A LONG-CHAIN SECONDARY ALCOHOL-DEHYDROGENASE FROM RHODOCOCCUS-ERYTHROPOLIS ATCC-4277

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 degrees 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-m value for NAD(+) with 2-octanol as the substrate was 260 mu M, whereas the apparent K-m 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 degrees C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols.