可溶性固定化木瓜蛋白酶的制备及其性质研究

Preparation and Properties of Soluble Immobilized Papain

目的制备一种可溶性固定化木瓜蛋白酶并对其部分酶学性质进行研究。方法将木瓜蛋白酶偶联在亲水性高分子聚合物羧甲基纤维素醋酸琥珀酸酯(AS-L)上制备得到水溶性固定化木瓜蛋白酶。在相同的条件下测定游离的木瓜蛋白酶和水溶性固定化木瓜蛋白酶的活力,得到两种状态的酶的最适作用pH值和最适作用温度,再于它们各自的最适条件下测定活力,得到各自的Km值;在相同的条件下测定两种状态的酶的热稳定性、酸碱稳定性、低温稳定性和回收稳定性。结果游离木瓜蛋白酶和可溶性固定化木瓜蛋白酶的最适作用pH值分别为6.0和5.0,最适作用温度分别为60℃和70℃,Km值分别为2.53、3.07mg·ml^(-1);可溶性固定化木瓜蛋白酶在60%条件下,保温12h,仍有62%的活性;在4℃条件下,30d后活性保留达90%以上;在pH6.0时稳定性最好,在pH4.0～7.0范围内也比较稳定(相对活性>80%)。结论可溶性固定化木瓜蛋白酶能够在pH5.5以上的溶液中完全溶解,最适作用pH值范围变窄,最适作用温度和Km值升高;热稳定性和酸碱稳定性均明显增强。

Objective To prepare a soluble immobilized papain and determine its enzymatic properties. Methods The soluble immobilized papain was obtained by conjugating the papain with the hydrophilic high molecular polymer carbexylmethylcellulose acetic succinate. Under the same condition, the activities of immobilized and soluble papains were determined to conclude their optimum pH and temperature, then under these respective optimum conditions, their activities were determined to conclude their Km value. At the same time, the thennndynamic stability, the acid- base stability, the low temperature stability and the operational stability were respectively detected. Results The optimun pH values of soluble and immobilized papain were 6.0 and 5.0. Their optimum temperature were 60℃ and 70℃. Their Km values were 2.53 and 3.07mg·ml^-1. Under 60℃, after 12 hours the relative activity of immobilized papain was 62%. Under 4℃, after 30 days the relative activity was still over 90%. Under the pH 6.0, after 72 hours the activity of immobilized papain didn＇t decrease, and betweeen pH 4.0 and 7.0 its stability was high （relative activity was over 80% ）. Conclusion Soluble immobilized papain can fully solubilize above the pH 5.5. The optimum pH range of papain was narrow, and its optimum temperature and Km value were all higher than before. Its thermodynamtic stability and the acid- base stability were also strengthened.