caa a22 4500 397494440 CHVBK 20180308164517.0 cr unu---uuuuu 161202e199505 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124804 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124804 Purification and Characterization of the 3-Ketosteroid-Δ1-Dehydrogenase of Arthrobacter simplex Produced in Streptomyces lividans [Elektronische Daten] [Kwang-Pil Choi, István Molnár, Mitsuo Yamashita, Yoshikatsu Murooka] The 3-ketosteroid-Δ1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly. The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyo-pearl HW55S from the supernatant of culture broth and cell-free extracts of S. lividans, and both preparations showed the same characteristics. The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence. Thus, the extracellular enzyme may derived from leakage of S. lividans cells during cultivation rather than secretion by processing of the signal sequence. The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (Mr 54,329). The optimum conditions for its activity were pH 10.0 and 40°C. The enzyme catalyzed the conversion of several 3-keto-steroids, but those containing 11α- or 11β-hydroxyl group were converted at low rates. The amino acid sequence of KS1DH from A. simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens. © Oxford University Press Regular Papers nationallicence Arthrobacter simplex nationallicence 3-ketosteroid-Δ1-dehydrogenase nationallicence Streptomyces lividans nationallicence Choi Kwang-Pil Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut Molnár István Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut Yamashita Mitsuo Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut Murooka Yoshikatsu Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut The Journal of Biochemistry Oxford University Press 117/5(1995-05), 1043-1049 0021-924X 117:5<1043 1995 117 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124804 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124804 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Choi Kwang-Pil Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut NATIONALLICENCE 700 1- Molnár István Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut NATIONALLICENCE 700 1- Yamashita Mitsuo Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut NATIONALLICENCE 700 1- Murooka Yoshikatsu Department of Fermentation Technology, Faculty of Engineering, Hiroshima University Kagamiyama-1, Higashi-Hiroshima, Hiroshima 724 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 117/5(1995-05), 1043-1049 0021-924X 117:5<1043 1995 117 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford