PURIFICATION AND CHARACTERIZATION OF A HALOALKANE DEHALOGENASE OF A NEW SUBSTRATE CLASS FROM A GAMMA-HEXACHLOROCYCLOHEXANE-DEGRADING BACTERIUM, SPHINGOMONAS-PAUCIMOBILIS UT26

The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene h alidohydrolase, which is involved in the degradation of gamma-hexachlo rocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403- 6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate -polyacrylamide gel, indicating that LinB is a monomeric enzyme. The o ptimal pH for activity was 8.2. Not only monochloroalkanes (C-3 to C-1 0) but also dichloroalkanes, bromoalkanes, and chlorinated aliphatic a lcohols were good substrates for LinB, suggesting that LinB is a haloa lkane dehalogenase with a broad range of substrate specificity. These results indicate that LinB shares properties with another haloalkane d ehalogenase, DhlA (S. Keuning, D. B. Janssen, and B. Witholt, J. Bacte riol. 163:635-639, 1985), which shows significant similarity to LinB i n primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kaz emier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of sub strate activities of various haloalkane dehalogenases suggested that L inB probably constitutes a new substrate specificity class within this group of enzymes.