| Autor: |
Wang L; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.; Key Laboratory of Industrial Fementation Microbiology, Ministry of Education Tianjin 300457, China., Zhao X; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.; Key Laboratory of Industrial Fementation Microbiology, Ministry of Education Tianjin 300457, China., Li J; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.; Key Laboratory of Industrial Fementation Microbiology, Ministry of Education Tianjin 300457, China., Ai J; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.; Key Laboratory of Industrial Fementation Microbiology, Ministry of Education Tianjin 300457, China., Sun J; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.; Key Laboratory of Industrial Fementation Microbiology, Ministry of Education Tianjin 300457, China., Mao S; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.; Key Laboratory of Industrial Fementation Microbiology, Ministry of Education Tianjin 300457, China. |
| Abstrakt: |
17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx 4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro . In addition, the effect of phosphorylation of the ferredoxin Adx 4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx 4-108 T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method. |
