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Zymographic detection of cinnamic acid decarboxylase activity
Journal of Microbiological Methods 51 (2002) 417 – 420 www.elsevier.com/locate/jmicmeth
Note
Zymographic detection of cinnamic acid decarboxylase activity Nu´ria Prim, F.I. Javier Pastor, Pilar Diaz * Department of Microbiology, Faculty of Biology, University of Barcelona, Avenue Diagonal 645, 08028 Barcelona, Spain Received 10 April 2002; received in revised form 7 May 2002; accepted 10 May 2002
Abstract The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Decarboxylase activity; Hydroxycinnamic acids; Vanillin; Zymogram
Cinnamic acid decarboxylases (4.1.1.-) are enzymes that catalyze the chain shortening of hydroxycinnamic acids into their 4-vinyl or 4-ethyl derivatives. Hydroxycinnamic acids are biologically important and abundant molecules in nature, possessing a potent in vitro antioxidant activity and showing potential health benefits, especially as components of the diet (Kroon and Williamson, 1999). Ferulic acid (4hydroxy-3-methoxy-trans-cinnamic acid), p-coumaric acid (4-hydroxy-trans-cinnamic acid) or caffeic acid (3,4-dihydroxycinnamic acid) constitute up to 1.5% by weight of the cell wall of plants, where they are esterified to polysaccharides (Kroon and Williamson, 1999). They play an important role as components of lignin and may act as precursors of several added value compounds. Among these, vanillin (4-hydroxy-3methoxybenzaldehyde), the most common flavor used *
Corresponding author. Tel.: +34-3-4034627; fax: +34-34034629. E-mail address: [email protected] (P. Diaz).
in foods, beverages, perfumes or pharmaceuticals, is of high economical importance (Priefert et al., 2001). The difference between prices of synthetic and natural vanillin, combined with the increasing demand for natural flavors, has led to a growing interest to produce natural vanillin by biotransformation of vegetal wastes (Thibault et al., 1998). For this purpose, knowledge of the enzymes involved in the catabolism of hydroxycinnamic acids is essential. Hydroxycinnamic acids can be released from vegetal cell walls by cinnamoyl ester hydrolases (Kroon and Williamson, 1999). Once released, they can be transformed by cinnamic acid decarboxylases into their corresponding volatile compounds 4-vinyl guaiacol (3-methoxy-4-hydroxystyrene) or 4-vinyl phenol (4-hydroxystyrene), considered as intermediates of vanillin production. Nevertheless, biotechnological production of vanillin has so far not been economical (Muheim and Lerch, 1999), and large screenings of microbial strains from natural sources must be performed for the isolation of microorganisms showing
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 1 0 9 - 4
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N. Prim et al. / Journal of Microbiological Methods 51 (2002) 417–420
the ability to either decarboxylate cinnamic acids or release acceptable amounts of vanillin (Torres and Rosazza, 2001). Several methods have been described for the determination of cinnamic acid decarboxylase activity, based mostly on UV spectroscopy or HPLC techniques (Cavin et al., 1997; Kermasha et al., 1995; Van Beek and Priest, 2000). These systems are reliable and accurate but require time-consuming sample processing or purification. For experiments involving the screening of a large number of strains or when nonpurified protein samples are assayed, the use of fast and simple techniques is very convenient. In this context, we have developed a direct method for the detection of cinnamic acid decarboxylase activity based on an increase of the pH originated by enzymatic decarboxylation of hydroxycinnamic acids. Detection of cinnamic acid decarboxylase activity was performed using LB agar plates containing 0.01% (w/v) bromocresol purple (Merck, Darmstadt, Germany), supplemented with 0.145% (w/v) ferulic, pcoumaric or caffeic acids (Sigma, St. Louis). The medium containing the pH indicator was sterilized, and the corresponding hydroxycinnamic acid was added from a 5% stock solution in iso-propanol before pouring onto the plates. The final pH of the plates was 5.25. A small aliquot (10 Al) of dense microbial cell suspensions, concentrated cell extracts prepared after culture centrifugation and sonication of the cells in 50 mM phosphate buffer pH 6.0, or culture supernatants (Prim et al., 2000) was laid on the surface of the plates and incubated for 1– 2 h at 37 – 45 jC. Hydroxycinnamic acid decarboxylase activity could readily be detected by a color shift from yellowish to purple as a result of a pH increase due to the decarboxylation of the cinnamic acid, which leads to an alkalization of the sample environment. As shown in Fig. 1, a color shift was observed for cell extracts of Escherichia coli bearing plasmid pUCPadA or pUCE3, both expressing Bacillus sp. BP-7 cinnamic acid decarboxylase PadA (unpublished). The enzyme PadA showed activity on all ferulic, p-coumaric and caffeic acids (Fig. 1), as confirmed by at least five independent replica assays. The same results were obtained for cell extracts of Bacillus sp. BP-7 previously grown in the presence of 0.023% ferulic acid for enzyme induction (not shown). On the contrary, no color change could be detected for cell suspensions of E. coli pUC19 or for noninduced
Fig. 1. Cinnamic acid decarboxylase activity detection from crude cell extracts. A 10 Al aliquot of concentrated cell extracts of each E. coli pUCPadA (1), E. coli pUCE3 (2) or E. coli pUC19 (3) in 50 mM phosphate buffer pH 6.0 was applied onto LB agar plates containing 0.01% bromocresol purple and 0.145% ferulic acid (A), p-coumaric acid (B), or caffeic acid (C). The plates were incubated for 2 h at 42 jC until a clear color shift of the pH indicator appeared. Decarboxylase activity could only be detected for E. coli pUCPadA (1) or E. coli pUCE3 (2), expressing Bacillus sp. BP-7 cinnamic acid decarboxylase PadA. No color shift appeared for control E. coli pUC19 samples lacking activity (3). E. coli pUCPadA (1) and E. coli pUCE3 (2) correspond to two recombinant clones containing different-length DNA inserts from Bacillus sp. BP-7. The stronger color shift found for p-coumaric acid-supplemented plates correlates well with the higher activity shown by PadA on this substrate when assayed by UV or HPLC techniques (unpublished).
cell extracts from Bacillus sp. BP-7, lacking cinnamic acid decarboxylase activity. The same results were obtained when UV or HPLC-based systems were used.
N. Prim et al. / Journal of Microbiological Methods 51 (2002) 417–420
Fig. 2. Zymographic analysis of cell extracts separated in polyacrylamide gel electrophoresis. Concentrated cell extracts of E. coli pUCPadA (1) and E. coli pUC19 (2) were separated on a nondenaturing polyacrylamide gel. The gel was washed in 50 mM phosphate buffer pH 6.0 for 20 min and overlaid on an LB agar plate containing 0.01% bromocresol purple and 0.145% ferulic acid. The gel was photographed after 80 min incubation at 45 jC. The darker broad band that appears in lane 1 corresponds to the color shift of the pH indicator caused by the decrease in the concentration of ferulic acid as a result of the activity of the cloned Bacillus sp. BP-7 cinnamic acid decarboxylase PadA. No color shift was detected in control E. coli pUC19 cell extracts (2), lacking hydroxycinnamic acid decarboxylase activity.
419
also used to locate protein bands with cinnamic acid decarboxylase activity after separation on polyacrylamide gels. Bacterial crude cell extracts, prepared as described above, were applied onto nondenaturing polyacrylamide gels and separated essentially as described previously (Diaz et al., 1999). After protein separation, the gels were washed in 50 mM phosphate buffer pH 6.0 for 20 min at room temperature with shaking and transferred by overlay onto LB agar plates prepared as described above. The overlays were incubated for 1– 2 h at 37– 45 jC until color shift appearance. Fig. 2 shows the zymographic analysis of crude cell extracts from E. coli bearing plasmid pUCPadA or pUC19. A color shift can be appreciated as a broad band for E. coli/pUCPadA cell extracts bearing cinnamic acid decarboxylase activity, while no color shift appeared in negative control cell extracts of E. coli/pUC19. The fast and simple assay for cinnamic acid decarboxylase activity described here provides an excellent method to identify those microorganisms with the ability to decarboxylate hydroxycinnamic acids, thus releasing some of the intermediates for the natural synthesis of vanillin or other added value compounds. The remarkable speed and simplicity of the system, together with no need for complex sample processing, may help to eliminate the tedious and time-consuming task of strain screening and sample processing for biotechnological purpose approaches.
Acknowledgements
After incubation of crude cell extracts of E. coli pUCPadA in the presence of the corresponding hydroxycinnamic acids, release of their respective decarboxylation products was confirmed by comparison to the spectra or chromatograms of the same products used as standards (not shown). No release of such decarboxylation products appeared after incubation of the substrates in the presence of crude cell extracts of E. coli pUC19, lacking hydroxycinnamic acid decarboxylase activity. Electrophoretic separation is a widely used technique for protein analysis. However, activity detection of nonpurified enzymes often requires zymographic analysis. The detection method described here was
This work was financed by the Scientific and Technological Research Council (CICYT, Spain), grant PPQ2001-2161-CO2-02, and by the III Pla de Recerca de Catalunya (Generalitat de Catalunya), grant 2001SGR 00143.
References Cavin, J.F., Barthelmebs, L., Guzzo, J., Van Beeumen, J., Samyn, B., Travers, J.-F., Divie`s, C., 1997. Purification and characterisation of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum. FEMS Microbiol. Lett. 147, 291 – 295. Diaz, P., Prim, N., Pastor, F.I.J., 1999. Direct fluorescence-based lipase activity assay. BioTechniques 27, 697 – 699.
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N. Prim et al. / Journal of Microbiological Methods 51 (2002) 417–420
Kermasha, S., Goetghebeur, M., Dumont, J., 1995. Determination of phenolic compound profiles in maple products by high-performance liquid chromatography. J. Agric. Food Chem. 43, 708 – 716. Kroon, P.A., Williamson, G., 1999. Hydroxycinnamates in plants and food: current and future perspectives. J. Sci. Food Agric. 79, 355 – 361. Muheim, A., Lerch, K., 1999. Towards a high-yield bioconversion of ferulic acid to vanillin. Appl. Microbiol. Biotechnol. 51, 456 – 461. Priefert, H., Rabenhorst, J., Steinbu¨chel, A., 2001. Biotechnological production of vanillin. Appl. Microbiol. Biotechnol. 56, 296 – 314. Prim, N., Blanco, A., Martı´nez, J., Pastor, F.I.J., Diaz, P., 2000. estA, a gene coding for a cell-bound esterase from Paenibacillus
sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases. Res. Microbiol. 151, 303 – 312. Thibault, J.F., Asther, M., Ceccaldi, B.C., Couteau, D., Delattre, M., Cardoso, J., Faulds, C., Heldt-Hansen, H.P., Kroon, P., LesageMeessen, L., Micard, V., Renard, C.M.G.C., Tuohy, M., van Hulle, S., Williamson, G., 1998. Fungal bioconversion of agricultural by-products to vanillin. Lebensm.-Wiss. Technol. 31, 530 – 536. Torres, J.L., Rosazza, J.P.N., 2001. Microbial transformations of pcoumaric acid by Bacillus megaterium and Curvularia lunata. J. Nat. Prod. 64, 1408 – 1414. Van Beek, S., Priest, F.G., 2000. Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whisky fermentation. Appl. Environ. Microbiol. 66, 5322 – 5328.
Note
Zymographic detection of cinnamic acid decarboxylase activity Nu´ria Prim, F.I. Javier Pastor, Pilar Diaz * Department of Microbiology, Faculty of Biology, University of Barcelona, Avenue Diagonal 645, 08028 Barcelona, Spain Received 10 April 2002; received in revised form 7 May 2002; accepted 10 May 2002
Abstract The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Decarboxylase activity; Hydroxycinnamic acids; Vanillin; Zymogram
Cinnamic acid decarboxylases (4.1.1.-) are enzymes that catalyze the chain shortening of hydroxycinnamic acids into their 4-vinyl or 4-ethyl derivatives. Hydroxycinnamic acids are biologically important and abundant molecules in nature, possessing a potent in vitro antioxidant activity and showing potential health benefits, especially as components of the diet (Kroon and Williamson, 1999). Ferulic acid (4hydroxy-3-methoxy-trans-cinnamic acid), p-coumaric acid (4-hydroxy-trans-cinnamic acid) or caffeic acid (3,4-dihydroxycinnamic acid) constitute up to 1.5% by weight of the cell wall of plants, where they are esterified to polysaccharides (Kroon and Williamson, 1999). They play an important role as components of lignin and may act as precursors of several added value compounds. Among these, vanillin (4-hydroxy-3methoxybenzaldehyde), the most common flavor used *
Corresponding author. Tel.: +34-3-4034627; fax: +34-34034629. E-mail address: [email protected] (P. Diaz).
in foods, beverages, perfumes or pharmaceuticals, is of high economical importance (Priefert et al., 2001). The difference between prices of synthetic and natural vanillin, combined with the increasing demand for natural flavors, has led to a growing interest to produce natural vanillin by biotransformation of vegetal wastes (Thibault et al., 1998). For this purpose, knowledge of the enzymes involved in the catabolism of hydroxycinnamic acids is essential. Hydroxycinnamic acids can be released from vegetal cell walls by cinnamoyl ester hydrolases (Kroon and Williamson, 1999). Once released, they can be transformed by cinnamic acid decarboxylases into their corresponding volatile compounds 4-vinyl guaiacol (3-methoxy-4-hydroxystyrene) or 4-vinyl phenol (4-hydroxystyrene), considered as intermediates of vanillin production. Nevertheless, biotechnological production of vanillin has so far not been economical (Muheim and Lerch, 1999), and large screenings of microbial strains from natural sources must be performed for the isolation of microorganisms showing
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 1 0 9 - 4
418
N. Prim et al. / Journal of Microbiological Methods 51 (2002) 417–420
the ability to either decarboxylate cinnamic acids or release acceptable amounts of vanillin (Torres and Rosazza, 2001). Several methods have been described for the determination of cinnamic acid decarboxylase activity, based mostly on UV spectroscopy or HPLC techniques (Cavin et al., 1997; Kermasha et al., 1995; Van Beek and Priest, 2000). These systems are reliable and accurate but require time-consuming sample processing or purification. For experiments involving the screening of a large number of strains or when nonpurified protein samples are assayed, the use of fast and simple techniques is very convenient. In this context, we have developed a direct method for the detection of cinnamic acid decarboxylase activity based on an increase of the pH originated by enzymatic decarboxylation of hydroxycinnamic acids. Detection of cinnamic acid decarboxylase activity was performed using LB agar plates containing 0.01% (w/v) bromocresol purple (Merck, Darmstadt, Germany), supplemented with 0.145% (w/v) ferulic, pcoumaric or caffeic acids (Sigma, St. Louis). The medium containing the pH indicator was sterilized, and the corresponding hydroxycinnamic acid was added from a 5% stock solution in iso-propanol before pouring onto the plates. The final pH of the plates was 5.25. A small aliquot (10 Al) of dense microbial cell suspensions, concentrated cell extracts prepared after culture centrifugation and sonication of the cells in 50 mM phosphate buffer pH 6.0, or culture supernatants (Prim et al., 2000) was laid on the surface of the plates and incubated for 1– 2 h at 37 – 45 jC. Hydroxycinnamic acid decarboxylase activity could readily be detected by a color shift from yellowish to purple as a result of a pH increase due to the decarboxylation of the cinnamic acid, which leads to an alkalization of the sample environment. As shown in Fig. 1, a color shift was observed for cell extracts of Escherichia coli bearing plasmid pUCPadA or pUCE3, both expressing Bacillus sp. BP-7 cinnamic acid decarboxylase PadA (unpublished). The enzyme PadA showed activity on all ferulic, p-coumaric and caffeic acids (Fig. 1), as confirmed by at least five independent replica assays. The same results were obtained for cell extracts of Bacillus sp. BP-7 previously grown in the presence of 0.023% ferulic acid for enzyme induction (not shown). On the contrary, no color change could be detected for cell suspensions of E. coli pUC19 or for noninduced
Fig. 1. Cinnamic acid decarboxylase activity detection from crude cell extracts. A 10 Al aliquot of concentrated cell extracts of each E. coli pUCPadA (1), E. coli pUCE3 (2) or E. coli pUC19 (3) in 50 mM phosphate buffer pH 6.0 was applied onto LB agar plates containing 0.01% bromocresol purple and 0.145% ferulic acid (A), p-coumaric acid (B), or caffeic acid (C). The plates were incubated for 2 h at 42 jC until a clear color shift of the pH indicator appeared. Decarboxylase activity could only be detected for E. coli pUCPadA (1) or E. coli pUCE3 (2), expressing Bacillus sp. BP-7 cinnamic acid decarboxylase PadA. No color shift appeared for control E. coli pUC19 samples lacking activity (3). E. coli pUCPadA (1) and E. coli pUCE3 (2) correspond to two recombinant clones containing different-length DNA inserts from Bacillus sp. BP-7. The stronger color shift found for p-coumaric acid-supplemented plates correlates well with the higher activity shown by PadA on this substrate when assayed by UV or HPLC techniques (unpublished).
cell extracts from Bacillus sp. BP-7, lacking cinnamic acid decarboxylase activity. The same results were obtained when UV or HPLC-based systems were used.
N. Prim et al. / Journal of Microbiological Methods 51 (2002) 417–420
Fig. 2. Zymographic analysis of cell extracts separated in polyacrylamide gel electrophoresis. Concentrated cell extracts of E. coli pUCPadA (1) and E. coli pUC19 (2) were separated on a nondenaturing polyacrylamide gel. The gel was washed in 50 mM phosphate buffer pH 6.0 for 20 min and overlaid on an LB agar plate containing 0.01% bromocresol purple and 0.145% ferulic acid. The gel was photographed after 80 min incubation at 45 jC. The darker broad band that appears in lane 1 corresponds to the color shift of the pH indicator caused by the decrease in the concentration of ferulic acid as a result of the activity of the cloned Bacillus sp. BP-7 cinnamic acid decarboxylase PadA. No color shift was detected in control E. coli pUC19 cell extracts (2), lacking hydroxycinnamic acid decarboxylase activity.
419
also used to locate protein bands with cinnamic acid decarboxylase activity after separation on polyacrylamide gels. Bacterial crude cell extracts, prepared as described above, were applied onto nondenaturing polyacrylamide gels and separated essentially as described previously (Diaz et al., 1999). After protein separation, the gels were washed in 50 mM phosphate buffer pH 6.0 for 20 min at room temperature with shaking and transferred by overlay onto LB agar plates prepared as described above. The overlays were incubated for 1– 2 h at 37– 45 jC until color shift appearance. Fig. 2 shows the zymographic analysis of crude cell extracts from E. coli bearing plasmid pUCPadA or pUC19. A color shift can be appreciated as a broad band for E. coli/pUCPadA cell extracts bearing cinnamic acid decarboxylase activity, while no color shift appeared in negative control cell extracts of E. coli/pUC19. The fast and simple assay for cinnamic acid decarboxylase activity described here provides an excellent method to identify those microorganisms with the ability to decarboxylate hydroxycinnamic acids, thus releasing some of the intermediates for the natural synthesis of vanillin or other added value compounds. The remarkable speed and simplicity of the system, together with no need for complex sample processing, may help to eliminate the tedious and time-consuming task of strain screening and sample processing for biotechnological purpose approaches.
Acknowledgements
After incubation of crude cell extracts of E. coli pUCPadA in the presence of the corresponding hydroxycinnamic acids, release of their respective decarboxylation products was confirmed by comparison to the spectra or chromatograms of the same products used as standards (not shown). No release of such decarboxylation products appeared after incubation of the substrates in the presence of crude cell extracts of E. coli pUC19, lacking hydroxycinnamic acid decarboxylase activity. Electrophoretic separation is a widely used technique for protein analysis. However, activity detection of nonpurified enzymes often requires zymographic analysis. The detection method described here was
This work was financed by the Scientific and Technological Research Council (CICYT, Spain), grant PPQ2001-2161-CO2-02, and by the III Pla de Recerca de Catalunya (Generalitat de Catalunya), grant 2001SGR 00143.
References Cavin, J.F., Barthelmebs, L., Guzzo, J., Van Beeumen, J., Samyn, B., Travers, J.-F., Divie`s, C., 1997. Purification and characterisation of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum. FEMS Microbiol. Lett. 147, 291 – 295. Diaz, P., Prim, N., Pastor, F.I.J., 1999. Direct fluorescence-based lipase activity assay. BioTechniques 27, 697 – 699.
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N. Prim et al. / Journal of Microbiological Methods 51 (2002) 417–420
Kermasha, S., Goetghebeur, M., Dumont, J., 1995. Determination of phenolic compound profiles in maple products by high-performance liquid chromatography. J. Agric. Food Chem. 43, 708 – 716. Kroon, P.A., Williamson, G., 1999. Hydroxycinnamates in plants and food: current and future perspectives. J. Sci. Food Agric. 79, 355 – 361. Muheim, A., Lerch, K., 1999. Towards a high-yield bioconversion of ferulic acid to vanillin. Appl. Microbiol. Biotechnol. 51, 456 – 461. Priefert, H., Rabenhorst, J., Steinbu¨chel, A., 2001. Biotechnological production of vanillin. Appl. Microbiol. Biotechnol. 56, 296 – 314. Prim, N., Blanco, A., Martı´nez, J., Pastor, F.I.J., Diaz, P., 2000. estA, a gene coding for a cell-bound esterase from Paenibacillus
sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases. Res. Microbiol. 151, 303 – 312. Thibault, J.F., Asther, M., Ceccaldi, B.C., Couteau, D., Delattre, M., Cardoso, J., Faulds, C., Heldt-Hansen, H.P., Kroon, P., LesageMeessen, L., Micard, V., Renard, C.M.G.C., Tuohy, M., van Hulle, S., Williamson, G., 1998. Fungal bioconversion of agricultural by-products to vanillin. Lebensm.-Wiss. Technol. 31, 530 – 536. Torres, J.L., Rosazza, J.P.N., 2001. Microbial transformations of pcoumaric acid by Bacillus megaterium and Curvularia lunata. J. Nat. Prod. 64, 1408 – 1414. Van Beek, S., Priest, F.G., 2000. Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whisky fermentation. Appl. Environ. Microbiol. 66, 5322 – 5328.