- Email: [email protected]
Improved Catalytic Performance of a 2-Haloacid Dehalogenase from Azotobacter sp. by Ion-Exchange Immobilisation
JOBNAME: BBRC 220#3 PAGE: 1 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 828–833 (1996)
0489
Improved Catalytic Performance of a 2-Haloacid Dehalogenase from Azotobacter sp. by Ion-Exchange Immobilisation A. Diez, M. I. Prieto, M. J. Alvarez, J. M. Bautista, A. Garrido, and A. Puyet1 Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain Received February 18, 1996 The stability and catalytic efficacy of the L-2-haloacid dehalogenase isolated from Azotobacter sp. RC26 were studied after immobilisation on a DEAE-Sephacel solid matrix. While the optimum temperature for the soluble dehalogenase falls in the range of 30–40°C, the activity of the immobilised enzyme shows a four-fold increase at 60°C. Immobilisation on a plug-flow bioreactor extends the range of usable substrate concentration. The improved catalytic characteristics after immobilisation of the haloacid dehalogenase may be relevant for its possible utilization in biotechnological applications ranging from waste treatment to synthesis of steroisomers. © 1996 Academic Press, Inc.
Dehalogenase activities found in many microorganisms may be used for the transformation of halogenated xenobiotic compounds, such as pesticides, herbicides, solvents and other industrial products (1,2). Most current applications rely on the utilization of degradative bacterial strains, either by inoculation of contaminated sites (3,4) or immobilisation in bioreactors (5–7). The use of bioreactors may be particularly useful in applications where containment or extensive control of the system is required, as is the case of utilization of genetically modified microorganisms. An alternative to the utilization of biofilm-based reactors is the immobilisation of the bio-catalyst. This would only be feasible when the dehalogenating reaction is catalysed by enzymes relatively resistant to unfavourable environmental conditions, and whose activity do not require the addition of expensive cofactors. Some enzymes involved in dehalogenation, like the 2-haloalkanoate dehalogenases (EC 3.8.1.2.), display regio- and chiral specificity which, in addition to optically active compounds, replacing expensive conventional chemistry (8). These enzymes catalyze hydrolytic dehalogenation of 2-chloropropionate (2MCPA) and monochloroacetate (MCA) to yield optically active lactic acid and glycolate respectively. Although strains of Pseudomonas producing 2-haloalkanoic dehalogenases are currently used for the production of D-chloropropionate and D-lactate from racemic mixtures of 2-MCPA (9), little information is available on the performance of bacterial dehalogenases after immobilisation on solid supports. The performance of a D-2-haloacid dehalogenase from Pseudomonas putida coupled to CNBr-controled pore glass has been studied recently (10). In this study, we have analyzed the behaviour after immobilisation of a L-2-haloalkanoic dehalogenase isolated from Azotobacter sp. strain RC26, both in batch and in a column reactor. MATERIALS AND METHODS Enzyme preparation. Cultures of Azotobacter sp. RC26 (11) were grown to stationary phase on 2 l of mineral medium (12) supplemented with 50 mM MCA. Cells were harvested in late exponential, resuspended in elution buffer (50 mM Tris-acetate, 0.4 mM EDTA, 1 mM b-MSH and 5% glycerol pH 8.5) and lysed by ultrasonication. Cell debris was eliminated by centrifugation at 100,000 xg for 30 min. Crude extracts were precipitated with ammonium sulphate at 55% saturation, solubilized in 5 ml of elution buffer and loaded in a Superdex 200 gel filtration column. Proteins were eluted with the same buffer. Fractions showing dehalogenase activity on MCA were pooled, loaded on a Mono-Q ionic exchange column and eluted with a 0–0.2 M gradient of Na2 SO4 on elution buffer. Dehalogenase activity eluted at approximately 1
Corresponding author. Fax: +341-3943824 828
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: BBRC 220#3 PAGE: 2 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.07 M Na2 SO4. Active fractions were pooled and desalted in a gel filtration column, and subsequently loaded in an hydroxylapatite column equilibrated with 10 mM potassium phosphate pH 7.4 buffer. Dehalogenase activity was not retained in the column. Approximately 200 mg of enzyme was isolated from 10 g (wet weight) of cells. Enzyme activity. Enzymatic activity on halogenated compounds was measured by chloride release detection as described (13) after incubation at 30°C for 30 min in TA buffer (Tris-acetate 0.1M pH 8.5) supplemented with 2 mM MCA. Activity of DEAE-bound dehalogenase in standard conditions was detected after removal of the Dehalogenase-DEAE-Sephacel complex by centrifugation, and quantitation of chloride in the supernatant. Immobilisation. Unless otherwise specified, adsorption of the 2-haloacid dehalogenase to the solid matrix was performed by incubation of approximately 0.1 mg of pure enzyme with 0.4 g of DEAE-Sephacel equilibrated in buffer TA at 25°C for 10 min. The mixture was centrifuged at 12000 rpm for 1 min and resuspended in the same buffer. Column reactor design. Approximately 0.2 mg of DEAE-immobilised RC26 haloacid dehalogenase were packed in a 1 ml column (4.6 × 60 mm). To test the efficacy of the system, TA buffer containing MCA was injected at 21°C with a constant flow of 5 ml/h. Chloride release in the effluent was monitored colorimetrically as described above. Enzyme kinetics. The kinetic constants were calculated by measuring initial velocities of halide production at varying initial substrate concentrations. A detailed description of the procedure has been outlined (11). Km values were derived from non-linear regression analysis with the program Enzfitter from Biosoft[PT].
RESULTS AND DISCUSSION Immobilisation of the RC26 haloacid dehalogenase DEAE-Sephacel. Based on the observed retention capacity by a DEAE-based chromatography matrix, and the good results obtained after immobilisation of several enzymes to ion-exchange solid supports (14,15), we examined the behaviour of RC26 haloacid dehalogenase after immobilisation on DEAE-Sephacel. The retention capacity of DEAE for the protein is shown in Fig. 1. Under the conditions tested (pH 8.5, 21°C), 3.5 g (wet weight) of DEAE-Sephacel were necessary to bind one milligram of protein. Saturation occurred at about 300 mg of protein per g of solid matrix. Routinely, 0.3 mg of purified enzyme were mixed with 1 g of DEAE-Sephacel equilibrated in TA buffer. At this protein:matrix ratio, less than 12% of the dehalogenase activity could be detected in the supernatant after sedimentation, indicating that the enzyme was successfully attached to the solid support. Effect of pH. The activity of the 2-haloacid dehalogenase at different pHs was inspected using 0.1 M acetate (pH 3–7), 0.1 M Tris-acetate (pH 7–10) and 0.1 M sodium bicarbonate (pH 10–12) buffers to attain acidic, neutral and basic buffered environment in the assay, respectively. The
FIG. 1. DEAE-Sephacel binding ability for RC26 haloacid dehalogenase. Increasing amounts of DEAE-Sephacel were added to 100 mg of purified enzyme, and dehalogenase activity on MCA was assayed in supernatants after sedimentation of the mixture. Enzyme activity values are shown as percentage of the total activity found in the soluble fraction (supernatant). 829
JOBNAME: BBRC 220#3 PAGE: 3 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
immobilised and soluble enzymes showed similar activity profiles from pH 3 to 12, with optimal values between pH 8 and 10 (not shown). The residual activity at pH 8, after incubation for 30 min at different pH, was also tested (Fig. 2). The results indicate that the immobilised enzyme is less affected by shifts at extreme alkaline pH than the soluble enzyme. Effect of temperature. The effect of temperature on the immobilised RC26 haloacid dehalogenase was analysed in two ways. Approximately mg of protein either soluble or immobilised were incubated for 30 min at different temperatures in the presence of 2 mM MCA. The profile of activity at different temperatures is shown in Fig. 3a. The soluble enzyme displays significant activity at temperatures ranging from 20°C to 60°C, with an optimal between 30°C–40°C. Remarkably, the optimal temperature for the bound protein shifted to 60°C, showing a linear increase from 30 to 50°C. The calculated activation energy, using the Arrhenius equation, was 10 kcal. Compared to the activity of the soluble protein at 30°C, a four-fold increase in chloride production is observed for the immobilised dehalogenase at 60°C. A second set of experiments were carried out to show the stability of the enzyme after incubation at different temperatures (Fig. 3b). Incubation at 50°C to 60°C for 10 min prior to the activity assay at 30°C reduce the activity of the soluble enzyme to approximately 50%; however, the DEAE attached dehalogenase appears to retain more residual activity after a heat shift up to 60°C, suggesting that the binding of the enzyme to the solid support favours its protection against inactivation or denaturation. Incubation at 70°C during 30 min. inactivates the enzyme, either soluble or immobilised. The enhancement of thermal and pH range stability upon immobilisation of enzymes has been frequently observed (16,17), probably caused by the formation of ionic bonds between the protein and DEAE may stabilise the conformation of the protein. The observed activity increase at high temperatures of the L-2-haloacid dehalogenase of RC26 after immobilisation may also be a consequence of the structure stabilisation. This change of optimal temperature was not observed for the D-2-haloacid dehalogenase of Pseudomonas putida AJ1/23 (10). Stability of the binding to DEAE-Sephacel. Protein loss due to the lability of the bonds is one of the main drawbacks to be considered in ionic-binding methods. The potential use of enzymes bound to solid supports rely on their long term storage stability and the possibility of recover and reuse the enzyme. A single batch of immobilised enzyme could be reused at least 7 times without significant loss of activity. After several washes in MCA-free buffer to eliminate substrate and products, the enzyme-DEAE-Sephacel complex showed 100% of the initial activity, indicating that
FIG. 2. Stability of immobilised dehalogenase at different pH. Dehalogenase activity at pH 8 after incubation for 30 min at the indicated pHs. V, soluble; ■, immobilised. 830
JOBNAME: BBRC 220#3 PAGE: 4 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Activity and stability at different temperatures. (a) Chloride released by the RC26 dehalogenase after 30 min incubation at different temperatures with 2 mM MCA; (b) activity at 30°C after incubation of the enzyme at the temperatures indicated for 10 min. 100% corresponds to the maximum activity observed. V, soluble; ■, immobilised.
the enzyme loss or inactivation is negligible under these conditions (Fig. 4). In addition, immobilised RC26 haloacid dehalogenase could be stored for two months at 4°C in TA buffer retaining 60% of its activity. Kinetics. The reaction kinetics for the bound enzyme showed a Km of the same order of magnitude (140 mM) than the soluble protein (107 mM), suggesting that the binding may not lead to major changes in protein conformation or active site accessibility. Similarly, both attached and soluble enzyme showed identical substrate range, being active on either mono- and dihaloacetate and L-2-chloropropionate. Activity of RC26 haloacid dehalogenase immobilised on a continuos flow column reactor. To evaluate the performance of the immobilised enzyme in a column reactor, dehalogenation of MCA
FIG. 4. Reutilization of immobilised dehalogenase. Activity of immobilised enzyme on 2 mM MCA. After 30 min incubation at 30°C, the mixture was sedimented and chloride was measured in the soluble fraction. The sediment was redissolved again in 2 mM MCA and this process was repeated 7 times except that MCA was omitted in cycles 3 to 5. 831
JOBNAME: BBRC 220#3 PAGE: 5 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 5. Activity in a plug-flow column reactor. Chloride release and substrate transformation efficiency of a column containing immobilised protein fed with increasing concentrations of MCA.
at different concentrations after injection in a 1 ml reactor was determined (Fig. 5). The results show that, under the conditions tested, the column-immobilised dehalogenase is able to fully transform up to 10 mM of MCA. This result indicates a significant improvement of the catalytic efficacy, since substrate concentrations higher than 2 mM MCA reduce the activity of either soluble or immobilised in batch RC26 haloacid dehalogenase because chlorine inhibition (11). This might be explained assuming a local reduction of the concentration of the product of dehalogenation (chlorine) as it flows along the column. In addition, saturation of the system rather than inhibition of the enzyme could be observed at substrate concentrations above 10 mM. There are few reports on immobilisation of enzymes with dehalogenase activity: earlier studies were carried on the dehalogenation of organohalides by porphyrines and corrins (18) and, recently, the behavior of a haloacid dehalogenase from group 1D immobilised on CNBr-controled pore glass has been studied (10). To our knowledge, this is the first report, on immobilisation of an haloacid dehalogenase of group 1L. The L-2-haloacid dehalogenase of the Azotobacter sp. RC26 is significantly resistant to relatively high temperatures and is active in a wide range of neutral to alkaline pH conditions. The immobilisation on a solid matrix of DEAE-Sephacel does not introduce changes in substrate affinity, while the activity is increased at high temperature and the stability at extreme alkaline conditions is improved. This system shows the feasibility of using immobilised enzymes for dechlorination of compounds used as herbicides (MCA) or substrates for chemical synthesis (2MCPA), and might be used for treatment of controlled waste fluids as well as for the design of reactors for the production of value added compounds, as the L and D isomers of Lactic acid or 2-monochloropropionic acid (8). ACKNOWLEDGMENTS Work supported by Grants PI900413 from the Commission of the European communities and BIO94-0471 from C.I.C.Y.T. Authors are grateful to M. Marin for technical help.
REFERENCES 1. Chaudry, G. R., and Chapalamadugu, S. (1991) Microbiol. Rev. 55, 59–79. 2. Fetzner, S., and Lingens, F. (1994) Microbiol. Rev. 58, 641–685. 3. Hickey, W. J., Searles, D. B., and Focht, D. D. (1993) Appl. Environ. Microbiol. 59, 1194–1200. 832
JOBNAME: BBRC 220#3 PAGE: 6 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Focht, D. D., and Shelton, D. (1987) Appl. Environ. Microbiol. 53, 1846–1849. Krumme, M. L., and Boyd, S. A. (1988) Water Res. 22, 171–177. Gälli, R. (1987) Appl. Microbiol. Biotech. 27, 206–213. Hartmans, S., and Tramper, J. (1991) Bioprocess Eng. 6, 83–92. Motosugi, K., Esaki, N., and Soda, K. (1984) Biotechnol. Bioeng. 26, 805–806. Taylor, S. C. (1987) in European patent application, Vol. CL.C12N9/14 EP 179, 603. Parker, K., and Colby, J. (1995) 6, 191–201. Diez, A., Alvarez, M. J., Prieto, M. I., Bautista, J. M., and Garrido-Pertierra, A. (1995) Can. J. Microbiol. 41, 730–739. Hareland, W. A., Crawford, R. L., Chapman, P. J., and Dagley, S. (1975) J. Bacteriol. 121, 272–285. Bergman, J. G., and Sanik, J. (1957) Anal. Chem. 29, 241–243. Ooshima, H., and Harano, Y. (1983) Biotechnol. Bioeng. 25, 143–155. Heng, M. H., and Glatz, C. E. (1994) Biotechnol. Bioeng. 44, 745–752. Dala, E., and Szajani, B. (1994) Appl. Biochem. Biotechnol. 49, 203–215. Cabral, J. M. S., Novais, J. M., and Cardoso, J. P. (1983) Chem. Eng. 27, B47. Marks, T. S., and Maule, A. (1992) Appl. Microbiol. Biotech. 38, 413–416.
833
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 828–833 (1996)
0489
Improved Catalytic Performance of a 2-Haloacid Dehalogenase from Azotobacter sp. by Ion-Exchange Immobilisation A. Diez, M. I. Prieto, M. J. Alvarez, J. M. Bautista, A. Garrido, and A. Puyet1 Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain Received February 18, 1996 The stability and catalytic efficacy of the L-2-haloacid dehalogenase isolated from Azotobacter sp. RC26 were studied after immobilisation on a DEAE-Sephacel solid matrix. While the optimum temperature for the soluble dehalogenase falls in the range of 30–40°C, the activity of the immobilised enzyme shows a four-fold increase at 60°C. Immobilisation on a plug-flow bioreactor extends the range of usable substrate concentration. The improved catalytic characteristics after immobilisation of the haloacid dehalogenase may be relevant for its possible utilization in biotechnological applications ranging from waste treatment to synthesis of steroisomers. © 1996 Academic Press, Inc.
Dehalogenase activities found in many microorganisms may be used for the transformation of halogenated xenobiotic compounds, such as pesticides, herbicides, solvents and other industrial products (1,2). Most current applications rely on the utilization of degradative bacterial strains, either by inoculation of contaminated sites (3,4) or immobilisation in bioreactors (5–7). The use of bioreactors may be particularly useful in applications where containment or extensive control of the system is required, as is the case of utilization of genetically modified microorganisms. An alternative to the utilization of biofilm-based reactors is the immobilisation of the bio-catalyst. This would only be feasible when the dehalogenating reaction is catalysed by enzymes relatively resistant to unfavourable environmental conditions, and whose activity do not require the addition of expensive cofactors. Some enzymes involved in dehalogenation, like the 2-haloalkanoate dehalogenases (EC 3.8.1.2.), display regio- and chiral specificity which, in addition to optically active compounds, replacing expensive conventional chemistry (8). These enzymes catalyze hydrolytic dehalogenation of 2-chloropropionate (2MCPA) and monochloroacetate (MCA) to yield optically active lactic acid and glycolate respectively. Although strains of Pseudomonas producing 2-haloalkanoic dehalogenases are currently used for the production of D-chloropropionate and D-lactate from racemic mixtures of 2-MCPA (9), little information is available on the performance of bacterial dehalogenases after immobilisation on solid supports. The performance of a D-2-haloacid dehalogenase from Pseudomonas putida coupled to CNBr-controled pore glass has been studied recently (10). In this study, we have analyzed the behaviour after immobilisation of a L-2-haloalkanoic dehalogenase isolated from Azotobacter sp. strain RC26, both in batch and in a column reactor. MATERIALS AND METHODS Enzyme preparation. Cultures of Azotobacter sp. RC26 (11) were grown to stationary phase on 2 l of mineral medium (12) supplemented with 50 mM MCA. Cells were harvested in late exponential, resuspended in elution buffer (50 mM Tris-acetate, 0.4 mM EDTA, 1 mM b-MSH and 5% glycerol pH 8.5) and lysed by ultrasonication. Cell debris was eliminated by centrifugation at 100,000 xg for 30 min. Crude extracts were precipitated with ammonium sulphate at 55% saturation, solubilized in 5 ml of elution buffer and loaded in a Superdex 200 gel filtration column. Proteins were eluted with the same buffer. Fractions showing dehalogenase activity on MCA were pooled, loaded on a Mono-Q ionic exchange column and eluted with a 0–0.2 M gradient of Na2 SO4 on elution buffer. Dehalogenase activity eluted at approximately 1
Corresponding author. Fax: +341-3943824 828
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: BBRC 220#3 PAGE: 2 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.07 M Na2 SO4. Active fractions were pooled and desalted in a gel filtration column, and subsequently loaded in an hydroxylapatite column equilibrated with 10 mM potassium phosphate pH 7.4 buffer. Dehalogenase activity was not retained in the column. Approximately 200 mg of enzyme was isolated from 10 g (wet weight) of cells. Enzyme activity. Enzymatic activity on halogenated compounds was measured by chloride release detection as described (13) after incubation at 30°C for 30 min in TA buffer (Tris-acetate 0.1M pH 8.5) supplemented with 2 mM MCA. Activity of DEAE-bound dehalogenase in standard conditions was detected after removal of the Dehalogenase-DEAE-Sephacel complex by centrifugation, and quantitation of chloride in the supernatant. Immobilisation. Unless otherwise specified, adsorption of the 2-haloacid dehalogenase to the solid matrix was performed by incubation of approximately 0.1 mg of pure enzyme with 0.4 g of DEAE-Sephacel equilibrated in buffer TA at 25°C for 10 min. The mixture was centrifuged at 12000 rpm for 1 min and resuspended in the same buffer. Column reactor design. Approximately 0.2 mg of DEAE-immobilised RC26 haloacid dehalogenase were packed in a 1 ml column (4.6 × 60 mm). To test the efficacy of the system, TA buffer containing MCA was injected at 21°C with a constant flow of 5 ml/h. Chloride release in the effluent was monitored colorimetrically as described above. Enzyme kinetics. The kinetic constants were calculated by measuring initial velocities of halide production at varying initial substrate concentrations. A detailed description of the procedure has been outlined (11). Km values were derived from non-linear regression analysis with the program Enzfitter from Biosoft[PT].
RESULTS AND DISCUSSION Immobilisation of the RC26 haloacid dehalogenase DEAE-Sephacel. Based on the observed retention capacity by a DEAE-based chromatography matrix, and the good results obtained after immobilisation of several enzymes to ion-exchange solid supports (14,15), we examined the behaviour of RC26 haloacid dehalogenase after immobilisation on DEAE-Sephacel. The retention capacity of DEAE for the protein is shown in Fig. 1. Under the conditions tested (pH 8.5, 21°C), 3.5 g (wet weight) of DEAE-Sephacel were necessary to bind one milligram of protein. Saturation occurred at about 300 mg of protein per g of solid matrix. Routinely, 0.3 mg of purified enzyme were mixed with 1 g of DEAE-Sephacel equilibrated in TA buffer. At this protein:matrix ratio, less than 12% of the dehalogenase activity could be detected in the supernatant after sedimentation, indicating that the enzyme was successfully attached to the solid support. Effect of pH. The activity of the 2-haloacid dehalogenase at different pHs was inspected using 0.1 M acetate (pH 3–7), 0.1 M Tris-acetate (pH 7–10) and 0.1 M sodium bicarbonate (pH 10–12) buffers to attain acidic, neutral and basic buffered environment in the assay, respectively. The
FIG. 1. DEAE-Sephacel binding ability for RC26 haloacid dehalogenase. Increasing amounts of DEAE-Sephacel were added to 100 mg of purified enzyme, and dehalogenase activity on MCA was assayed in supernatants after sedimentation of the mixture. Enzyme activity values are shown as percentage of the total activity found in the soluble fraction (supernatant). 829
JOBNAME: BBRC 220#3 PAGE: 3 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
immobilised and soluble enzymes showed similar activity profiles from pH 3 to 12, with optimal values between pH 8 and 10 (not shown). The residual activity at pH 8, after incubation for 30 min at different pH, was also tested (Fig. 2). The results indicate that the immobilised enzyme is less affected by shifts at extreme alkaline pH than the soluble enzyme. Effect of temperature. The effect of temperature on the immobilised RC26 haloacid dehalogenase was analysed in two ways. Approximately mg of protein either soluble or immobilised were incubated for 30 min at different temperatures in the presence of 2 mM MCA. The profile of activity at different temperatures is shown in Fig. 3a. The soluble enzyme displays significant activity at temperatures ranging from 20°C to 60°C, with an optimal between 30°C–40°C. Remarkably, the optimal temperature for the bound protein shifted to 60°C, showing a linear increase from 30 to 50°C. The calculated activation energy, using the Arrhenius equation, was 10 kcal. Compared to the activity of the soluble protein at 30°C, a four-fold increase in chloride production is observed for the immobilised dehalogenase at 60°C. A second set of experiments were carried out to show the stability of the enzyme after incubation at different temperatures (Fig. 3b). Incubation at 50°C to 60°C for 10 min prior to the activity assay at 30°C reduce the activity of the soluble enzyme to approximately 50%; however, the DEAE attached dehalogenase appears to retain more residual activity after a heat shift up to 60°C, suggesting that the binding of the enzyme to the solid support favours its protection against inactivation or denaturation. Incubation at 70°C during 30 min. inactivates the enzyme, either soluble or immobilised. The enhancement of thermal and pH range stability upon immobilisation of enzymes has been frequently observed (16,17), probably caused by the formation of ionic bonds between the protein and DEAE may stabilise the conformation of the protein. The observed activity increase at high temperatures of the L-2-haloacid dehalogenase of RC26 after immobilisation may also be a consequence of the structure stabilisation. This change of optimal temperature was not observed for the D-2-haloacid dehalogenase of Pseudomonas putida AJ1/23 (10). Stability of the binding to DEAE-Sephacel. Protein loss due to the lability of the bonds is one of the main drawbacks to be considered in ionic-binding methods. The potential use of enzymes bound to solid supports rely on their long term storage stability and the possibility of recover and reuse the enzyme. A single batch of immobilised enzyme could be reused at least 7 times without significant loss of activity. After several washes in MCA-free buffer to eliminate substrate and products, the enzyme-DEAE-Sephacel complex showed 100% of the initial activity, indicating that
FIG. 2. Stability of immobilised dehalogenase at different pH. Dehalogenase activity at pH 8 after incubation for 30 min at the indicated pHs. V, soluble; ■, immobilised. 830
JOBNAME: BBRC 220#3 PAGE: 4 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Activity and stability at different temperatures. (a) Chloride released by the RC26 dehalogenase after 30 min incubation at different temperatures with 2 mM MCA; (b) activity at 30°C after incubation of the enzyme at the temperatures indicated for 10 min. 100% corresponds to the maximum activity observed. V, soluble; ■, immobilised.
the enzyme loss or inactivation is negligible under these conditions (Fig. 4). In addition, immobilised RC26 haloacid dehalogenase could be stored for two months at 4°C in TA buffer retaining 60% of its activity. Kinetics. The reaction kinetics for the bound enzyme showed a Km of the same order of magnitude (140 mM) than the soluble protein (107 mM), suggesting that the binding may not lead to major changes in protein conformation or active site accessibility. Similarly, both attached and soluble enzyme showed identical substrate range, being active on either mono- and dihaloacetate and L-2-chloropropionate. Activity of RC26 haloacid dehalogenase immobilised on a continuos flow column reactor. To evaluate the performance of the immobilised enzyme in a column reactor, dehalogenation of MCA
FIG. 4. Reutilization of immobilised dehalogenase. Activity of immobilised enzyme on 2 mM MCA. After 30 min incubation at 30°C, the mixture was sedimented and chloride was measured in the soluble fraction. The sediment was redissolved again in 2 mM MCA and this process was repeated 7 times except that MCA was omitted in cycles 3 to 5. 831
JOBNAME: BBRC 220#3 PAGE: 5 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 5. Activity in a plug-flow column reactor. Chloride release and substrate transformation efficiency of a column containing immobilised protein fed with increasing concentrations of MCA.
at different concentrations after injection in a 1 ml reactor was determined (Fig. 5). The results show that, under the conditions tested, the column-immobilised dehalogenase is able to fully transform up to 10 mM of MCA. This result indicates a significant improvement of the catalytic efficacy, since substrate concentrations higher than 2 mM MCA reduce the activity of either soluble or immobilised in batch RC26 haloacid dehalogenase because chlorine inhibition (11). This might be explained assuming a local reduction of the concentration of the product of dehalogenation (chlorine) as it flows along the column. In addition, saturation of the system rather than inhibition of the enzyme could be observed at substrate concentrations above 10 mM. There are few reports on immobilisation of enzymes with dehalogenase activity: earlier studies were carried on the dehalogenation of organohalides by porphyrines and corrins (18) and, recently, the behavior of a haloacid dehalogenase from group 1D immobilised on CNBr-controled pore glass has been studied (10). To our knowledge, this is the first report, on immobilisation of an haloacid dehalogenase of group 1L. The L-2-haloacid dehalogenase of the Azotobacter sp. RC26 is significantly resistant to relatively high temperatures and is active in a wide range of neutral to alkaline pH conditions. The immobilisation on a solid matrix of DEAE-Sephacel does not introduce changes in substrate affinity, while the activity is increased at high temperature and the stability at extreme alkaline conditions is improved. This system shows the feasibility of using immobilised enzymes for dechlorination of compounds used as herbicides (MCA) or substrates for chemical synthesis (2MCPA), and might be used for treatment of controlled waste fluids as well as for the design of reactors for the production of value added compounds, as the L and D isomers of Lactic acid or 2-monochloropropionic acid (8). ACKNOWLEDGMENTS Work supported by Grants PI900413 from the Commission of the European communities and BIO94-0471 from C.I.C.Y.T. Authors are grateful to M. Marin for technical help.
REFERENCES 1. Chaudry, G. R., and Chapalamadugu, S. (1991) Microbiol. Rev. 55, 59–79. 2. Fetzner, S., and Lingens, F. (1994) Microbiol. Rev. 58, 641–685. 3. Hickey, W. J., Searles, D. B., and Focht, D. D. (1993) Appl. Environ. Microbiol. 59, 1194–1200. 832
JOBNAME: BBRC 220#3 PAGE: 6 SESS: 5 OUTPUT: Thu May 2 04:51:48 1996 /xypage/worksmart/tsp000/69829b/61
Vol. 220, No. 3, 1996 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Focht, D. D., and Shelton, D. (1987) Appl. Environ. Microbiol. 53, 1846–1849. Krumme, M. L., and Boyd, S. A. (1988) Water Res. 22, 171–177. Gälli, R. (1987) Appl. Microbiol. Biotech. 27, 206–213. Hartmans, S., and Tramper, J. (1991) Bioprocess Eng. 6, 83–92. Motosugi, K., Esaki, N., and Soda, K. (1984) Biotechnol. Bioeng. 26, 805–806. Taylor, S. C. (1987) in European patent application, Vol. CL.C12N9/14 EP 179, 603. Parker, K., and Colby, J. (1995) 6, 191–201. Diez, A., Alvarez, M. J., Prieto, M. I., Bautista, J. M., and Garrido-Pertierra, A. (1995) Can. J. Microbiol. 41, 730–739. Hareland, W. A., Crawford, R. L., Chapman, P. J., and Dagley, S. (1975) J. Bacteriol. 121, 272–285. Bergman, J. G., and Sanik, J. (1957) Anal. Chem. 29, 241–243. Ooshima, H., and Harano, Y. (1983) Biotechnol. Bioeng. 25, 143–155. Heng, M. H., and Glatz, C. E. (1994) Biotechnol. Bioeng. 44, 745–752. Dala, E., and Szajani, B. (1994) Appl. Biochem. Biotechnol. 49, 203–215. Cabral, J. M. S., Novais, J. M., and Cardoso, J. P. (1983) Chem. Eng. 27, B47. Marks, T. S., and Maule, A. (1992) Appl. Microbiol. Biotech. 38, 413–416.
833