Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Appl Microbiol Biotechnol. 2010 Jun;87(2):595-607. doi: 10.1007/s00253-010-2472-z. Epub 2010 Feb 26.
The oxidizing activity of CYP109B1 from Bacillus subtilis was reconstituted in vitro with various artificial redox proteins including putidaredoxin reductase and putidaredoxin from Pseudomonas putida, truncated bovine adrenodoxin reductase and adrenodoxin, flavodoxin reductase and flavodoxin from Escherichia coli, and two flavodoxins from B. subtilis (YkuN and YkuP). Binding and oxidation of a broad range of chemically different substrates (fatty acids, n-alkanes, primary n-alcohols, terpenoids like (+)-valencene, alpha- and beta-ionone, and the steroid testosterone) were investigated. CYP109B1was found to oxidize saturated fatty acids (conversion up to 99%) and their methyl and ethyl esters (conversion up to 80%) at subterminal positions with a preference for the carbon atoms C11 and C12 counted from the carboxyl group. For the hydroxylation of primary n-alcohols, the omega(-2) position was preferred. n-Alkanes were not accepted as substrates by CYP109B1. Regioselective hydroxylation of terpenoids alpha-ionone (approximately 70% conversion) and beta-ionone (approximately 91% conversion) yielded the allylic alcohols 3-hydroxy-alpha-ionone and 4-hydroxy-beta-ionone, respectively. Furthermore, indole was demonstrated to inhibit fatty acid oxidation.
枯草芽孢杆菌 CYP109B1 的氧化活性在体外与各种人工氧化还原蛋白（包括来自恶臭假单胞菌的 Putidaredoxin 还原酶和 Putidaredoxin、截短的牛肾上腺皮质酮还原酶和肾上腺皮质酮、来自大肠杆菌的 Flavodoxin 还原酶和 Flavodoxin，以及来自枯草芽孢杆菌的两种 Flavodoxin（YkuN 和 YkuP））一起被重新构建。研究了各种化学性质不同的底物（脂肪酸、正烷烃、伯醇、类胡萝卜素（如（+）-Valencene、α-和β-紫罗兰酮以及甾体睾丸激素）的结合和氧化。发现 CYP109B1 可在亚末端位置氧化饱和脂肪酸（转化率高达 99%）及其甲酯和乙酯（转化率高达 80%），优先考虑从羧基数起的第 11 和 12 个碳原子。对于伯醇的羟基化，ω(-2)位优先。CYP109B1 不接受正烷烃作为底物。类胡萝卜素α-紫罗兰酮（转化率约为 70%）和β-紫罗兰酮（转化率约为 91%）的区域选择性羟化生成相应的烯丙醇 3-羟基-α-紫罗兰酮和 4-羟基-β-紫罗兰酮。此外，吲哚被证明可以抑制脂肪酸的氧化。
