固定化胰蛋白酶的性质研究

Properties of immobilized trypsin

目的 测定固定化胰蛋白酶的催化特性和部分稳定性。方法 在相同的条件下,测定固定化和可溶性胰蛋白酶的活力,得其最适作用pH值和最适作用温度,再测定活力,得到各自的米氏常数(Km)值;测得两种状态酶的热稳定性和酸碱稳定性。同时测定了固定化胰蛋白酶的低温稳定性。结果 固定化和可溶性胰蛋白酶的最适作用pH值分别为8 .0～9. 0和7. 0～9.0 ,最适作用温度分别为6 0℃和5 5℃,Km值分别为2 . 32 6、1 42. 9mg·ml-1 ;固定化胰蛋白酶在5 5℃条件下,保温5h ,仍有78 92 %的活性;在4℃条件下,8d后,活性才略微下降,两周活性保留达90 %以上;在pH 9.0的活性未下降,在pH 7.0～1 0 .0范围内也比较稳定(相对活性>80 %)。结论 胰蛋白酶经固定化后,最适作用pH值范围变窄,最适作用温度和Km值升高;

OBJECTIVE To determine the catalytic properties and stabilities of immobilized trypsin. METHODS The activities of immobilized and soluble trypsins were determined in the same condition to get their optimum pH and temperature, and then in the optimum condition, their activities were determined to conclude their Km value. Further more, the low temperature stability was determined. RESULTS The optimun pH of immobilized and soluble trypsin were 8.0-9.0 and 7.0-9.0,optimum temperatures were 60℃ and 55℃,Km values were 2.326 and 1.429 mg·ml -1. Under 55℃, after 5 hours the relative activity of immobilized trypsin was 78.92%. Under 4℃, after 8 days the activity of immobilized trypsin started to decrease, and the relative activity was still over 90% at the end of the second week. Under the pH 9.0, after 72 hours the activity of immobilized trypsin didn't decrease, and betweeen pH 7.0 and 9.0 its stability was high(relative activity was over 80%). CONCLUSION After immobilization the optimum pH range of trypsin decreases, and its optimum temperature, Km value and stabilities are all higher than before.