3α-羟类固醇脱氢酶的亲和纯化及化学修饰提高酶稳定性

Affinity Purification of 3α-Hydroxysteriod Dehydrogenase and Enzyme Stability Improvement by Chemical Modification

研究了3α-羟类固醇脱氢酶(3α-HSD)经化学修饰后的稳定性变化。已构建的基因工程菌E.coli BL21(DE3)/p ET28a-hsd经诱导表达、镍柱亲和纯化得到电泳纯的3α-HSD酶,酶蛋白得率是16.5%,活性回收率为64.7%,纯化倍数约为3.8,15%SDS-PAGE结果显示3α-HSD酶为单一条带,相对分子质量在28 000左右。采用邻苯二甲酸酐(PA)作为修饰剂,对3α-HSD纯酶进行化学修饰,与对照组相比,修饰酶抵抗p H波动的能力有所增强,50℃水浴10 min后酶的热稳定性明显提高,37℃贮藏40 h后修饰酶的贮藏稳定性比原酶提高9倍。

The stability change of 3α-hydroxysteroid dehydrogenase(3α-HSD) after chemical modification was studied. The purified 3α-HSD was obtained by a induced expression of constructed genetic engineering bacteria E.coli BL21(DE3)/p ET28a-hsd at first and then by an affinity purification using Ni2+-Sepharose column with 16.5% of protein yield,64.5% of recovery yield and3.8 times of specific activity. The SDS-PAGE(15%,reducing conditions) analysis showed that3α-HSD was composed of a single polypeptide of ~28 k Da. The chemical modification of 3α-HSD was done using phthalic anhydride(PA). After modification,the p H stability of 3α-HSD increased within p H 6.0~p H 11.0 compared with the control group and its heat stability significantly increased after incubation at 50 ℃ for 10 min. When stored at 37 ℃ for 40 h,the storage stability of modified3α-HSD was improved about 9-fold than that of native enzyme.