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

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.