Hydrolysis of diethyl diferulates by a tannase from Aspergillus oryzae
´ M.-T. Garcıa-Conesa a,*, P. Østergaard b, S. Kauppinen b, G. Williamson a a Phytochemicals Team, Division of Diet, Health & Consumer Sciences, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK. b ´ Screening Biotechnology, Enzyme Research Novo Nordisk A/S, Novo Alle, bldg. 1BM1.05 DK-2880 Bagsvaerd, Denmark.
Abstract Diferulic acid forms cross-links in naturally occurring plant cell wall polymers such as arabinoxylans and pectins. We have used model ethyl esteriﬁed substrates to ﬁnd enzymes able to break these cross-links. A tannase from Aspergillus oryzae exhibited esterase activity on several synthetic ethyl esteriﬁed diferulates. The efﬁciency of this esterase activity on most diferulates is low compared to that of a cinnamoyl esterase, FAEA, from Aspergillus niger. Of the diferulate substrates assayed, tannase was most efﬁcient at hydrolysing the ﬁrst ester bond of the 5–5- type of dimer. Importantly and unlike the cinnamoyl esterase, tannase from A. oryzae is able to hydrolyse both ester bonds from the 8–5-benzofuran dimer, thus forming the corresponding free acid product. These results suggest that tannases may contribute to plant cell wall degradation by cleaving some of the cross-links existing between cell wall polymers. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Aspergillus oryzae; cell wall polymers; Diethyl diferulates
1. Introduction Micro-organisms need to produce a combination of enzymes, primarily carbohydrases and ‘esterases’ (able to remove side chain substituents) that act synergistically, in order to increase digestibility of the plant cell wall. Crosslinking of the cell wall polymers by ferulic acid dehydrodimers: 8–5-, 8–O–4-, 5–5- and 8–8- diFAs (Ralph, Quideau, Grabbber & Hatﬁeld, 1994) (Fig. 1), is a major obstacle which limits the
References: Aoki, K., Shinke, R., & Nishira, H. (1976). Puriﬁcation and some properties of yeast tannase. Agricultural and Biological Chemistry, 40, 79–85. Barthomeuf, C., Regerat, F., & Pourrat, H. (1994). Production, puriﬁcation and characterisation of a tannase from Aspergillus niger LCF 8. Journal of Fermentation and Bioengineering, 77, 320–323. Bartolome, B., Faulds, C. B., Kroon, P. A., Waldron, K. W., Gilbert, H. J., Hazlewood, G., & Williamson, G. (1997). An Aspergillus niger esterase (FAE-III) and a recombinant Pseudomonas ﬂuorescens subsp. cellulose esterase (XYLD) release a 5–5-ferulic dehydrodimer (‘diferulic acid’) from barley and wheat cell walls. Applied Environmental Microbiology, 63, 208–212. Beverini, M., & Metche, M. (1990). Identiﬁcation, puriﬁcation and physicochemical properties of tannase of Aspergillus oryzae. Sciences des Aliments, 10, 807–816. Bradford, M. (1976). A rapid and sensitive method for the quantiﬁcation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemestry, 72, 248–254. Deschamps, A. M., Otuk, G., & Lebeault, J. M. (1983). Production of tannase and degradation of chestnut tannin by bacteria. Journal of Fermentation Technolology, 61, 55–59. Faulds, C. B., & Williamson, G. (1991). The puriﬁcation and characterisation of 4-hydroxy-3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes. Journal of General Microbiology, 137, 2339–2345. Faulds, C. B., & Williamson, G. (1994). Puriﬁcation and characterisation of a ferulic acid esterase (FAE-III) from Aspergillus niger: speciﬁcity for the phenolic moiety and binding to micro-crystalline cellulose. Microbiology, 144, 779–787. ´ Garcıa-Conesa, M. T., Plumb, G. W., Kroon, P. A., Wallace, G., & Williamson, G. (1997). Antioxidant properties of ferulic acid dimers. Redox Report, 3, 239. ´ Garcıa-Conesa, M. T., Kroon, P. A., Ralph, J., Mellon, F. A., Colquhoun, I. J., Saulnier, L., Thibault, J-F., & Williamson, G. (1999). An esterase from Aspergillus niger (FAEA) can break plant cell wall cross-links without release of free diferulic acids. European Journal of Biochemistry, 266, 644–652. Grabber, J. H., Hatﬁeld, R. D., & Ralph, J. (1998a). Diferulate cross-links impede the enzymatic degradation of non-ligniﬁed maize walls. Journal of the Science of Food and Agriculture, 77, 193–200. Grabber, J. H., Ralph, J., & Hatﬁeld, R. D. (1998b). Diferulate cross-links limit the enzymatic degradation of synthetically ligniﬁed primary walls of maize, . Journal of Agriculture and Food Chemistry, 46, 2609–2614. ´ Kroon, P. A., Garcıa-Conesa, M. T., Fillingham, I. J., Hazlewood, G. P., & Williamson, G. (1999). Release of ferulic acid dehydrodimers from plant cell walls by feruloyl esterases. Journal of the Science of Food and Agriculture, 79, 428–434. Lekha, P. K., & Lonsanne, B. K. (1997). Production and application of tannin acyl hydrolase: state of the art. Advances in Applied Microbiology, 44, 215–260. Nelson, E., Pell, A. N., Schoﬁeld, P., & Zinder, S. (1995). Isolation and characterisation of an anaerobic ruminal bacterium capable of degrading hydrolysable tannins. Applied Environmental Microbiology, 61, 3293–3298. Niehaus, J. U., & Gross, G. G. (1997). A gallotannin degrading esterase from leaves of pedunculate oak. Phytochemistry, 45, 1555–1560. Ralph, J., Quideau, S., Grabber, J. H., & Hatﬁeld, R. D. (1994). Identiﬁcation and synthesis of new ferulic acid dehydrodimers present in grass cell walls. Journal of the Chemical Society, Perkin Transactions 1, 3485–3498. ´ Ralph, J., Garcıa-Conesa, M. T., & Williamson, G. (1998). Simple preparation of 8–5-coupled diferulate. Journal of Agriculture and Food Chemistry, 46, 2531–2532. Richitzenhain, H. (1949). Enzymatische versuche zur entstehung des lignins. Chemische Berichte, 82, 447–453. Scalbert, A. (1991). Antimicrobial properties of tannins. Phytochemistry, 30, 3875–3883. Waldron, K. W., Parr, A. J., Ng, A., & Ralph, J. (1996). Cell wall esteriﬁed dimers: identiﬁcation and quantiﬁcation by reverse phase high performance liquid chromatography and diode array detection. Phytochemical Analysis, 7, 305–312.