Novel Methodology for Enzymatic Removal of Atrazine from Water by

The Otto Warburg Center for Agricultural Biotechnology,. Faculty of Agriculture ... expression of two fusion proteins which dechlorinate atrazine whil...
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Environ. Sci. Technol. 2000, 34, 1292-1296

Novel Methodology for Enzymatic Removal of Atrazine from Water by CBD-Fusion Protein Immobilized on Cellulose CARL KAUFFMANN AND ODED SHOSEYOV* The Kennedy Leigh Center for Horticultural Research and The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel ETAI SHPIGEL CBD-Technologies Ltd., 2 Pekeris Street, Park Tamar, P.O. Box 199, Rehovot 76100, Israel EDWARD A. BAYER Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel RAPHAEL LAMED Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel YUVAL SHOHAM Department of Food Engineering and Biotechnology, Technion - IIT, Haifa 32000, Israel RAPHI T. MANDELBAUM Institute of Soil, Water and Environmental Sciences, Volcani Research Institute, Bet Dagan 50250, Israel

s-Triazines including atrazine are heavily used agricultural herbicides, and their extensive removal from industrial wastewater is required before these effluents can be disposed. The use of enzymes for this purpose is an important potential alternative to conventional methods of detoxification. The purpose of the present study was to develop an enzymebased technology for the treatment of atrazine contaminated water. In this paper we describe the construction and expression of two fusion proteins which dechlorinate atrazine while being firmly bound to an insoluble cellulose matrix. Dechlorination of atrazine produces in one enzymatic step the nonregulated compound hydroxyatrazine from the regulated mother compound, atrazine.

Introduction Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] is a commonly used herbicide. It is currently one of the two most widely used agricultural pesticides in the United States and is registered worldwide in more than 70 countries. Atrazine is considered to be somewhat persistent in water and mobile in soil. For these reasons, the use of atrazine has been prohibited in most European Union countries. In * Corresponding author phone: (972)-8-948-1084; fax: (972)-8946-2283; e-mail: [email protected]. 1292

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America, however, it is still among the most frequently detected pesticides in groundwater (1). Extensive removal of atrazine from wastewater produced by atrazine manufacturing plants is required before these effluents can be disposed. Removal and/or detoxification of pollutants can be accomplished by physical, chemical, or biological means, the latter being of special interest because of its potential low cost and its nominal effect on the environment. To date, removal of atrazine from production effluents is primarily achieved by adsorption to granular activated carbon, followed by biological treatment. The use of enzymes to detoxify industrial wastewater failed to attract much attention due to the high cost of enzymebased systems. However, as toxic wastewater treatment is becoming increasingly expensive and efficient enzyme production methods are being developed, new interest is being paid to enzymatic wastewater treatment. The idea of applying peroxidase, laccase, and/or tyrosinase for the removal of phenols and aromatic amines from drinking water and wastewater emerged in the 1980s, and research in this field is continuing (2-6). The use of nitrile hydratase and amidase for removal of the highly toxic and carcinogenic acrylonitrile is another area where the use of enzymes for detoxification is sought (7). Immobilization of biologically active proteins is of significant industrial importance. In the immobilized state, enzymes are often more stable and can be recycled, thus reducing operational costs (8). In recent years the use of genetic engineering to construct fusion proteins (chimeric proteins) containing a functional domain (catalytic or otherwise) together with an affinity tag (a binding domain) has received much attention. Several gene fusion systems have been published in recent years (see ref 9 for review). However to date, affinity tags such as six histidine residues (10), glutathione S-transferase (11), or mannose-binding protein (12) have all required relatively expensive affinity matrices, limiting their commercial application in an industrial setting. Cellulose-binding domains (CBDs) provide a new class of affinity tags with appealing attributes. These include the following: (i) effective immobilization of CBD fusion proteins to cellulose-based matrices without the need for covalent cross-linking and (ii) a readily available, low-cost affinity matrix (cellulose) with inherently low, nonspecific proteinbinding characteristics. CBDs thus provide a specific means for linking enzymes or other proteins on cellulose (13-19). Here we report the construction, expression, and immobilization on cellulose of two different CBD fusion proteins. One construct consisted of CBDct (a CBD isolated from Clostridium thermocellum) fused to atrazine chlorohydrolase (AtzA), a novel enzyme which dechlorinates atrazine to the nonregulated compound hydroxyatrazine (20). The second construct comprised three components: Protein-A fused to CBDcc (a CBD isolated from Clostridium cellulovorans) fused to AtzA. The two CBD fusion proteins dechlorinated atrazine while being immobilized on cellulose.

Experimental Section E. coli DH5R and E. coli XL-1 Blue were used for standard cloning, while expression was carried out in E. coli BL21(DE3) and E. coli BL21(DE3)pLysS, as indicated; the plasmid used was pET-3d (all from Novagen Inc., Madison, WI). Restriction enzymes were from MBI Fermentas, Vilnius, Lithuania. Atrazine, [∆-14C]hydroxyatrazine, and [∆-14C]atrazine were gifts from Novartis, Greensboro, NC. All other 10.1021/es990754h CCC: $19.00

 2000 American Chemical Society Published on Web 03/03/2000

chemicals were purchased from standard sources and were of the best available grade. M9 minimal medium was 41 mM Na2HPO4, 22 mM KH2PO4, 8.4 mM NaCl, 18 mM NH4Cl, 0.4% (w/v) glucose, 2 mM MgSO4, 0.1 mM CaCl2 and 1 mM thiamine-HCl. TB medium was 1.2% (w/v) Bacto-tryptone, 2.4% (w/v) Bacto-yeast extract, 0.4% (v/v) glycerol, 72 mM K2HPO4, and 17 mM KH2PO4. LB-medium was 1% (w/v) Bacto-tryptone, 1% (w/v) NaCl, and 0.5% (w/v) Bacto-yeast extract, with the pH adjusted to 7.5 with 5 M NaOH. Construction of pET-atzA and pET-CBDcc‚atzA. Pseudomonas sp. strain ADP (21) was grown in LB-medium, and the DNA was extracted from the cells applying standard methods (22). The atzA gene (20) was PCR-amplified using Pfu DNA polymerase (Stratagene, La Jolla, CA) and primers (BTG Ltd., Rehovot, Israel), designed to add an NcoI site at the N-terminus of the atzA gene and a BamHI site at the C-terminus. The N-terminal primer was 5′ AAA ACC ATG GCG CAA ACG CTC AGC ATC C 3′, and the C-terminal primer was 5′ AAA AGG ATC CTA GAG GCT GCG CCA AG 3′. The 1.5 kb (kilo bases) PCR product was digested with NcoI/ BamHI and ligated into pET-3d and pET-CBD180 (19) (predigested with the same enzymes) resulting in pET-atzA and pET-CDBcc‚atzA, respectively. Construction of pET-CBCct‚atzA. The CBDct coding sequence was PCR-amplified using pCBD (23) as a DNA template and VENT DNA polymerase (New England Biolabs, Beverly, MA). The T7 Promoter Primer (Promega, Madison, WI) was used as the N-terminal primer. The C-terminal primer, designed to add an NcoI site to the C-terminus, was 5′ AAA ACC ATG GAT ACT ACA CTG CCA CCG 3′. The 0.5 kb PCR product was digested with NcoI and ligated into pETatzA, which had been predigested with the same enzyme, thus resulting in pET-CBDct‚atzA. Construction of pET-Protein-A‚CBDcc‚atzA. The pETCDBcc‚atzA plasmid was digested with NdeI/BamHI, and a 1.7 kb fragment, consisting of atzA and 0.2 kb of the C-terminus of the CBDcc gene, was isolated from the digest. This fragment was ligated into pET-Protein-A‚CBD (Shoseyov, O., unpublished results), which had been digested with the same enzymes, resulting in pET-Protein-A‚CBDcc‚atzA. The presence of the cloned genes was confirmed by restriction-site analysis, by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of the proteins expressed by the four different clones and by activity tests of the recombinant proteins. Dechlorination of Atrazine by CBDcc•atzA Inclusion Bodies. E. coli BL21(DE3)pET-CBDcc‚atzA was grown overnight in M9 minimal medium containing 50 µg/mL ampicillin. After 1:80 dilution in TB medium containing 100 µg/mL ampicillin, cells were grown to 1.5 OD600 nm at 37 °C. At this point, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM. After an additional 4 h, the cells were harvested by centrifugation (6000g, 10 min, 4 °C) and resuspended in 50 mM Tris buffer, pH 8. The cells were lysed by three passages through a homogenizer (type 8.30H, Rannie, Denmark) applying an internal pressure of 10 000 psi. Inclusion bodies were isolated by centrifugation (9000g, 10 min, 4 °C) and washed once with 1% Triton X-100 and three times with water. Inclusion bodies (14 mg/mL), CoSO4 (1 mM), atrazine (33 ppm with ∆-14C atrazine added to a final concentration 56 000-60 000 CPM/mL), and phosphate buffer (50 mM, pH 7.5) were mixed in 1.5 mL microtubes. Samples (200 µL) were taken at t ) 0 h and t ) 22 h and extracted with ethyl acetate (200 µL). After vortex and centrifugation (10 000g, 5 min, room temperature), aliquots (80 µL) from the aqueous and from the organic phases were added to a channeled TLCplate with preadsorbent zone (Uniplate, Silica Gel, HLF, 20 × 20 cm, Analtech, Newark, DE). The TLC plate was developed

using a chloroform-methanol-formic acid-water (75:20: 4:2, vol/vol) solvent system. After developing, the plates were scanned using a model BAS1000 BioImaging Analyzer System (Fugix Co., Japan). Dechlorination of Atrazine by Cellulose-Immobilized Enzyme Constructs. E. coli BL21(DE3)pLysS host strains, harboring either the pET-atzA, pET-CBDct‚atzA or pETProtein-A‚CBDcc‚atzA plasmid, respectively, were grown in 2 mL cultures with agitation (37 °C, 250 rpm) in LB medium, containing 34 µg/mL of chloramphenicol and 100 µg/mL of of ampicillin. When the cultures reached a turbidity (OD600 nm) of 2.4-3.4, the cells were harvested and stored overnight at 4 °C. The cells were then collected by centrifugation (10 000g, 1 min, room temperature) and resuspended in fresh LB medium, containing the same antibiotics. Each culture was used to inoculate 100 mL of fresh medium. The cells were then incubated with agitation (250 rpm) for 3 1/2 h at 37 °C. At this point, the temperature was lowered to 30 °C, IPTG was added to a final concentration of 0.4 mM, and the fermentation was continued for an additional 2 h. The fermentation flasks were placed on ice for 15 min before the cells were harvested by centrifugation (4000g, 5 min, 4 °C). The cells were resuspended in 0.25 culture volume of cold 50 mM MOPS (3-[N-morpholino]propanesulfonic acid), pH 7, 2 mM EDTA (ethylenediaminetetraacetic acid) and centrifuged as above. The supernatant fluids were removed, and the cells were stored at -20 °C. The cells were thawed, whereby the cell-associated lysozyme (pLysS) was released, resulting in cell lysis. The protein was extracted by adding 10 mL of 50 mM MOPS pH 7, 1 mM CoSO4, and 0.5 mM PMSF (phenylmethylsulfonyl fluoride) to each frozen pellet and by rotating the suspensions for 20 min at room temperature. The cell lysates were centrifuged (30 000g, 30 min, 4 °C), and the supernatant fluids were used for subsequent cellulose-binding and activity experiments. To bind the CBD fusion proteins to cellulose, 4 mg of cellulose (SigmaCell Type 20, Sigma, St. Louis, MO) was added to 2 mL of clarified crude cell extract, and the suspension was rotated slowly for 1 h at room temperature. The suspensions were then centrifuged (4000g, 5 min, 4 °C), and the supernatant fluids were decanted. The cellulose pellets were washed three times: once with 2 mL of 1 M NaCl, 50 mM MOPS, pH 7, and twice with 50 mM MOPS, pH 7. After each wash, performed by slow rotation of the tube for 30 min at room temperature, the suspensions were centrifuged (4000g, 5 min, 4 °C), and the supernatant fluids were discarded. After the last wash, the cellulose pellets were resuspended in 5 mL of 50 mM MOPS, pH 7. The suspensions were transferred to fresh tubes and centrifuged, and the supernatant fluids were decanted. The cellulose-bound cell fraction, the nonbound fraction, and the initial crude cell extract were examined for atrazine-dechlorinating activity. Equivalent amounts of the desired bound, nonbound, or crude samples were brought to a final volume of 4 mL of substrate solution (30 ppm atrazine in 50 mM MOPS, pH 7, containing 1 mM CoSO4). The assays were incubated with slow rotation at room temperature, and the activity was determined by measuring the disappearance of atrazine and the concomitant appearance of hydroxyatrazine. One unit (U) was defined as the conversion of 1 µmol of atrazine to hydroxyatrazine per minute. Aliquots (0.6 mL) were taken from the reaction assays at t ) 0 min, 10 min, 30 min, 1 h, and 20 h and quenched by rapid mixing with an equal volume of methanol. The quenched samples were stored at -20 °C until analyzed for atrazine and hydroxyatrazine. The concentration of atrazine and hydroxyatrazine was determined by HPLC as described previously (24). Stability Assay of Cellulose-Immobilized CBDct‚AtzA. CBDct‚AtzA was produced, bound to cellulose, and assayed VOL. 34, NO. 7, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. TLC chromatogram showing the conversion of atrazine to hydroxyatrazine by CBDcc‚AtzA inclusion bodies. Marker A ) authentic atrazine standard. Marker H ) authentic hydroxyatrazine standard. The control experiment was performed without the addition of inclusion bodies.

FIGURE 1. Schematic representation of the four constructs, containing the atzA, CBDcc, CBDct, and/or Protein-A gene, cloned in the multiple cloning site of the pET-3d vector downstream to the T7 promoter. for atrazine dechlorinating activity as described above with the following modifications: 5 mg of cellulose (beaded cellulose, 80-100 µm, Sigma, St. Louis, MO) were reacted with 1.1 mL of clarified crude extract; the cellulose-bound fraction was assayed for 1 h once a day for 5 successive days each time with 1 mL of substrate solution (15 ppm atrazine, otherwise as described above); the cellulose-bound fraction was maintained in substrate solution on a rotary shaker at room temperature between the activity assays.

Results and Discussion

FIGURE 3. SDS-PAGE analysis of proteins expressed in E. coli transformed with pET-AtzA, pET-CBDct‚AtzA, or pET-Protein-A‚ CBDcc‚AtzA. A: Total proteins (soluble and insoluble). B: Soluble proteins. C: Cellulose bound proteins. Lane 1: pET-AtzA. Lane 2: pET-CBDct‚AtzA. Lane 3: pET-Protein-A‚CBDcc‚AtzA. M ) high molecular weight standard mixture (Sigma Chemical Co., St. Louis, MO).

In this study, the potential for dechlorination of atrazine in aqueous solutions was examined, using cellulose-bound, CBD-tagged enzymes. Four different constructs: atzA, CBDcc‚ atzA, CBDct‚atzA, and Protein-A‚CBDcc‚atzA (Figure 1) were cloned and expressed in E. coli using the pET-3d vector. The atzA is a gene which encodes the newly described enzyme, atrazine chlorohydrolase (20). The CBDcc‚atzA construct includes a CBD from the cellulolytic mesophile, C. cellulovorans, fused to the latter enzyme. CBDct‚atzA, is a similar construct, in which an analogous CBD from the thermophilic strain, C. thermocellum, is substituted for the CBD from C. cellulovorans. The final construct includes a Protein-A component fused to CBDcc‚atzA. The Protein-A component imparts a degree of solubility to the expressed protein (25). Dechlorination of Atrazine to Hydroxyatrazine by CBDcc‚AtzA Inclusion Bodies. The expression of the initial fusion protein, CBDcc‚AtzA, appeared to result in the exclusive formation of insoluble inclusion bodies. Efforts to refold CBDcc‚AtzA into a soluble and fully active, bifunctional protein were unsuccessful. However, the CBDcc‚AtzA inclusion bodies exhibited low but measurable levels of atrazine dechlorination activity. The conversion of [∆-14C]atrazine to [∆-14C]hydroxyatrazine by CBDcc‚AtzA inclusion bodies is illustrated in Figure 2. Atrazine was fully converted to hydroxyatrazine after 22 h, while no conversion was detected in the negative control. Positive controls (not shown), in which similar amounts of soluble AtzA was included in the assay system, commonly resulted in total conversion of atrazine to hydroxyatrazine within 15 min. Expression and Binding to Cellulose of CBDct‚AtzA and Protein-A‚CBDcc‚AtzA. Soluble and active AtzA, CBDct‚ AtzA, and Protein-A‚CBDcc‚AtzA were obtained by reducing the total fermentation time to 5 1/2 h and by lowering the postinduction fermentation temperature to 30 °C. Figure 3

shows the SDS-PAGE analysis of the expression of AtzA, CBDct‚AtzA, Protein-A‚CBDcc‚AtzA and of the immobilization of the latter two fusion proteins to cellulose. Figure 3A illustrates total cell protein (soluble plus insoluble) and shows strong expression of AtzA (predicted size 50 kDa) and of CBDct‚AtzA (predicted size 67 kDa), while no significant overexpression of Protein-A‚CBDcc‚AtzA (predicted size 110 kDa) is indicated. Figure 3B illustrates total soluble proteins. While no obvious protein bands of CBDct‚AtzA and ProteinA‚CBDcc‚AtzA were observed, a protein band corresponding to the expected molecular weight of AtzA was detected. Total protein concentration in the soluble fractions were 0.91, 1.4, and 1.3 mg/mL for atzA, CBDct‚atzA, and Protein-A‚CBDcc‚ atzA clones, respectively. Figure 3C illustrates the protein fractions from each extract that were bound to cellulose. After washing the cellulose extensively, both CBDct‚AtzA, and Protein-A‚CBDcc‚AtzA remained bound (Figure 3C, lanes 2 and 3), while no AtzA was found to be bound to the cellulose (Figure 3C, lane 1). Seventeen and 18 µg/mL CBDct‚AtzA and Protein-A‚CBDcc‚AtzA were bound to the cellulose, respectively. Dechlorination of Atrazine by Cellulose-Immobilized Enzyme Constructs. Cellulose-bound CBDct‚AtzA and Protein-A‚CBDcc‚AtzA degraded atrazine to hydroxyatrazine. The conversion of atrazine to hydroxyatrazine by immobilized Protein-A‚CBDcc‚AtzA is illustrated in Figure 4. The figure shows three overlapping HPLC chromatograms, illustrating the disappearance of atrazine and concomitant appearance of hydroxyatrazine during the initial 30 min of an activity assay with Protein-A‚CBDcc‚AtzA bound to cellulose. After 1 h 98% of the atrazine had been depleted, and the concentration was found to be 0.7 ppm or 0.003 mΜ. After 20 h, atrazine was no longer detectable in our assay

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FIGURE 6. Time course analysis of relative activity and relative amount of immobilized protein in a 5 day stability assay of celluloseimmobilized CBDct‚AtzA. Values are shown as percentages of values at day one. FIGURE 4. Time course analysis by HPLC of atrazine dechlorination to hydroxyatrazine by Protein-A‚CBDcc‚AtzA bound to cellulose. The overlapping graphs show the concentration of atrazine and hydroxyatrazine at times 0, 10, and 30 min. The concentration of atrazine decreased from 0.11 µM (at t ) 0 min) to 0.03 µM (at t ) 30 min), while the concentration of hydroxyatrazine increased from 0.00 µM (at t ) 0 min) to 0.07 µM (at t ) 30 min).

FIGURE 5. Enzyme specific activities of crude soluble and cellulose bound recombinant proteins produced in E. coli transformed with pET-AtzA, pET-CBDct‚AtzA, or pET-Protein-A‚CBDct‚AtzA. conditions. The conversion of atrazine to hydroxyatrazine by immobilized CBDct‚AtzA proceeded in a similar manner (data not shown). Figure 5 shows the comparative specific activities of the crude soluble and cellulose-bound proteins of the of the three constructs. The results clearly demonstrate that fusion of AtzA to both CBDs enabled the immobilization of active enzymes on insoluble cellulose, while AtzA alone was unable to bind to the cellulose. The increase in the specific activities of the CBD-fused enzymes when bound to the cellulose is attributed to the purification factor rather than the true specific activity of the enzyme. The specific activity of CBDct‚ AtzA bound to the cellulose found in this work was 3 orders of magnitude higher than that reported previously for the same enzyme immobilized in sol-gel glass (26) and 5 times higher than that obtained with an improved matrix of the same sol-gel system (24). This indicates that the immobilization of AtzA via CBD to cellulose is comparable or superior to previously reported methods. Stability Assay of Cellulose-Immobilized CBDct‚AtzA. Cellulose-bound CBDct‚AtzA maintained a significant part of its activity throughout the experiment (5 days). Figure 6 illustrates the activity of cellulose-bound CBDct‚AtzA and the amount of bound protein relative to the values at day one in a 5 day experiment. After an initial loss of activity of about 45% from day one to day two, the cellulose-bound CBDct‚AtzA maintained its activity over the remaining 4 days without any further loss. The loss of activity in the first day is probably related to instability of the enzyme and cannot

be attributed to enzyme leakage from the cellulose matrix since it was shown that the amount of the immobilized enzyme was constant throughout the experiment. This indicates that CBD is an effective immobilization tag. We have demonstrated the feasibility of applying an enzyme-based system to convert atrazine, an important herbicide and a significant source of environmental pollution, to its dechlorinated and nonregulated hydroxy derivative. Hydroxyatrazine is nonherbicidal (27) and does not leach from soil as readily as atrazine (28). Therefore, enzymatic dechlorination should be considered for treating industrial wastewater that contains high levels of atrazine (20). The enzyme, atrazine chlorhydrolase, was attached to an inexpensive insoluble resin (cellulose) by virtue of a CBD tag. The use of the resultant fusion protein is an example of CBD platform technology for purification and immobilization of biologically active proteins. Under the experimental conditions of this study the CBD from C. thermocellum appeared more soluble than the CBD from C. cellulovorans. Addition of Protein-A to CBDcc rectified its inherent insolubility. Further development of CBD-fusion enzymes may open the possibility for efficient production of cellulose based biofilters for enzymatic remediation of atrazine-polluted water and possibly other toxic compounds.

Acknowledgments C.K. wishes to thank the Danish Natural Science Research Council for financial support.

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Received for review July 7, 1999. Revised manuscript received January 3, 2000. Accepted January 5, 2000. ES990754H