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 Dehydrogenaseand Enzyme Stability Improvement by Chemical Modification

摘要:

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）/ pET28a-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pH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。

Abstract:

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）/ pET28a-hsd at first and then by an affinity purification using Ni2+-Sepharose column with 16.5% of protein yield，64.5% of recovery yield and 3.8 times of specific activity. The SDS-PAGE（15%，reducing conditions） analysis showed that 3α-HSD was composed of a single polypeptide of ~28 kDa. The chemical modification of 3α-HSD was done using phthalic anhydride（PA）. After modification，the pH stability of 3α-HSD increased within pH 6.0~pH 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 modified 3α-HSD was improved about 9-fold than that of native enzyme.