Ammonium Sulfate Precipitation Concentration (90% Recovery) → Sepharcyl S-100 Gel Filtration (Recovery 93%) → Pure Product (Total Recovery = 91% × 80% × 82 % × 90% × 90% × 93% = 45%, purity 95.1%). [Conclusion] This study can provide reference for the further industrialization of isolation and purification of hirudin.