A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols.
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机译:NAD依赖的仲醇脱氢酶已从降解烷烃的细菌红球菌ATCC 4277中纯化。发现该酶对多种底物具有活性,特别是长链仲脂肪族醇。尽管在含6至11个碳原子的直链2-醇中观察到了最佳活性,但只要2-十四烷醇被氧化的速率为中级醇的25%,则仲醇被氧化。纯化的酶对2-辛醇的S-(+)立体异构体具有特异性,并且在pH 9和37°C,25的条件下对2-辛醇的比活度超过200μmol/ min / mg的蛋白质。比以前报道的任何S-(+)仲醇脱氢酶高2倍。含3至13个碳原子的直链伯醇的利用效率比相应的仲醇低20至40倍。以2-辛醇为底物的NAD(sup +)的表观K(infm)值为260μM,而2-醇的表观K(infm)值范围从2-戊醇的5 mM以上到更低。 2-十四烷醇的浓度小于2μM。该酶表现出中等的热稳定性(在60℃下半衰期为4小时),可能潜在地用于合成光学纯的仲醇立体异构体。
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