枯草芽孢杆菌中性蛋白酶的固定化研究

Studies on Immobilization of Neutral Protease fromBacillus subtilis

DOI：10.3969/j.issn.1673-1689.2019.10.014

中文关键词: 枯草芽孢杆菌中性蛋白酶 葡萄糖酸冼必泰 固定化酶

英文关键词: neutral protease,chlorhexidine,immobilized enzyme

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中文摘要:

采用酰胺交联法将葡萄糖酸冼必泰修饰在载体上，并优化了修饰后的载体对中性蛋白酶的结合条件及催化活性的影响。结果表明，葡萄糖酸冼必泰对载体的修饰条件及修饰后的载体对中性蛋白酶的固定条件为：葡萄糖酸冼必泰的添加量为0.095 mmol、树脂活化时间为1 h、载体羧基与1-（3-二甲氨基丙基）-3-乙基碳二亚胺（EDC）摩尔比为1∶3、中性蛋白酶的添加量为600 U，酶活化时EDC的添加量为13.7 mg。在此条件下，固定化中性蛋白酶的酶活性可达398 U/g树脂。经傅立叶红外分析，枯草芽孢杆菌中性蛋白酶成功的交联到修饰有亲和配基的亲和树脂载体上。经过固定后，固定化酶的米氏常数Km值为2.9 mg/mL，游离酶的Km值为1.7 mg/mL，随意固定化酶的Km值为7.1 mg/mL。

英文摘要:

Chlorhexidine was modified on carrier by cross-linking protocol and the effects of modified carriers on neutral proteases were studied. The reaction conditions were optimized. The additive amount of Chlorhexidine was 0.095 mmol；the activate time of resins was 1 h；the molar ratio of carboxyl to EDC was 1∶3；the addition of neutral protease was 600 U and the addition of EDC was 13.7 mg on the process of activating enzymes. The maximal neutral protease activity was 398 U/g resin. The neutral protease was cross-linked to the affinity carrier through the Fourier transform infrared detection. TheKmvalues of immobilized，free and randomized immobilized neutral protease were 2.9，1.7 and 7.1 mg/mL，respectively.

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