Y Li1, F Lu, T Sun, L Du. 1. Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, P. R. China.
Abstract
AIMS: To improve KSDH enzyme activity and the transformation level for androst-4-ene-3,17-dione. METHODS AND RESULTS: 3-ketosteroid-Delta(1)-dehydrogenase gene from Arthrobacter simplex was expressed in Bacillus subtilis under the control of P43 promoter. The molecular weight of expressed enzyme was about 55 kDa by SDS-PAGE analysis. The activities of intracellular and extracellular soluble enzymes examined by spectrophotometrical method were 110 +/- 0.5 mU mg(-1) and 15 +/- 0.6 mU mg(-1) of protein, respectively. The transformation rate of androst-4-ene-3,17-dione was 45.3% in the B. subtilis recombinant cells. CONCLUSIONS: The enzyme activity of KSDH expressed in B. subtilis was improved about 30-fold compared with that of Arthrobacter simplex, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: The recombinant B. subtilis cells used for biotransformation of steroids provide a new method for production of steroid medicine. The time required for transformation of B. subtilis is much shorter than that of other bacteria, which means it will have wider usage in biopharmaceutical industry.
AIMS: To improve KSDH enzyme activity and the transformation level for androst-4-ene-3,17-dione. METHODS AND RESULTS: 3-ketosteroid-Delta(1)-dehydrogenase gene from Arthrobacter simplex was expressed in Bacillus subtilis under the control of P43 promoter. The molecular weight of expressed enzyme was about 55 kDa by SDS-PAGE analysis. The activities of intracellular and extracellular soluble enzymes examined by spectrophotometrical method were 110 +/- 0.5 mU mg(-1) and 15 +/- 0.6 mU mg(-1) of protein, respectively. The transformation rate of androst-4-ene-3,17-dione was 45.3% in the B. subtilis recombinant cells. CONCLUSIONS: The enzyme activity of KSDH expressed in B. subtilis was improved about 30-fold compared with that of Arthrobacter simplex, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: The recombinant B. subtilis cells used for biotransformation of steroids provide a new method for production of steroid medicine. The time required for transformation of B. subtilis is much shorter than that of other bacteria, which means it will have wider usage in biopharmaceutical industry.
Authors: Chloé Grazon; R C Baer; Uroš Kuzmanović; Thuy Nguyen; Mingfu Chen; Marjon Zamani; Margaret Chern; Patricia Aquino; Xiaoman Zhang; Sébastien Lecommandoux; Andy Fan; Mario Cabodi; Catherine Klapperich; Mark W Grinstaff; Allison M Dennis; James E Galagan Journal: Nat Commun Date: 2020-03-09 Impact factor: 14.919