Vol. 169, No. 9JOURNAL OF BACTERIOLOGY, Sept. 1987, p. 4049-40540021-9193/87/094049-06$02.00/0Copyright © 1987, American Society for Microbiology

Purification and Properties of Haloalkane Dehalogenase fromCorynebacterium sp. Strain m15-3

TOYOKAZU YOKOTA,* TOSHIO OMORI, AND TOHRU KODAMA

Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan

Received 20 January 1987/Accepted 18 June 1987

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 C2to C12 mono- and dihalogenated alkanes, some haloalcohols, and haloacids. The K,,, value of the enzyme for1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimumpH 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. Strainm2C-32 dehalogenated a variety of halogenated compounds,such as 1-chlorobutane, 1,9-dichlorononane, 4-chlorobu-tanol, and 3-chloropropionic acid, under aerobic conditions;however, under anaerobic conditions the strain dehalo-genated only haloalkanes. Those halogenated compounds,except for haloacids, were dehalogenated by the cell extractof strain m2C-32. The cell extract, having a broad substratespecificity, dehalogenated C2 to Cg haloalkanes to producealcohols (33).Two kinds of haloacid dehalogenases, haloacetate,

dehalogenase (EC 3.8.1.3) (8, 13, 14, 28) and 2-haloaciddehalogenase (EC 3.8.1.2) (9, 17, 21), have been found.Furthermore, a DL-2-haloacid dehalogenase was purifiedfrom a Pseudomonas sp. (22). A number of dehalogenaseswhich are active with haloacids have been purified andcharacterized, whereas only two enzymes which dehalo-genate short-carbon-chain haloalkanes have been demon-strated. A. glutathione-dependent dehalogenase which actson dihalomethanes was purified from Hyphomicrobium sp.strain DM2 (16), and a haloalkane dehalogenase whichcatalyzed hydrolytic dehalogenation of n-halogenated C1 toC4 alkanes was purified from Xanthobacter autotrophicusGJ10 (15). The dehalogenase of X. autotrophicus GJ10 wasfound to be constitutively produced (12), and the dehalo-genases of 1-chlorobutane-utilizing strains m2C-32 andm15-3 were found to be inducible. The substrate specificitiesof all of the dehalogenases which have ever been purifiedwere rather narrow, whereas the dehalogenase of strainm2C-32 seemed to have a very wide range of substratespecificity. However, we could not purify the dehalogenaseof strain m2C-32 to homogeneity. On the other hand, theresting cells and cell extract of strain m15-3 also showedalmost the sam.e substrate specificity as did those of strainm2C-32, Which suggested the presence of a haloalkanedehalQgenase with a broad substrate specificity in the cellextract of straih m15-3. Therefore, in this paper we describethe purification and properties of a haloalkane dehalogenasefrom the 1-chlorobutane-utilizing bacterium strain m15-3.

* Corresponding author.

MATERIALS AND METHODS

Materials. DEAE-cellulose (Whatman DE-52) was ob-tained from Whatman Ltd.; hydroxyapatite was fromSeikagaku Kogyo. Butyl-Toyopearl, DEAE-Toyopearl, andToyopearl HW55S were purchased from Toyo Soda Manu-facturing Co. Ampholine was from LKB Products AB,Bromma, Sweden. pl marker proteins and marker proteinsfor SDS-polyacrylamide gel electrophoresis were from Ori-ental Yeast Co. Standard proteins for gel filtration were fromBio-Rad Laboratories, Richmond, Calif. Other chemicalswere of analytical grade.Organisms and growth conditions. 1-Chlorobutane-utiliz-

ing strains m2C-32 and nm15-3 were isolated from soil (33).The organisms were grown aerobically at 30°C in a mediumcontaining the following (per liter): KH2PO4, 1.5 g;Na2HPO4- 12H20, 1.5 g; NH4NO3, 4.0 g; MgSO4 .7H20,0.2 g; CaSO4. 2H20, 10 mg; FeSO4 7H20, 5 mg; and yeastextract, 50 mg (pH 7.2). For solid culture, 17 g of agar wasadded. The carbon source, 1-chlorobutane, was sterilizedseparately by filtration through a membrane filter (type FG;pore size, 0.2 ,um) (Millipore Corp., Bedford, Mass.) andadded to yield a concentration of 0.2% (vol/vol).DNA was extracted from cells by the method of Marmtir

(19). The Tm value was determined by the method of DeLeyand Shell (6), and, on the -basis of the Tm value, the G+Ccontent was calculated by using the formula described byMarmur and Doty (20). Cell walls were prepared by themethod of Cummins and Harris (4) and analyzed by themethod of Staneck and Roberts (26).For the cultivation of strain m15-3 in a fermentor, 1%

sodium succinate was used as a carbon source owing to thepoor growth of strain m15-3 in a liquid culture with 1-chlorobutane. Cultures (20 liters each) were grown in afermentor operated at a stirring rate of 600 rpm and an airrate of 1 liter/liter of medium per min at 30°C. The growthwas monitored by measuring the A550 of the culture.

Cells harvested in the late exponential phase of growthwere washed twice with 50 mM phosphate buffer, pH 7.2,and resuspended in the same buffer (60 g [wet weight] of cellsper liter of buffer). KH2PO4-NaOH buffer was used as aphosphate buffer throughout this study. To induce thedehalogenase, 500 ml of the cell suspension was incubated

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with 1 ml of 1-chlorobutane in a 5-liter Erlenmeyer flask at30°C on a rotary shaker for more than 9 h, until the amountof released chloride ion increased up to about 100 ,ugIml. Thecells were harvested by centrifugation, washed with thephosphate buffer, and stored at -80°C in a freezer.

Induction of dehalogenase. The resting cells of strainsm2C-32 and mlS-3 were prepared from cells which weregrown on sodium succinate, suspended in 50 mM phosphatebuffer (pH 7.2), and incubated with or without 1-chloro-butane (0.2%, vol/vol) in the presence or absence of chloram-phenicol (0.5 mg/ml). After incubation at 30°C for 12 h, thecells were harvested by centrifugation, and cell extractswere prepared as previously described (33). Cell extractswere also prepared from cells grown on 1-chlorobutane inliquid culture. Dehalogenase activity in cell extracts wasdetermined.Enzyme purification. The dehalogenase was purified from

strain m15-3. All operations were carried out at 0 to 4°C,unless otherwise stated.

(i) Preparation of cell extract. Cell extract was preparedfrom 40 g of strain mlS-3 cells, as previously described (33).

(ii) Ammonium sulfate fractionation. To the cell extract,7.5% streptomycin sulfate solution in 50 mM phosphatebuffer (pH 7.2) was added7 at a final concentration of 1.5%(wt/vol). The precipitate formed was removed by centrifu-gation at 15,000 x g for 15 min. Solid ammonium sulfate wasadded to the supernatant solution to 45% saturation, and theresulting precipitate was renmoved by centrifugation at 20,000x g for 10 min, The supernatant solution was brought to 65%saturation with additional ammonium sulfate. The precipi-tate was collected by centrifugation at 20,000 x g for 15 min,redissolved in a minimal volume of 100 mM phosphatebuffer, pH 7.0, and dialyzed against the same buffer.

(iii) DEAE-cellulose column chromatography. The dialyzedenzyme solution was applied to a DEAE-cellulose column(1.8 by 19 cm) which was equilibrated with the same phos-phate buffer. The enzyme was eluted with a linear gradient ofphosphate buffer (100 to 140 mM in a total volume of 400 ml;pH 7.0), and active fractions were pooled.

(iv) Butyl-Toyopearl column chromatography. The enzymesolution was brought to 30% saturation with ammoniumsulfate and applied to a butyl-Toyopearl column (1.8 by 9cm) which was equilibrated with 30% ammonium sulfate-50mM phosphate buffer (pH 7.0). After the column waswashed with the same buffer, the enzyme was eluted with adouble linear gradient of 30 to 0% ammonium sulfate-50 to 5mM phosphate buffer (pH 7.0) in a total volume of 200 ml.Active fractions were pooled and dialyzed against 100 mMphosphate buffer (pH 7.0).

(v) flEAE-Toyopearl column chromatography. The dia-lyzed enzyme solution was applied to a DEAE-Toyopearlcolumn (1.4 by 20 cm) which was equilibrated with 100 mMphosphate buffer (pH 7.0). The column was washed with thesame buffer, and the enzyme was eluted with a lineargradient of 300 ml of 100 to 140 mM phosphate-buffer (pH7.0). Active fractions were pooled and dialyzed against 10mM phosphate buffer (pH 6.8).

(vi) Hydroxyapatite column chromatography. The dialyzedenzyme solution was applied to a hydroxyapatite column(1.4 by 15 cm) which was equilibrated with 10 mM phosphatebuffer (pH 6.8) and washed with the same buffer. Theenzyme was eluted with a linear gradient of phosphate buffer(10 to 50 mM in a total volume of 200 ml; pH 6.8). Activefractions were pooled and concentrated by ultrafiltration.

(vii) Toyopearl HW55S gel filtration. The enzyme solution(about 10 ml) was applied to Toyopearl HWS5S column (4.4

by 60 cm) which was equilibrated with 50 mM phosphatebuffer (pH 7.2), and the enzyme was then eluted with thesame buffer at a flow rate of 1.5 ml/min. Dehalogenaseactivity coincided exactly with the protein peak. Activefractions were pooled and stored at -80°C.Enzyme assay. Dehalogenase activity was assayed as

follows, unless otherwise stated. Enzyme solution was di-luted with 50 mM phosphate buffer, pH 7.2, to yield a finalvolume of 1.0 ml in a microtube and incubated with 10 ,molof 1-chlorobutane at 30'C. One unit of enzyme activity wasdefined as the amount of enzyme required in order to release1 ,umol of chloride ion per min. In the study of substratespecificity, the concentration of substrate was always 10,umol/ml and incubation was always performed for 1 h. Theamount of halide ion released was determined spectropho-tometrically at 460 nm with mercuric thiocyanate and ferricammonium sulfate by the method of Iwasaki et al. (10).

Protein assay. The protein concentration was determinedby using the Bio-Rad protein assay with ovalbumin as astandard.

Disc electrophoresis. Polyacrylamide gel electrophoresiswas performed by the general procedures of Davis (5) byusing the gel and reservoir buffers of Williams and Reisfeld(31). A sample was applied to a column of 7% polyacryl-amide gel (pH 8.0) and run at a constant current of 2 mA percolumn in a Tris-barbital buffer, pH 7.0, at <4°C. Proteinbands were stained with Coomassie brilliant blue R-250.

Determination of molecular weight. (i) Gel filtration onToyopearl HW55S. Gel filtration was done at 4°C on aToyopearl HW55S column (4.4 by 60 cm) equilibrated with50 mnM phosphate buffer, pH 7.2. Proteins were eluted withthe same buffer at a flow rate of 1.5 ml/min. Thyroglobulin(molecular weight, 670,000), immunoglobulin G (molecularweight, 158,000), ovalbumin (molecular weight, 44,000), andmyoglobin (molecular weight, 17,000) were used as refer-ence proteins.

(ii) SDS-polyacrylamide gel electrophoresis. Sodium dode-cyl sulfate (SDS)-polyacrylamide gel electrophoresis wasperformed by the method of Weber and Osborn (30).Cytochrome c hexamer (molecular weight, 74,400), tetramer(molecular weight, 49,600), trimer (molecular weight,37,200), dimer (molecular weight, 24,800), and monomer(molecular weight, 12,400) were used as standard proteins.

Isoelectric focusing. To estimate the pI value of purifiedenzyme, gel disc isoelectric focusing (32) was performed on5% polyacrylamide gel containing 2% Ampholine (pH 3.5 to10) with modifications as indicated. As anode and cathodesolutions, 0.02 M H3P04 and 1 M NaOH, respectively, wereused. Focusing was continued for 5 h at a constant voltage of200 V, with cooling at 0°C. The gels were immersed inCoomassie brilliant blue G-250-3.5% perchloric acid solu-tion to stain protein bands. The pl values of the standardproteins, which consisted of equine cytochrome c andacetylated cytochrome c, were 10.6, 9.7, 8.3, 6.4, 4.9, and4.1.Amino acid analysis. The purified enzyme was hydrolyzed

in 6 N HCl at 110°C in a vacuum for 20, 48, and 70 h. Thehydrolysates were analyzed for amino acid composition on aHitachi model 835 amino acid analyzer. Tryptophan wasdetermined spectrophotometrically by the method of Edel-hoch (7).

Determination of product. Five milliliters of enzyme solu-tion (50 mM phosphate buffer; pH 7.2) was incubated with100 ,ul of substrate at 30°C for 12 h in a Thunberg tube inwhich the atmosphere was replaced with nitrogen gas. Thereaction mixture was extracted with ether, and the product

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TABLE 1. Induction of dehalogenase

Strain/growth Induction conditions Sp act (mU/substrate Chloramphenicol' 1-Chlorobutaneb mg of protein)

m2C-32Succinate - - 0

- + 58.1+ + 0

1-Chlorobutane 85.0

m15-3Succinate - - 0

- + 11.0+ + 1.8

1-Chlorobutane 72.1a Concentration: +, 0.5 mg/ml; -, 0 mg/ml.b Concentration: +, 0.2% (vol/vol); -, 0% (vol/vol).

was determined by gas-liquid chromatography (Gaskuro-pack 54 (2 m), 180°C; injector, 230°C), as previously de-scribed (33).

RESULTS

Characterization of 1-chlorobutane-utilizing strains m2C-32and m15-3. Both strains m2C-32 and mlS-3 are aerobic,gram-positive, oxidase-negative, and nonmotile bacteria.They are rods (0.4 to 0.6 ,um by 1 to 3 ,um) and are not acidfast. They are not spore-formers. The diamino acid of thecell wall peptidoglycan was meso-diaminopimelic acid. TheG+C content of the DNA was 62.3% for strain m2C-32 and63.4% for strain mlS-3. Those properties showed that theorganisms should be classified as Corynebacterium sp.strains (25). Owing to the lack of information concerning thepathogenicity of the strains, we could not identify m2C-32and m15-3 to the species level.

Induction of dehalogenase. The dehalogenase activities inthe cell extracts of strains m2C-32 and mlS-3 are given inTable 1. Dehalogenase activities of both strains were notfound in the cell extracts prepared from cells grown onsodium succinate. However, the cell extracts showeddehalogenase activities when the cells grown on sodiumsuccinate were incubated with 1-chlorobutane. Chloram-phenicol appeared to inhibit the production of dehalogenasein the cells during incubation with 1-chlorobutane. Theseresults suggest that the dehalogenases of both strains wereproduced inducibly during incubation with 1-chlorobutane.Enzyme purification. A summary of the enzyme purifica-

tion procedure is shown in Table 2. The enzyme was purifiedabout 400-fold, with a yield of 10% from the cell extract ofstrain m15-3.The purified enzyme showed a single protein band upon

both polyacrylamide disc gel electrophoresis (Fig. 1) andSDS-polyacrylamide gel electrophoresis (data not shown).

Molecular weight. The molecular weight of the enzymewas estimated to be 33,000 by gel filtration and 36,000 bySDS-polyacrylamide gel electrophoresis, indicating that theenzyme consists of a single peptide chain with a molecularweight of 36,000.

Effects of pH and temperature. Maximum enzyme activitywas observed at pH 9.4. The enzyme was stable in the pHrange from 7.5 to 8.5 when assayed after incubation atvarious pHs (pH 6.0 to 8.0, 50 mM phosphate buffer; pH 8.5to 10.5, 100 mM glycine-NaOH buffer) for 24 h at 4°C. Theamount of released chloride ion was measured after incuba-

TABLE 2. Purification of haloalkane dehalogenaseProtein Activity Sp act Purification Yield

Purification step (mg) (U) (U/mg) (fold) (%)Cell extract 2,424 13.48 0.0056 1.0 100(NH4)2SO4. 1,129 8.79 0.0078 1.4 6545-65%

DEAE-cellulose 29.0 5.82 0.201 35.9 43Butyl-Toyopearl 13.0 4.23 0.324 57.9 31DEAE-Toyopearl 3.55 2.20 0.621 111 16Hydroxyapatite 0.70 1.44 2.06 368 11Toyopearl HWSSS 0.56 1.40 2.48 443 10

tion for 1 h at various temperatures in 100 mM glycine-NaOH buffer (pH 9.4) plus 1-chlorobutane. Maximum activ-ity was observed at 30 to 35°C. The enzyme in 50 mMphosphate buffer (pH 7.2) could be incubated for 1 h withoutappreciable loss of activity at 0 or 30°C; however, whenheated for 1 h at 40 and 50°C, it retained 60 and 12%,respectively, of initial activity. The enzyme retained 60% ofinitial activity after incubation for 12 h at 30°C in 50 mMphosphate buffer (pH 7.2).Enzyme kinetics. The Michaelis constant for 1-chloro-

butane was determined with the purified enzyme in 100 mMglycine-NaOH buffer (pH 9.4). Measurement of the rates ofchloride liberation with different concentrations of 1-chlorobutane revealed a Km of 0.18 mM and a Vmax of 1.5,umol/min per mg of protein.

Identification of the reaction product. The gas-liquidchromatogram of the product from 1-chlorobutane showed amajor peak corresponding to n-butanol, suggesting that thepurified enzyme converted 1-chlorobutane into n-butanolunder anaerobic conditions. This result confirmed that theenzyme was a hydrolytic dehalogenase.

Substrate specificity. The substrate specificity of the en-zyme was investigated. A broad substrate specificity of thedehalogenase was demonstrated; the relative values of therate of halide release are shown in Table 3. The enzyme

FIG. 1. Polyacrylamide gel electrophoresis of purified haloal-kane dehalogenase. The stained band represents 5 ,ug of enzyme.

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TABLE 3. Substrate specificity of the enzyme

RelativeSubstrate activity (%)a

Monohalogenated alkanesBromoethane.......................................1-Chloropropane .....................................

1-Chlorobutane ......................................

1-Chloropentane .....................................

1-Chlorohexane ......................................

1-Chloroheptane .....................................

1-Chlorooctane.......................................1-Chlorononane ......................................

1-Chlorododecane ....................................

1-Bromobutane ......................................

1-lodobutane .........................................

1-Chloro-2-methylpropane ...........................

1-Chloro-3-methylbutane .............................

2-Chlorobutane ......................................

2-Chlorooctane.......................................

Dihalogenated alkanes1,2-Dibromoethane ...................................

1,3-Dichloropropane .................................

1,4-Dichlorobutane ...................................

1,5-Dichloropentane..................................1,6-Dichlorohexane ..................................

1,7-Dichloroheptane ..................................

1,8-Dichlorooctane ...................................

1 ,9-Dichlorononane ..................................

1,2-Dichlorobutane ...................................

1,3-Dichlorobutane ...................................

2,3-Dichlorobutane ...................................

Haloalcohols3-Chloropropanol ....................................

4-Chlorobutanol ......................................

HaloacidsFluoroacetic acid ....................................

Chloroacetic acid ....................................

3-Chloropropionic acid...............................

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a The rates of halide ion liberation from different substrates are expressedas percentages of the rate observed with 1-chlorobutane.

dehalogenated monohalogenated n-alkanes from bromo-ethane to 1-chlorododecane, dihalogenated n-alkanes from1,2-dibromoethane to 1,9-dichlorononane, chloroalkaneswith a side chain, and 2-chloroalkanes. The order of reac-tivity was determined by using chloro-, bromo-, andiodobutane and was as follows: bromo- > chloro- > iodo-.The enzyme was active with some chloroalcohols andweakly active with some haloacids. No activity was foundwith 1-chlorohexadecane, 1-bromohexadecane, 3-chloro-hexane, 1,2-dichloroethane, 1,1-dichloroethane, 1, 1-dichloro-butane, 2-chloroethanol, 6-chlorohexanol, 2-chloropropionicacid, 2-chlorobutyric acid, 3-chlorobutyric acid, chlorocyclo-hexane, or chlorobenzene.

Isoelectric point and amino acid composition. The isoelec-tric point of the purified enzyme was estimated to be pH 4.5by isoelectric focusing. The amino acid composition of theenzyme is shown in Table 4. The predominant residues were

proline, aspartic acid (or asparagine), glutamic acid (orglutamine), and leucine.

DISCUSSION

The cells grown on sodium succinate did not produce thedehalogenase, but those cells incubated with 1-chlorobutanein buffer produced the dehalogenase inducibly, indicatingthat the purified dehalogenase of strain m15-3 is used forgrowth on 1-chlorobutane. A high level of haloalkanedehalogenase induction was not observed in strain m15-3compared with that observed in strain m2C-32 (Table 1), andit was difficult to induce a constant level of dehalogenase instrain m15-3 (data not shown). One of the reasons for thepoor growth of strain m15-3 on 1-chlorobutane in liquidculture may have been the low level of dehalogenase induc-tion in the strain.A haloalkane dehalogenase was purified to electrophoretic

homogeneity from Corynebacterium sp. strain m15-3. Theenzyme hydrolytically dehalogenated various haloalkanesand some related halocompounds. The dehalogenase ofstrain m15-3 seemed to liberate easily the halogen on theterminal carbon of haloalkanes. Most of the haloalkaneswhich were used in this study as substrates to determine thesubstrate specificity of the enzyme are insoluble or practi-cally insoluble in water (for example, the solubility of1-chlorobutane is 0.066%). Thus, 10 ,umol of the substratesper ml added to the reaction mixture could not be dissolve tothe extent of 10 mM, except for bromoethane, 1-chloropro-pane, 1-chloro-3-methylbutane, 1,2-dibromoethane, 1,1-dichloroethane, haloalcohols, and haloacids. We must con-sider the influence of substrate solubility on the relativeactivity of the enzyme shown in Table 3.

In the dehalogenation of dihalogenated alkanes by thehaloalkane dehalogenase, corresponding haloalcohols were

supposed to be formed, and substrate specificity of theenzyme (Table 3) suggested that some of the producedhaloalcohols could be further dehalogenated. However, inthe case of the cell extract of strain m2C-32 1,3-dichloro-propane and 1,4-dichlorobutane were transformed into thecorresponding chlorohydrins, but diol compounds were notdetected (33).

TABLE 4. Amino acid composition of the enzyme

Amino acid Content (Mol%)a No. of residues/enzyme moleculeb

Aspartic acidc 10.1 33Threonined 4.1 13Serined 5.8 19Glutamic acidc 9.7 31Proline 10.7 35Glycine 8.5 27Alanine 8.4 27Half-cystine 0.5 2Valine 5.8 19Methionine 0.6 2Isoleucine 4.9 16Leucine 9.2 30Tyrosine 2.1 7Phenylalanine 5.0 16Lysine 3.1 10Histidine 3.3 11Arginine 5.5 18Tryptophan 2.8 9

a Values are averages for 20-, 48-, and 70-h hydrolysis.I Values were calculated on the basis of a molecular weight of 36,000.c Values given for these amino acids are sums of the acids and the amides.d Values given for these amino acids were corrected to time zero of

hydrolysis.

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Though the strain m15-3 resting cells showed a dehalo-genation activity with 3-haloacids such as 3-chloropropionicacid and 3-chlorobutyric acid (33), the purified enzymeshowed very low or no activity with 3-haloacids. Thissuggested that another dehalogenase was involved in thedehalogenation of 3-haloacids by the strain m15-3 restingcells.

Extensive investigations of microbial dehalogenation ofhaloacids have demonstrated the reaction mechanisms, andthe enzymes have been characterized. Two kinds of dehalo-genases, which act on haloacids, have been found: halo-acetate dehalogenase (EC 3.8.1.3) (which dehalogenatesexclusively haloacetate [8, 13, 14, 28]) and 2-haloaciddehalogenase (EC 3.8.1.2) (which catalyzes dehalogenationof 2-haloalkanoic acids [9, 17, 21]). Both of them arehydrolytic enzymes. These enzymes are evidently differentfrom the dehalogenase described here because they actsolely on L-2-haloacids.The enzymes which have been observed to catalyze the

dehalogenation of chlorinated aromatic compounds (18, 23)have not been purified. On the basis of substrate specificity,the dehalogenase of strain m15-3 is different from thoseenzymes.There are many reports concerning biological dehalogena-

tion of haloalkanes. Haloalkanes with long carbon chains,such as 1,9-dichlorononane, were dehalogenated oxidatively(24, 33), whereas haloalkanes with short carbon chains, suchas dichloromethane, 1,2-dichloroethane, and 1-chlorobu-tane, were dehalogenated hydrolytically (11, 33) or by glu-tathione-dependent reaction (27). Also, haloalkanes withshort carbon chains, such as carbon tetrachloride andtrichloroethylene, were dehalogenated under methanogenicconditions (1, 29) or denitrification conditions (2). A gluta-thione-dependent dehalogenase purified from Hyphomi-crobium sp. strain DM2 acts only on dihalomethanes (16)and is clearly different from the enzyme described here.Keuning et al. (15) purified a haloalkane dehalogenase whichcatalyzes the dehalogenation of halogenated C1 to C4 n-alkanes from X. autotrophicus GJ10. The purified haloalkanedehalogenase of Corynebacterium sp. strain m15-3 appearsto have characteristics similar to those of X. autotrophicusGJ10. Each of these enzymes catalyzes hydrolytic dehalo-genation of haloalkanes and is composed of a single peptidechain with a molecular weight of 36,000; however, they showmany distinctly different characteristics. One purifieddehalogenase, from Corynebacterium sp. strain m15-3, is aninducible enzyme, and the other, from X. autotrophicusGJ10, is a constitutive enzyme. The former enzyme, havinga broad substrate specificity, dehalogenated C2 to C12haloalkanes, even some haloalcohols, and, though weakly,some haloacids. The latter enzyme hydrolyzed haloalkaneswith chains of less than five carbons and did not act onhaloacids. The pl values of the enzymes were 4.5 and 5.4,respectively.

Haloalkanes, which are produced industrially and notfound in nature, are called xenobiotics and are consideredenvironmental pollutants. On the other hand, several bacte-rial strains which can degrade haloalkanes have been iso-lated from the environment (3, 12, 24, 27, 33), and, asmentioned above, some dehalogenases which act on halo-alkanes have been purified. Organisms which possess thedehalogenase that acts on haloalkanes seem to play animportant role in the degradation of environmental pollut-ants. It would be interesting to investigate the physiologicalrole of the enzymes that act on xenobiotics and to elucidatehow the bacteria could acquire those enzymes.

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7. Edelhoch, H. 1967. Spectroscopic determination of tryptophanand tyrosine in proteins. Biochemistry 6:1948-1954.

8. Goldman, G. 1965. The enzymatic cleavage of the carbon-fluorine bond in fluoroacetate. J. Biol. Chem. 240:3434-3438.

9. Goldman, P., G. W. A. Milne, and D. B. Keister. 1968. Carbon-halogen bond cleavage. Ill. Studies on bacterial halidohydro-lases. J. Biol. Chem. 243:428-434.

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