JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 88, No. 1, 7-11. 1999 Gene Cloning and Characterization of Aldehyde Dehydrogenase from a Petroleum-Degrading Bacterium, Strain HD-1 NAOKO OKIBE,’ KEI AMADA,’ SHIN-ICHI HIRANO,’ MITSURU HARUKI,’ TADAYUKI IMANAKA,2 MASAAKI MORIKAWA,’ AND SHIGENORI KANAYA’* Department of Material and Life Science, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka X5-0871’ and Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyo to 606-8501 ,2 Japan Received 24 February 1999/Accepted 9 April 1999 The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD+- dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recom- binant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel Jiltration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various allphatic aldehyde substrates. The Km values of the enzyme were 6.4 $4 for acetaldehyde, 4.2 ,uM for hexanal, 2.8 ,&f for octanal, and 0.84 fl for decanal, whereas the k,, values for these substrates were nearly equal (51-64 min-I). These results indicate that hd-ALDH acts preferentially on long-chain allphatic aldehydes. [Key words: aldehyde dehydrogenase, petroleum-degrading bacterium, gene cloning, substrate specificity, kinetics] We previously isolated strain HD-1, which degrades aliphatic and aromatic hydrocarbons under both aerobic and anaerobic conditions, from an oil spring in Shizuoka (1). This strain degrades tetradecane and produces l- dodecene as one of the metabolic intermediates under anaerobic conditions (2). However, the alkane degrada- tion pathway of strain HD-1 remains to be elucidated. As to the aerobic alkane degradation pathway, alkanes are usually oxidized in a stepwise manner to fatty alco- hol, fatty aldehyde, and then fatty acid (or fatty acyl- CoA). This final product is metabolized by the p-oxida- tion pathway and the subsequent TCA cycle (3, 4). Ac- cording to intensive studies on the alkane metabolic path- way of Pseudomonas oleovolans, alkane hydroxylase, alcohol dehydrogenase, aldehyde dehydrogenase (ALDH), and acyl-CoA synthetase are essential for this pathway (4, 5). Therefore, it would be informative to examine whether these enzymes are involved in the alkane degra- dation pathway of strain HD-1. ALDH (EC 1.2.1.3) is distributed in a wide range of living organisms and detoxifies various harmful xenobio- tic compounds sometimes in cooperation with the cyto- chrome P-450 system (6). Xanthobacter autofrophicus can metabolize 1,2-dichloroethane and a reactive intermedi- ate, 2-chloroacetaldehyde (CAA), is further oxidized by CAA dehydrogenase (7). The gene encoding this enzyme has recently been cloned and the deduced amino acid sequence revealed that CAA dehydrogenase is a member of the NAD+-dependent ALDH group (8). Rhodococcus sp. strain NI86/12 is utilized as a biosafener, which protects plants against the herbicide S-ethyl dipropylcar- bamothioate (EPTC). It is reported that cytochrome P- 450 and ALDH play important roles in the degradation of EPTC (9). Thus, ALDH is recognized as an im- portant enzyme for oxidation of various compounds, * Corresponding author. including hydrocarbons. However, only limited informa- tion is available regarding ALDH from oil-degrading bac- teria. In this report, we have cloned the gene encoding ALDH from strain HD-1, expressed it in E. coli, and biochemically characterized the recombinant enzyme. MATERIALS AND METHODS Cells and plasmids Strain HD-1 was isolated from an oil spring in Shizuoka, Japan (1). E. coli DH5a [F-, $80, lacZAM15, recA1, endAl, gyrA96, t&l, hsdR17(rkp, mkf), SupE44, relA1, deoR, A(lacZYA-argF’)U169, 1-1 and plasmid pBluescript KS(+) for DNA manipula- tions were obtained from Toyobo Co. Ltd (Kyoto). E. coli HMS174(DE3)pLysS [F-, recA1, hSdR(rk12-mk12+), RifR(DE3), pLysS(CmR)] and plasmid PET-25b for gene expression were obtained from Novagen (Madison, WI, USA). All transformants were grown in L broth, in the presence of 50 mg/l ampicillin, unless otherwise stated. Cloning of the hd-aid gene The genomic DNA of strain HD-1 was extracted and purified by CsCl-ethidium bromide equilibrium density gradient ultracentrifugation as previously described (10). A part of the hd-ald gene was amplified by PCR with a combination of forward (5’-ATHGTHCCXTGGAAYTTYCC-3’) and reverse (5’- GGXCCRAADATYTC YTC-3’) primers (H =A+C+T, R=A+G, X=A+C+G+T and Y=C+T), which were constructed according to the amino acid sequence in highly conserved regions among various aldehyde de- hydrogenases. For E. coli ALDH, these primers encom- passed the sequences coding for amino acids 161-167 (IVPWNFP) and 401-406 (EEIFGP) (11). The resultant 73%bp DNA fragment, which was the only DNA frag- ment amplified by PCR, was used as the probe for Southern blotting and colony hybridization to clone the entire ald gene. PCR was performed in 30 cycles using a 7