Résumé
To improve 3-ketosteroid-△1-dehydrogenase (KSDH) activity and the transformation level for androst-4-ene-3,17-dione,3-ketosteroid-△1 -dehydrogenase gene (ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110 ± 0.5mU and 15 ± 0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B.subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.
Résumé
To improve 3-ketosteroid-△1-dehydrogenase (KSDH) activity and the transformation level for androst-4-ene-3,17-dione,3-ketosteroid-△1 -dehydrogenase gene (ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110 ± 0.5mU and 15 ± 0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B.subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.
Résumé
The selective side-chain cleavage of phytosterol to 4-androstene-3,17-dione(4-AD)and 1,4-androstadiene-3,17-dione(ADD)by Mycobacterium sp.was described.Because of the similarity in chemical structure between 4-AD and ADD,it is difficult to separate them from the fermentation broth.So far,it has been verified that the ADD can be produced by dehydrogenation of 4-AD.In this reaction,3-Ketosteriod-1-Dehydrogenase(ksdD)plays an important role.The gene knocking out method was used to solve the problem.Partial sequence of ksdD was obtained by PCR which was 631bp in length.Then,a targeting vector pUC19-MK was constructed,which was electroporate into the original strain Mycobacterium neoauru.The method of homologous recombination was used to knock out ksdD gene located in the chromosome of Mycobacterium neoauru.In this way,ksdD would lose its enzyme activity.In the result,5 transformants were screened.The experiments of steroid transformation by the transformants were carried out.The productivity of 4-AD reached 17.52% after 144h,which is 192% higher than the original strain.Meanwhile,the productivity of ADD reached 6.12%,which is 89.9% lower than the original strain.
Résumé
To improve 3-ketosteroid-△1-dehydrogenase(KSDH) activity and the transformation level for androst-4-ene-3,17-dione, 3-ketosteroid-△1-dehydrogenase gene(ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110?0.5mU and 15?0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.