The Clone And Expression Of 3-ketosteroid-1(2)-dehydrogenase From Arthrobacter Simplex And Mycobacterium Sp.

In the steroid pharmaceutical industry, the 3-ketosteroid-1(2)-dehydrogenation catalyzed by microorganism is very important. It is the key reaction to make cortin with high anti-phlogisticthe activity. It can double or triple the anti-phlogisticthe activity of steroid medicine.In this paper, we got the 3-ketosteroid-1(2)-dehydrogenase gene from Arthrobacter simplex which is used in industry and Mycobacterium sp., which had a high 3-ketosteroid-1(2)-dehydrogenase activity. All the two KSDDs were expressed in E.coliDH5a, and their activities was compared.First, according to the genome sequence of Arthrobacter simplex in NCBI, we designed the primers, and got the whole sequence of 3-ketosteroid-1(2)-dehydrogenase. It is precisely the same with the 3-ketosteroid-1(2)-dehydrogenase gene of Arthrobacter simplex in NCBI.Through analyzing the genome sequence of 3-ketosteroid-1(2)-dehydrogenase in Mycobacterium, we designed the primers, and used Mycobacterium sp. which is stored in our lab as the template and got the conservative region. It is about 1500bp long. Through genome walking, we got the sequence after the conservative region. It includes the open reading frame of 1701 bp, coding 567 amino acids.It exhibits 82% DNA sequence homology with the 3-ketosteroid-1(2)-dehydrogenase gene of Mycobacterium smegmatis str. MC2155 (Genbank CP000480)First we used pET22 to construct pTQ004, pTQ005 as the expression vector, and both the KSDDs were expressed in E.coli BL21(DE3)/plysS. Induced by IPTG, we can see the protein expressed was mainly inclusion, and no enzyme activity can be detected.We used pUC18 to construct pTQ002, pTQ003 as the expression vector, and both the KSDDs were expressed in E.coliDH5a. Induced by IPTG, the enzyme activity was measured at 30℃. The 3-ketosteroid-1(2)-dehydrogenase activity of Arthrobacter simplex was 0.82±0.09U, while the 3-ketosteroid-1(2)-dehydrogenase activity of Mycobacterium sp. was 0.37±0.05U, about half of the former one. pUC18 is not a strong promoter, so there is not much expressed protein in the fermentation broth, and the protein band can not be seen in the SDS-PAGE.