化学修饰木瓜蛋白酶的固定化及性质研究

Study on the Immobilization of the Chemically Modified Papain and its Properties

在底物保护和无底物保护下,用丁二酸酐对木瓜蛋白酶进行化学修饰,以三硝基苯磺酸法测定修饰酶的平均氨基修饰度,以棉布为载体,戊二醛为交联剂,对修饰前后的木瓜蛋白酶分别进行固定化。考察了温度、pH和表面活性剂SDS对化学修饰的固定化木瓜蛋白酶活力的影响,并与固定化天然木瓜蛋白酶进行了比较。研究表明,化学修饰固定化木瓜蛋白酶的最适反应温度为80℃;最适pH为9.0;在SDS浓度为20mg/mL时酶活也仍能保持在40%左右;米氏常数为187g/L。与天然的固定化酶相比,化学修饰的固定化木瓜蛋白酶的热稳定性、耐碱性和耐洗涤性得到了显著提高。

Under the substrate protecting and non-substrate protecting papain was modified by using succinic anhydride. The average ratio of modified-NH2 was measured by acid （TNBS） method. The modified papain was immobilized on the cotton fabric with the aid of glutaraldehyde. The factors related with the activity of the immobilized modified papain, such as temperature, pH and the concentration of SDS were studied and compared with native immobilized unmodified papain. The results showed that the optimal reaction temperature and pH of immobilized modified papain were 80℃ and 9.0, respectively. When the concentration of SDS was 20mg/mL, the activity of the immobilized modified papain maintained about 40%. The Km of immobilized chemical modified papain was 187g/L. Compared with immobilized nature papin, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were improved obviously.