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 accessibility of main chain-degrading enzymes to the structural polysaccharides and reduces cell wall digestibility (Grabber, Hatﬁeld & Ralph, 1998a,b). The breakage of one or both ester bonds from these dehydrodimer cross-links between plant cell wall polymers is essential for degradation of plant cell wall. An esterase from Aspergillus niger, FAEA, is able to cleave both ester bonds from the 5–5-diferulate and the 8–O–4-diferulate and can release the corresponding free acids from xylanase solubilised plant material (Bartolome ´ et al., 1997; Kroon, Garcıa-Conesa, Fillingham, Hazlewood & Williamson, 1999). However, this esterase was able to cleave only one ester bond from the 8–5-benzofuran type of dimer and, although this may be sufﬁcient to facilitate the subsequent action of main polymers-degrading enzymes, it * Corresponding author. Tel.: 44-1603-507723; fax: 44-1603255267. ´ E-mail address: email@example.com (M.-T. Garcıa-Conesa). 0144-8617/01/$ - see front matter PII: S0144-861 7(00)00248-4
´ was not possible to release the free acid (Garcıa-Conesa et al., 1999). To date, there are no reports on the release of this dimer from plant material. Microbial esterases are a broad group of enzymes able to hydrolyse the ester bond of a variety of naturally occurring esters of ﬂavonoids and hydroxycinnamates present in plants. Deﬁnition and characterisation of these enzymes are based mainly on their speciﬁcity towards a range of synthetic and puriﬁed substrates. The availability of new substrates provides researchers with a tool to re-examine the speciﬁcity of esterases for hydrolysing the variety of components present in plant cell walls. For example, synthetic dehydrodiferulates (Ralph et al., 1994; Ralph, ´ Garcıa-Conesa & Williamson, 1998) can be used to search for other esterases able to cleave plant cell wall cross-links, perhaps even more speciﬁc than FAEA from A. niger, and in particular, to search for esterases able to cleave the second ester bond from the...