ZHANG Jinyou,XIN YanjuanCAO XupengXUE Song,and ZHANG Wei

1) Marine Bioproducts Engineering Group,Dalian Institute of Chemical Physics,CAS,Dalian 116023,P. R.China

2) Flinders Centre for Marine Bioproducts Development (FCMB2),Medical Biotechnology,School of Medicine,Adelaide, SA 5042, Australia

3) University of Chinese Academy of Sciences,Beijing 100049, P. R.China

1 Introduction

To date,over 3800 natural organohalogen compounds,in addition to myriad xenobiotic compounds,have been identified or synthesized (Gribble,2003; Gribble,2012).Many of these compounds were synthesized by means of chemical methods and extensively utilized as pharmaceuticals,agrochemicals,and synthetic precursors.However,the wide application of such halogenated compounds caused severe environmental pollution.The bioremediation of these halogenated organic pollutants by microorganisms is an environmentally friendly approach to mitigate these problems.In the endeavors of bioremediation,many kinds of dehalogenases have been investigated and reported; for instance,2-haloacid dehalogenases can dehalogenate the halogenated 2-haloalkanoic acid (Fetzner and Lingens,1994).

The 2-haloacid dehalogenases have been purified and identified extensively in bacteria and were typically classified into four groups (Fetzner and Lingens,1994; Sodaet al.,1996): L-2-haloacid dehalogenase (L-DEX),D-2-haloacid dehalogenase (D-DEX),DL-2-haloacid dehalogenase (configuration inversion) (DL-DEXi),and DL-2-haloacid dehalogenase (configuration retention) (DLDEXr).L-DEX and D-DEX specifically act on the L and D isomers of 2-chloropropionate (2-CPA),respectively.Further study of the reaction mechanism L-DEX showed that the L-DEX firstly formed an ester intermediate with the substrate of L-2-CPA and then the intermediate formed the product and enzyme by attacking with a molecular water.While,the DL-DEXi firstly activate a molecular of water,then the activated water attacks the substrate of L and/or D-2-CPA to produce the product of D-lactate and/or L-lactate (Sodaet al.,1996).To date,the reaction mechanisms of DL-DEXr and D-DEX are still not clear.

Beside one archaeobacteriaSulfolobus tokodaii(Littlechild,2011),many bacteria which produce 2-haloacid dehalogenase have been isolated from the terrestrial environment (Diezet al.,1996; Leighet al.,1988; Motosugiet al.,1982; Van der Ploeget al.,1991),while few have been isolated from the marine environment (Fetzner and Lingens,1994; Huanget al.,2011b).The marine bacteria have been found to be the important resource for novel enzymes,chemical compounds,inhibitors,and so on.Thus increasing attention has been devoted to the study of marine bacteria.So far,many of those novel enzymes from maine bacteria have been proved to possess novel properties,such as favorable thermal stability,catalytic efficiency,tolerance to high salinity (Sarkaret al.,2010;Zhang and Kim,2010).Nowadays,the 2-haloacid dehalogenases that have been purified and characterized are produced by the microorganism which is isolated from the terrestrial environment,while the properties of 2-haloacid dehalogenases that are produced by bacteria isolated from the marine environment have not yet been studied.In our laboratory,bacteria possessing dehalogenation activities and being isolated from an intertidal marine spongeHymeniacidon perlevishave been identified and characterized (Huanget al.,2011a),but information about the dehalogenase from them remains unclear.Among them,DEH99 was identified as the first strain fromParacoccusand postulated to own a novel 2-haloacid dehalogenase (Huanget al.,2011b).In this study,we describe the successful purification and characterization of one novel 2-haloacid dehalogenase from marine bacteriumParacoccussp.DEH99.

2 Materials and Methods

2.1 Organism and Culture Conditions

StrainParacoccussp.DEH99 was isolated from marine spongeH.perlevis,using DL-2-chloropropionic acid(DL-2-CPA) as the sole carbon source (Huanget al.,2011 b).The bacterial cells were grown at 30℃ on a medium containing 12 g L?1Na2HPO4·12H2O; 1 g L?1NaH2PO4·2H2O;30 g L?1NaCl; 0.1 g L?1Yeast extract; 2.0 g L?1(NH4)2SO4;0.1 g L?1MgSO4·7H2O,1% trace element solution; and supplemented with 10 mmolL?1DL-2-CPA.Cells were collected by centrifugation (5857×g,4℃) at the late logarithmic phase and stored at ?80℃ after washing twice with potassium phosphate buffer (50 mmolL?1,pH 7.5)

2.2 Preparation of Crude Enzyme Extract

The frozen cells were resuspended in potassium phosphate buffer (pH 7.5,50 mmolL?1) with dithiothreitol(DTT,0.65 mmolL?1),and disrupted by sonication (350 W) for 180 cycles (each cycle consisted of working for 5 s and pausing for 5 s).Following centrifugation (14000 r min-1,30 min,4℃),the supernatant was obtained as crude enzyme extract for further experiments.

2.3 Dehalogenase Purification

All the purification steps were carried out at 4℃.All buffers contained 0.65 mmolL?1DTT.

The cell-free extract was salted out with ammonium sulfate at 40% and 60% saturation,respectively.The precipitate from the 40% saturation was removed via centrifugation at 14000 r min-1for 10 min.The precipitate from the 60% saturation was collected by centrifugation under the same conditions,and was resuspended in a small volume of potassium phosphate buffer (50 mmolL?1,pH 7.5).The resuspendant was then desalted and concentrated by ultrafiltration membrane with molecular weight cut off at 10 kDa.The concentrated resuspendant was loaded onto the Q-Sepharose HP column (0.7×2.5 cm)previously equilibrated with the potassium phosphate buffer (50 mmolL?1,pH7.5).After washing with 10 ml potassium phosphate buffer (50 mmolL?1,pH 7.5) and 10 mL potassium phosphate buffer (50 mmolL?1,pH 7.5)containing 0.03 molL?1Na2SO4,the elution was conducted with a linear gradient of 0.03?0.25 mmolL?1Na2SO4in potassium phosphate buffer (50 mmolL?1,pH 7.5).The flow rate was 1 mLmin?1.Fractions containing dehalogenase were pooled and concentrated.The concentrated dehalogenase was loaded on the Superdex 200 column(1.6×60 cm) and eluted at a flow rate of 0.5 mLmin?1.Fractions containing dehalogenase were pooled and concentrated by ultracentrifuge.Then,the concentrate was purified again by Superdex 200 column under the conditions described above.

2.4 Assay of Dehalogenase Activity

The activity of 2-haloacid dehalogenase was assayed according to the method of Huang (Huanget al.,2011b).The standard assay system (1 mL) contained 10 mmol L?1DL-2-CPA,100 mmolL?1Glycine-NaOH (pH 10.0),and the enzyme preparation.Following incubation at 30℃ for 30 min,the reaction was terminated by adding 10 μL phosphoric acid (85%).The assay system was then centrifuged (14000 r min-1,10 min) and the supernatant was used to determine the content of DL-2-CPA.One unit of the dehalogenase activity was defined as the amount of the enzyme that catalyzed 1 μmol of substrate per min.

The configuration of the lactate was determined by monitoring NADH production using a lactic acid detection kit (Megazyme,Megazyme International Ireland Ltd.,Ireland).

2.5 Determination of Protein Content

The protein concentration was determined using the method of Brandford (Bradford,1976),using bovine serum albumin (BSA) as a standard.

2.6 Determination of the Molecular Weight

The molecular weight of the native dehalogenase was determined by gel filtration using a Superdex 200 column(1.6×60 cm) on an FPLC system (AkataprimeTMPlus,GE healthcare,Sweden).The samples were eluted with potassium phosphate (50 mmolL?1,pH 7.5) containing 0.65 mmolL?1DTT at 0.5 mLmin?1.The following markers were used for calibration: dextran blue (2000 kDa),BSA (66.4 kDa),ovalbumin (44.3 kDa),and ribonuclease A (13.7 kDa).

The subunit molecular weight of the dehalogenase was determined on SDS-PAGE with 12% separating gels and 5% stacking slab gels.Following electrophoresis separa-tion,the gels were stained with Coomassie brilliant blue R-250.The molecular weight (MW) reference markers used were phosphorylase (97.2 kDa),BSA (66.4 kDa),ovalbumin (44.3 kDa),carbonic anhydrase (29.0 kDa),and lysozyme (14.3 kDa).

2.7 Effects of pH and Temperature

Within the pH range of 6.0?12.0 in the following buffers: K2HPO4-KH2PO4(pH 6.0?7.5),Tris-H2SO4(pH 8.0?9.5),and Glycine-NaOH (pH 9.5?12.0),the optimal pH for the dehalogenase was determined under standard assay conditions with a reaction time of 66 min.After incubating the dehalogenase at each desired pH for 24 h,the pH stability of the dehalogenase was examined by measuring the residual activity under standard assay conditions with a reaction time of 120 min.The optimal temperature of the dehalogenase was measured under the same standard assay conditions within the temperature range of 20?60℃.After incubating the dehalogenase at each desired temperature for 30 min,the thermal stability of the dehalogenase was assayed by determining the residual activity with a reaction time of 120 min.

2.8 Substrate Specificity

The substrate specificity of the dehalogenase was tested using a range of organohalogen substrates.The reaction system contained Glycine-NaOH (100 mmolL?1,pH 10.0),substrates (10 mmolL?1),and the enzyme in a total volume of 1 mL,and was incubated at 30℃ for 80 min.The residual amount of substrate was measured for each reaction by HPLC.

2.9 Effect of Inhibitors

The effect of several inhibitors on purified dehalogenase activity was investigated.The reaction system contained 10 mmolL?1DL-2-CPA,10 mmolL?1Glycine-NaOH (pH 10.0),inhibitors (1 mmolL?1),and the enzyme.Following incubation at 30℃ for 120 min,dehalogenase activity was determined.The inhibitors were as follows:EDTA,DTT,Cu2+,Zn2+,and Co2+.

2.10 Km and Vmax of Dehalogenase

Kinetic studies of the dehalogenase were carried out with different concentrations of L-2-CPA,and the rate of the dehalogenase reactions was determined.The Lineweaver-Burk double reciprocal plot was conducted according to the Michaelis-Menten equation using L-2-CPA concentrations of 0.5,1,2.5,5,7.5,10,and 15 mmolL?1L-2-CPA.

3 Results

3.1 Molecular Weight of the Dehalogenase

With a four-step approach,the 2-haloacid dehalogenase of Deh99 was purified from the marine bacteriaParacoccussp.DEH99.On the SDS-PAGE,a single band of purified Deh99 was observed at 25.0 kDa (Fig.1).The relative molecular weight for the native Deh99 was estimated to be 50.0 kDa by gel filtration on Superdex 200 column (Fig.2).The data indicated that the Deh99 was a dimer.

Fig.1 SDS-PAGE of dehalogenase.Lane 1,Marker; Lane 2,purified Deh99.

Fig.2 Relative molecular weight of the dehalogenase estimated by Superdex 200.1,Ribonuclease A (13.7 kDa); 2,Ovalbumin (44.3 kDa); 3,BSA (66.4 kDa).

3.2 Effects of pH and Temperature

The effect of pH on the activity of the dehalogenase was examined between pH 6.0 and 11.0.The dehalogenase showed that the optimal pH for the best activity was 10 (Fig.3).After 24 h incubation,the enzyme activity was stable within the range of pH 6.0?7.0,and declined rapidly at pH levels greater than 7.0 (Fig.3).The optimal temperature for dehalogenase activity was 40℃ at pH 10.0.The dehalogenase was stable between 4 – 30℃,and its activity declined rapidly at temperatures greater than 40℃ (Fig.4).

Fig.3 Effect of pH on the activity and stability of the dehalogenase.

Fig.4 Effect of temperature on the activity and stability of the dehalogenase.

3.3 Substrate Specificity

It was found that the Deh99 specifically acts on L-2-haloalkanic acid (Table 1).The Deh99 catalyzed dehalogenation of 2-haloalkanoic acid,but not halogenated propionamide nor 3-chlopropionate,indicating that halogen atoms at the C-2 position and a free carboxyl group are required for the substrates.All the monohaloalkanic acids,except 2-floropropionic acid,were dehalogenated by Deh99 effectively.The Deh99 showed the highest activity on the monochloroacetate.The dehalogenated product of the L-2-CPA was determined to be D-lactate (Table 2).

Table 1 Substrate specificity of dehalogenase from Paracoccus sp.DEH99

Table 2 Stereospecificity of Deh99

3.4 Kinetics of the Dehalogenase

The dehalogenase activity followed Michaelis-Menten kinetics over a substrate range of 0.5?10 mmolL?1for L-2-CPA,with an apparentKmvalue of 0.21 mmolL?1and aVmaxvalue of 2.5 μmol min?1mg?1.

3.5 Effect of Inhibitors

The effect of inhibitors on the dehalogenase activity was examined under standard assay conditions （Table 3）.The dehalogenase activity was strongly inhibited by Cu2+and Zn2+,and slightly impaired by DTT and EDTA.

Table 3 The effect of inhibitors on Deh99

4 Discussion

2-haloacid dehalogenases have been isolated and characterized from various bacteria,such asPseudomonassp.(Joneset al.,1992; Little and Wiliams,1971; Liuet al.,1994; Morsbergeret al.,1991; Tsanget al.,1988),Rhizobiumsp.(Cairnset al.,1996),Moraxellasp.(Kawasakiet al.,1992).Most of the bacteria were screened from the terrestrial environment,while few have been screened from the marine environment (Huanget al.,2011a).A bacteriumParacoccussp.DEH99,which possesses 2-haloacid dehalogenase activity,has been isolated and identified in our laboratory (Huanget al.,2011a,2011b).The 2-haloacid dehalogenase fromParacoccussp.DEH99,named Deh99,was purified in this study using a four-step process,and its homogeneity was confirmed by SDS-PAGE analysis.The dehalogenase activities of Deh99 were found in the bound fraction after Q-Sepharose FF anion exchange chromatography (pH 6.0,data not shown),which indicated that the isoelectric point of the purified enzyme may be below 6.0.The purified dehalogenase could act stereospecifically on L-2-CPA,and the product was D-lactate.According to the substrate specificity,the dehalogenase fromParacoccussp.DEH99 belongs to the L-DEX family of dehalogenases.

Members of the L-DEX family of dehalogenases possess some common features; for instance,they have an optimal pH range of 9?11.The L-DEX family of dehalogenases includes monomers (Diezet al.,1996; Little and Wiliams,1971; Motosugiet al.,1982),dimers (Liuet al.,1994; Morsbergeret al.,1991; Schneideret al.,1991;Tsanget al.,1988),and tetramers (Joneset al.,1992),with the molecular weights of subunits between 15 kDa and 29 kDa.The purified Deh99 is a dimer with a subunit molecular weight of 25.0 kDa,which is close to those of the dimer dehalogenases fromPseudomonassp.strain YL(27 kDa) (Liuet al.,1994),Pseudomonassp.strain CBS3(29 kDa) (Morsbergeret al.,1991; Schneideret al.,1991),andPseudomonas cepaciaMBA4 (25.9 kDa) (Tsanget al.,1988).The activity of Deh99 on halogenated acetate is higher than that on the halogenated propionate,which resembles the characteristics of dehalogenase (HdlIVa)fromP.cepaciaMBA4 (Tsanget al.,1988),dehalogenase (DehII) fromPseudomonassp.strain CBS3(Morsbergeret al.,1991; Schneideret al.,1991),and dehalogenase (L-DEX) fromPseudomonassp.strain YL(Liuet al.,1994).However,the activities of Deh99 on monochloroacetate and dichloroacetate are higher than the known L-DEX above mentioned on monobromoacetate.The estimatedKmof the Deh99 is 0.21 mmolL?1for L-2-CPA,which is close to that of the 2-haloacid dehalogenases (Km=0.3?0.4 mmolL?1) (Little and Wiliams,1971;Liuet al.,1994).

The Deh99 is hardly affected by DTT and EDTA,which is similar to the cases with 2-haloacid dehalogenases fromPseudomonassp.strain YL (Liuet al.,1994),Pseudomonassp.strain CBS3 (Schneideret al.,1991),andP.cepaciaMBA4 (Tsanget al.,1988).The activity of Deh99 can be inhibited strongly by Cu2+while that of other dimmer dehalogenases can not (Tsanget al.,1988).Although the activities of 2-haloacid dehalogenases fromAzotobactersp.strain RC26 andP.putida109 were also inhibited greatly by Cu2+,those two dehalogenases were monomer (Diezet al.,1996; Motosugiet al.,1982).Furthermore,the activity of Deh99 was inhibited 29.5% and 87% by Co2+and Zn2+respectively,whereas activity of HdlIVa fromP.cepaciaMBA4 was inhibited up to 30% by Co2+and Zn2+(Tsanget al.,1988).

In summary,2-haloacid dehalogenase (Deh99),for the first time,was purified and characterized from marine bacteriaParacoccussp.DEH99.Deh99 showed some novel properties with regard to substrate specificity and inhibitory effects.Considering its promising chiral selectivity and dehalogenation,the Deh99 present in this study may have potential applications in the areas of environmental protection and fine chemistry.

Acknowledgements

This work was financially supported by National Basic Research Program of China (973 program,Grant No.2009CB724700),the Hundred Talent Program of the Chinese Academy of Sciences (A1097) and National Natural Science Foundation of China (No.31100092).

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