基因重组蛋清溶菌酶的分离纯化

Isolation and purification of recombinant egg white lysozyme

以毕赤氏基因重组酵母发酵液为研究对象,经过离心分离、超滤、膜分离、阳离子交换树脂吸附后,得到的纯化液,真空干燥得到酶活为20000U·mg^(-1)的蛋清溶菌酶粉末。研究结果表明:发酵液在5000r/min、25℃下离心15min,得到离心液溶菌酶活性为360.1U·mL^(-1),菌体细胞的去除率达99.99%;超滤最佳操作条件:压力为0.15MPa,pH为6.5;一级超滤得到的溶菌酶活性为455.4U·mL^(-1),收率为90.6%,纯化程度提高了23.7%;二级超滤得到的溶菌酶活性为648U·mL^(-1),收率为61.2%,纯化程度提高了42.3%; D152阳离子交换树脂对料液进行离子交换层析,得溶菌酶的酶活性为770.5U·mL^(-1),收率达90%,纯化程度提高了18.9%。

After centrifugation,ultrafiltration,membrane separation and cation exchange resin adsorption,the purified solution obtained from Pichia pastoris yeast fermentation broth with Pichia pastoris gene was dried in vacuum to obtain the lysozyme powder with enzyme activity of 20000U mg^-1.The results showed that the fermentation broth was centrifuged at 5000r/min and 25℃or 15min,and the lysozyme activity of the centrifuge was 360.1U ·mL^-1.The removal rate of the cells was 99.99%.The optimal operating conditions for ultrafiltration were :pressure was 0.15MPa and pH value was 6.5;lysozyme activity obtained by first-stage ultrafiltration was 455.4U·mL^-1,the yield was 90.6%,and the purification degree increased by 23.7%;lysozyme activity obtained by second-stage ultrafiltration was 648U·mL^-1,yield was 61.2%,and the purification degree increased by 42.3%;D152 cation exchange resin for ion exchange chromatography,the enzyme activity of lysozyme was 770.5U·mL^-1,the yield was 90%,and the purification degree increased by 18.9%.