微小毛霉凝乳酶的分离纯化研究

Isolation and Purification of Chymosin from Mucor pusillus

研究微小毛霉(HL-1)凝乳酶的分离纯化条件及方法。研究酶的最适浸提温度、酶的浸提pH值和最适浸提时间,探讨离子浓度、加水量对浸提效率的影响,利用高速冷冻离心法、有机溶剂沉淀法,膜分离法和层析法等对粗酶液进行了分离。利用光谱法对纯化样品进行检测。酶的最适浸提温度为30℃;最适pH为6.0;浸提10 h活力最高;1%的氯化钠有利于酶的分离,加水比例为15时有利于提取,在10 000 r/min下离心10min澄清效果最好,95%的酒精沉淀效果最好,利用0.2μm的微滤膜可除去发酵液中的菌体,8 000的超滤膜可拦截凝乳酶蛋白,S300的填料可有效分离凝乳酶,纯度达95%以上。

Conditions and methods to isolate and purify chymosin from Mucor pusillus（HL-1） were studied.The optimum extraction temperature,pH value and time of the enzyme were studied.The effects of ion concentration and the amount of water added on the extraction efficiency were investigated;high-speed refrigerated centrifuge,organic solvent precipitation,membrane separation and chromatographic methods were used to separate the crude enzyme liquid.The results showed that The optimum extraction temperature was at 30 ℃;pH 6.0;the activity of the enzyme was the highest when extracted for 10 hours;1% of sodium chloride was conducive to the separation of the enzyme,water adding ratio at 15 was conducive to the extraction,centrifuge at 10 000 r/min for 10 min had the best clear effect,95% alcohol had the best precipitation effect,using 0.2 micron filter membrane could remove the thalamus in the fermentation broth,8 000 ultra filter membrane could completely block out chymosin protein,and filler S300 could effectively separate the chymosin with over 95% of purity.Therefore the optimum isolation and purification conditions of chymosin from Mucor pusillus（HL-1） were obtained.