Purification and properties of haloalkane dehalogenase from Corynebacterium sp. strain m15-3

Abstract

A haloalkane dehalogenase was purified to electrophoretic homogeneity from cell extracts of a 1-chlorobutane-utiizing strain, m15-3, which was identified as a Corynebacterium sp. The enzyme hydrolyzed C2 to C12 mono-and dihalogenated alkanes, some haloalcohols, and haloacids. The K,,, value of the enzyme for 1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimum pH for enzyme activity was found to be 9.4, and the optimum temperature was 30 to 35°C. The enzyme was stable for 1 h at temperatures ranging from 4 to 30°C but was progressively less stable at 40 and 50°C.1-Chlorobutane-utilizing bacteria, Cornybacterium sp. strains tn2C-32 and m15-3, are able to grow with 1-chlorobutane as the sole carbon and energy source. Strain m2C-32 dehalogenated a variety of halogenated compounds, such as 1-chlorobutane, 1,9-dichlorononane, 4-chlorobutanol, and 3-chloropropionic acid, under aerobic conditions; however, under anaerobic conditions the strain dehalogenated only haloalkanes. Those halogenated compounds, except for haloacids, were dehalogenated by the cell extract of strain m2C-32. The cell extract, having a broad substrate specificity, dehalogenated C2 to Cg haloalkanes to produce alcohols (33).Two kinds of haloacid dehalogenases, haloacetate, dehalogenase (EC 3.8.1.3) (8, 13, 14, 28) and 2-haloacid dehalogenase (EC 3.8.1.2) (9, 17, 21), have been found. Furthermore, a DL-2-haloacid dehalogenase was purified from a Pseudomonas sp. (22). A number of dehalogenases which are active with haloacids have been purified and characterized, whereas only two enzymes which dehalogenate short-carbon-chain haloalkanes have been demonstrated. A. glutathione-dependent dehalogenase which acts on dihalomethanes was purified from Hyphomicrobium sp. strain DM2 (16), and a haloalkane dehalogenase which catalyzed hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes was purified from Xanthobacter autotrophicus GJ10 (15). The dehalogenase of X. autotrophicus GJ10 was found to be constitutively produced (12), and the dehalogenases of 1-chlorobutane-utilizing strains m2C-32 and m15-3 were found to be inducible. The substrate specificities of all of the dehalogenases which have ever been purified were rather narrow, whereas the dehalogenase of strain m2C-32 seemed to have a very wide range of substrate specificity. However, we could not purify the dehalogenase of strain m2C-32 to homogeneity. On the other hand, the resting cells and cell extract of strain m15-3 also showed almost the sam.e substrate specificity as did those of strain m2C-32, Which suggested the presence of a haloalkane dehalQgenase with a broad substrate specificity in the cell extract of straih m15-3. Therefore, in this paper we describe the purification and properties of a haloalkane dehalogenase from the 1-chlorobutane-utilizing bacterium strain m15-3. * Corresponding author. MATERIALS AND METHODSMaterials. DEAE-...

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

“…erythropolis Y2 possesses one of the broadest substrate ranges reported to date (cf. Scholtz et al, 1987;Yokota et al, 1987;Janssen et al, 1988). This halidohydrolase showed little or no activity towards terminally halogenated C14, CI6 and C,8 alkanes, but the organism could utilize these haloalkanes as substrates for growth.…”

“…erythropolis Y2 halidohydrolase closely resembled those of enzymes from Corynebacterium sp. m-15-3 (data from Yokota et al, 1987) and Arthrobacter sp. HA1 (data from Scholtz et al, 1987).…”

“…Those haloalkane-degrading organisms which have been characterized usually possess a single enzyme active towards a broad range of haloalkanes and related compounds (Scholtz et al, 1987;Yokota et al, 1987;Janssen et al, 1988). However, Scholtz et al (1988) provided evidence for the presence of three haloalkane dehalogenases in Arthrobacter sp.…”

Haloalkane-UtilizingRhodococcusStrains Isolated from Geographically Distinct Locations Possess a Highly Conserved Gene Cluster Encoding Haloalkane Catabolism

“…Haloalkane dehalogenases active against long-chain haloaliphatic compounds are present in the gram-positive haloalkane-utilizing bacterial strains Y2, isolated in the United Kingdom (16), m15-3, isolated in Japan (25), HA1, isolated in Switzerland (20), GJ70, isolated in The Netherlands (7), and NCIMB13064, isolated in the United Kingdom (2). These enzymes are different from the extensively studied haloalkane dehalogenase (DhlA) found in several gram-negative 1,2-dichloroethane-utilizing bacteria of the genera Xanthobacter and Ancylobacter (8,23).…”

The Plasmid-Located Haloalkane Dehalogenase Gene from Rhodococcus Rhodochrous NCIMB 13064

“…Haloalkane dehalogenases have been found in Gram-negative (Keuning et al, 1985) and Gram-positive haloalkane-utilizing bacteria (Keuning et al, 1985;Yokota et al, 1987;Scholtz et al, 1987;Sallis et al, 1990). Haloalkane dehalogenases are unique enzymes which catalyse hydrolytic dehalogenation of corresponding hydrocarbons without participation of oxygen or coenzymes.…”