Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Enzyme and Microbial Technology 31 (2002) 1000–1005

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472 Estelle Bonnin a,∗ , Luc Saulnier a , Magali Brunel a , Cécile Marot a , Laurence Lesage-Meessen b,1 , Marcel Asther b,1 , Jean-François Thibault a a b

Unité de Recherche sur les Polysaccharides, Leurs Organisations et Interactions, INRA, BP 71627, 44316 Nantes Cedex 03, France Unité de Biotechnologie des Champignons Filamenteux, INRA, CP 925, 136 Avenue de Luminy, 13288 Marseille Cedex 09, France Received 22 November 2001; received in revised form 22 July 2002; accepted 6 September 2002

Abstract Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Aspergillus niger; Sugar beet pulp; Maize bran; Polysaccharide-degrading enzymes; Ferulic acid esterases

1. Introduction Ferulic acid, the most abundant hydroxycinnamic acid in cell wall polysaccharides, is widely distributed in higher plants. It represents about 1% of sugar beet pulp or wheat bran, and up to 4% in maize bran. It is ester-linked to O-5 of arabinose residues in arabinoxylans from cereals [1], whereas, it is associated almost exclusively with the pectic side chains of sugar beet, and is found linked at 50–60% to the O-2 of arabinose residues and at 40–50% to the O-6 of galactose residues [2,3]. Due to its chemical similarity with vanillin, ferulic acid is a suitable precursor for vanillin and led us to set up a biotechnological way to transform it into vanillin. In the process, ferulic acid was released from raw materials by enzymatic treatment [4,5], and was biotransformed into vanillin by two different white-rot basidiomycetes [6]. Aspergillus niger first transformed ferulic acid into vanillic acid, and vanillic acid was then metabolized into vanillin by Pycnoporus cinnabarinus. Vanillin obtained in this manner could be consider as “natural” according to the European and US legislations, i.e. originating from a natural source and ob∗

Corresponding author. Tel.: +33-240-675000; fax: +33-240-675066. E-mail address: [email protected] (E. Bonnin). 1 Tel.: +33-491-828600; fax: +33-491-828601.

tained by enzymatic or fungal transformation (EC directive 88/388, OJ No. L 184, July 15, 1988). The release of ferulic acid from various raw materials was studied with some commercial enzyme mixtures. Thus, SP 584 (Novozymes A/S, Denmark) was demonstrated to be able of solubilizing a high percentage of ferulic acid present in sugar beet pulp to give both free and esterified forms [4]. Ferulic acid was also efficiently released from wheat bran by a mixture of Trichoderma viride xylanase and A. niger ferulate esterase, FAE III [5]. Besides, the release of ferulic acid from maize bran by commercial enzymes was low and autoclaving treatment of the bran improved the solubilization of feruloylated oligosaccharides, which are substrate for feruloyl esterases [7]. However, the use of cell wall-degrading enzymes and feruloyl esterases involved either a dependence to commercially available mixtures or numerous steps of purification of the enzymes, as in the case of FAE III [5]. In our previous paper [8], the strain of A. niger selected for ferulic acid conversion, namely A. niger I-1472, was shown to produce large amounts of various polysaccharide-degrading enzymes, including feruloyl esterases, when it is grown on sugar beet pulp as carbon source. In the present paper, the enzymes produced by A. niger have been evaluated in the saccharification of sugar beet pulp or maize bran to release ferulic acid before its bioconversion into vanillic acid.

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 3 6 - 3

E. Bonnin et al. / Enzyme and Microbial Technology 31 (2002) 1000–1005

30 ◦ C. After 3 days of culture, cell-free supernatants were removed by filtration on glass-fiber filters (GF/D Whatman, Maidstone, UK). Crude enzymes were obtained by dialysis and freeze-drying of culture supernatant.

2. Materials and methods 2.1. Raw materials Sugar beet pulp from Sofalia (Chappes, France) was ground in a Forplex hammer mill and sieved between 2 and 0.08 mm. Commercial micronized (particle size < 80 ␮m) maize bran were provided by ULICE (Riom, France) and was destarched as previously described [7]. Destarched maize bran (5 g) was then suspended in 50 ml of deionized water and heated for 1 h at 160 ◦ C under magnetic stirring in a laboratory autoclave [7]. Supernatant was recovered by centrifugation, residue was washed twice with 50 ml of water. Supernatant and washings were pooled and then freeze-dried. A total of 3.2 g of autoclaved fraction from maize bran (AFMB) were recovered. The composition of sugar beet pulp, maize bran, and AFMB is given in the Table 1. 2.2. Enzymes SP 584 and Novozym 342 were from Aspergillus sp. and Humicola insolens, respectively, and were provided by Novozymes (Bagsvaerd, Denmark). 2.3. Fungal strain and culture conditions Aspergillus niger I-1472 originated from the Collection Nationale de Culture de Microorganismes, Institut Pasteur (Paris, France). It was grown as previously described [6] except that the culture was carried out in 3.5 l liquid medium and 15 g/l sugar beet pulp+2.5 g/l maltose were used as carbon sources in the presence of 0.5 g/l Tween 80. Culture was inoculated with 105 A. niger conidiospores and incubated at

Table 1 Composition (mg/g dry matter) of the different raw materials used as ferulic acid sources [8] Sugar beet pulp

Rhamnose Fucose Arabinose Xylose Mannose Galactose Glucose Uronic acids Methanol Acetic acid Ferulic acid Diferulic acid Proteins Ash

24 2 209 17 11 51 211 211 18 39 8 0.4 113 36

1001

Maize bran Initial

AFMB

0 2 154 276 3 51 248 42

0 2 208 386 2 73 39 47

nd nd 31 32

nd nd 34 0

50 10

8 8

AFMB: autoclaved fraction from maize bran; nd: not determined.

2.4. Substrates for enzymatic assays Polygalacturonic acid from orange peel, carboxymethylcellulose, xylan from oat spelt, p-NP-␣-l-rhamnopyranoside (pNP-Rhap), p-NP-␤-d-galactopyranoside (pNP-Galp), and p-NP-␣-l-arabinofuranoside (pNP-Araf) were from Sigma Chemicals (St. Louis, MO). Rhamnoglacturonan, ␣-1,5-arabinan, and type I galactan were prepared from sugar beet pulp and potato pulp, respectively, as previously described [8–10]. Feruloylated oligosaccharides 5-O-(trans-feruloyl)-lAraf (FA) and O-␤-d-Xylp(1 2)-[5-O-(trans-feruloyl)-␣l-Araf] (XFA) were isolated from maize bran [11] and [2-O-(trans-feruloyl)-␣-l-Araf]-(1 5)-l-Araf (A2 F) from sugar beet pulp [12]. 2.5. Enzyme assays Enzymatic activities towards polymeric substrates were calculated from the increase in reducing ends [13], using appropriate sugars for standard curves. Substrates (5 g/l in acetate buffer 50 mmol/l, pH 4.5) were incubated with appropriately diluted enzymes at 40 ◦ C. Glycosidases activities were performed on p-nitrophenylglycosides (2 mmol/l final concentration in acetate buffer, 50 mmol/l, pH 5.0) by incubation with suitably diluted crude enzyme at 40 ◦ C. They were calculated from the release of p-nitrophenol measured spectrophotometrically at 400 nm [14]. Assays for feruloyl esterase activities were performed as already described [8,15]. Enzymatic activities were all expressed in nkatal (nkat), one nkat being defined as the amount of enzyme that catalyzes the release of 1 nmol of reducing end, p-nitrophenol or free ferulic acid per second in the conditions described above. Total protein was estimated by using the Coomassie protein assay reagent (Bio-Rad). Enzyme activities and protein determinations were performed in duplicate, with appropriate blanks to allow correction for any background reactions. Standard error was less than 5%. 2.6. Enzymatic degradation of raw materials Degradation were performed in duplicate on 100 mg dry matter in the presence of 1 mg enzymatic proteins in 10 ml water at 40 ◦ C. After 24-h incubation, the mixtures were boiled in a water bath for 15 min, and centrifuged (20,000 × g, 20 min). Residues were freeze-dried, weighed and ground in liquid nitrogen (Spex Feezer Mill), whereas supernatants were freezed before analysis.

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2.7. Chemical characterization of raw materials and degradation products

Table 2 Specific activities (nkat/mg) exhibited by A. niger grown on sugar beet pulp and the commercial preparations SP 584 and Novozym 342

Dry matter was determined by drying the sample at 120 ◦ C for 2 h. Data were expressed on a moisture-free basis. Ashes were weighed after overnight incineration of samples at 550 ◦ C followed by 1 h at 900 ◦ C. Individual neutral sugars in the extracted fractions were determined after hydrolysis (4 N sulfuric acid, 100 ◦ C, 2 h), derivatization in their alditol acetates [16], and analysis by gas liquid chromatography on a DB-225 (J&W Scientific; 30 m×0.32 mm, i.d.) fused-silica capillary column. Uronic acid content was colorimetrically determined by the automated m-hydroxydiphenyl method with galacturonic acid as standard [17,18]. Standard error between replicated assays was less than 5%. Monomers originating from cell wall polysaccharides were estimated by HPAEC on a Carbopac PA1 column (4 mm × 250 mm), eluted with H2 O (A), NaOAc 0.1 mol/l (B), and NaOH 0.1 mol/l (C) in the following conditions: t = 0 min, 85:0:15 (A:B:C); t = 23 min, 85:0:15 (A:B:C); t = 45 min, 40:30:30 (A:B:C). Detection was carried out by pulsed amperometry (Dionex). Phenolic acids were determined after alkaline treatment with NaOH (2 M) at 35 ◦ C for 30 min. o-Coumaric acid was added as an internal standard, the mixture was acidified to pH 2.0 with HCl, and then extracted with Et2 O. Et2 O-extracts were evaporated to dryness at 40 ◦ C. Samples were dissolved in MeOH/H2 O (50:50, v/v), and analyzed by HPLC on a C18 column (Purospher, Merck, Germany) as previously described [19]. Free ferulic acid was determined as follows: internal standart (o-coumaric acid) was added to supernatant, then NaCl solution was added to supernatant (final NaCl concentration 2 mol/l) and the mixture was acidified to pH 2.0 with HCl, and then extracted with Et2 O. Et2 O-extracts were then treated and injected on the HPLC system as described above. Ester-linked ferulic acid was calculated as the difference between total and free ferulic acid.

Substrates

A. niger enzymes

SP 584

Novozym 342

Arabinan Xylan Galactan I Rhamnogalacturonan CMC Polygalacturonic acid pNP-Rhap pNP-Galp pNP-Araf FAX FA A2 F

120.6 125.5 32.9 53.4 13.6 86.0 12.9 19.8 266.6 9.2 5.8 2.4

291.1 62.2 943.9 256.7 4.7 2400.2 0.2 84.8 619.5 0.1 0.1 0.3

7.2 104.3 2.8 nd 24.9 0.4 0.0 0.0 0.3 0.8 0.2 nd

3. Results 3.1. Enzymatic activities in commercial mixtures and in A. niger I-1472 culture broth Pectin-, hemicellulose-, and cellulose-degrading enzymes were tested with model substrates in the different sources of enzymes (Table 2). The highest activities in SP 584 were polygalacturonase, galactanase, arabinofuranosidase, arabinanase, and rhamnogalacturonase, all related to degradation of pectins. Nevertheless, SP 584 was almost devoid of feruloyl esterases, whatever the substrate tested. In Novozym 342, xylanase and CMCase were the highest activities. No activity was found towards any of the pNPderivatives assayed. On the other hand, feruloyl esterase

nd: not determined.

activity in Novozym 342 was low towards FA and slightly higher towards FAX. Aspergillus niger I-1472 exhibited all these activities at the same time, the most important being, in the decreasing order arabinofuranosidase, xylanase, polygalacturonase, and arabinanase. Feruloyl esterases were much more active than in Novozym 342. Even if some activities were much lower in A. niger culture broth than in SP 584, A. niger produced a larger spectrum of polysaccharide-degrading enzymes. The enzymatic activities were slightly different to those previously published for the same strain of Aspergillus [8]. This could be ascribed to a much higher volume of culture (3.5 l instead of 250 ml) inducing some differences in the culture supernatant composition. As the activities in the Table 1 are expressed in specific activity, it could come from a slight difference in the protein content. 3.2. Solubilizing effect of crude enzymes or SP 584 on sugar beet pulp Sugar beet pulp mainly contained sugars (72.3% of dry mattter) with equal amounts of arabinose, glucose, and uronic acids (Table 1). Glucose was previously shown to originate from cellulose [4]. The pulp contained also almost 1% of ferulic acid and could then be considered as a potential source of ferulic acid. Enzymatic degradations of sugar beet pulp were carried out in the presence of 10 mg enzymes per g of dried pulp. Table 3 shows that using either SP 584 or crude enzymes from A. niger yielded the same amount of residue. The composition of sugar fractions in the residues were also about the same, showing that most of the pectic monomers were removed by the enzymatic degradations, whatever the source of enzymes. For example, galacturonic acid content represented 7.4 and 9.1% in the residues remained by SP 584 and A. niger culture broth, respectively. Moreover, the analysis of the supernatants showed that the sugars were totally solubilized under a monomeric form

E. Bonnin et al. / Enzyme and Microbial Technology 31 (2002) 1000–1005 Table 3 Yield and composition of the residues obtained after enzymic degradation of sugar beet pulp by SP 584 and the enzymes from A. niger I-1472

Yield (mg/g) Composition (mg/g) Rhamnose Arabinose Xylose Galactose Glucose Uronic acids

SP 584

A. niger enzymes

391

416

8 33 27 16 446 40

9 35 27 22 440 46

The degradations were carried out in duplicate with a ratio of enzyme/substrate (1/100). Values are means of replicated dosages after each degradation.

after degradation by SP 584 and A. niger culture supernatant, except xylose and glucose (Table 4). The main difference between the two degradations was the solubilization of cellulose, as A. niger culture broth was less efficient than SP 584 in releasing monomeric glucose. Nevertheless, this result was favorable to our process, since cellulosic residue obtained after enzymatic degradation of the pectic fraction could be used for an enzymatic production of cellobiose, that enhanced the yield in vanillin when it is added in the culture medium of P. cinnabarinus [20]. Ferulic acid analysis in the supernatants showed that SP 584 released 78.9% of ferulic acid, including 49.4% of free ferulic acid, whereas crude enzymes from A. niger released 70.2% of ferulic acid including 38.7% of free ferulic acid (Table 5). Therefore, A. niger culture supernatant was not as efficient as SP 584 in the release of ferulic acid from sugar beet pulp, although activity measured towards A2 F, isolated from sugar beet pulp, was higher in A. niger enzymes than in SP 584. As ferulic acid in sugar beet pectin is linked partly to arabinose and partly to galactose [2,3], it is likely that the activity on feruloylated galactose was different in the two enzyme sources. However, it was not possible to assay this activity since the substrate is not available. 3.3. Solubilizing effect of crude enzymes or Novozym 342 on autoclaved maize bran The composition of destarched maize bran and AFMB is given in Table 1. Maize bran was mainly composed of

1003

Table 4 Analysis of sugars present as monomer after degradation of sugar beet pulp and AFMB by either SP 584, Novozym 342, or enzymes from A. niger I-1472

Rhamnose Arabinose Xylose Galactose Glucose Uronic acids

Sugar beet pulp

AFMB

SP 584

A. niger enzymes

Initial

Novozym 342

A. niger enzymes

90.1 109.7 28.6 104.6 10.0 97.3

91.7 98.8 22.0 93.4 4.8 94.3

0.0 57.8 15.2 29.3 0.0 nd

0.0 55.6 23.0 27.9 87.0 nd

0.0 62.3 29.5 52.9 70.3 nd

nd: not determined. Degradations were carried out with a ratio of enzymes/substrate (1/100). Each monomer is expressed as a percentage of its initial content in sugar beet pulp or AFMB. Values are means of replicated dosages after each degradation.

xylose, arabinose, galactose, and glucuronic acid. It also contained significant amount of glucose that originated from cellulose [19] and 3.1% (w/w) of ferulic acid. AFMB was mainly composed of arabinose and xylose, but also contained galactose, glucuronic acid, and some glucose. Arabinose present in AFMB was present mainly in monomeric form (Table 4), but xylose and galactose or glucose were mainly present as oligomers. AFMB was also rich in ferulic acid, which was fully esterified to sugars. Therefore, AFMB contained a mixture of feruloylated oligosaccharides. Enzymatic degradations of maize bran and AFMB were carried out with Novozym 342 or A. niger culture supernatant. As previously reported [7], Novozym 342 was only able to solubilize about 19% of the ferulic acid initially present in the bran. Furthermore, due to its low feruloyl esterase activity, about 30% of the ferulic acid solubilized by Novozym 342 was still esterified to sugars (Table 5). A. niger supernatant had a very limited action on maize bran as only 3.4% of initial ferulic acid was solubilized (Table 5). However, ferulic acid was totally present in its free form, suggesting that A. niger culture supernatant was able to de-esterify the feruloyl esters and could be an interesting source of feruloyl esterases. When AFMB was treated with Novozym 342, the enzyme mixture was only able to release one third of the ferulic acid esterified to sugars, due to its very low feruloyl esterase activity. Novozym 342 had no rhamnosidase and galactosi-

Table 5 Solubilization of ferulic acid from raw materials by SP 584, Novozym 342, or enzymes from A. niger I-1472 Sugar beet pulp

Maize bran

AFMB

SP 584

A. niger enzymes

Novozym 342

A. niger enzymes

Novozym 342

A. niger enzymes

Free ferulic acid Esterified ferulic acid

49.4 29.5

38.7 31.5

13.6 5.8

3.4 0.0

31.9 65.4

90.3 5.2

Total

78.9

70.2

19.4

3.4

97.3

95.5

Enzymic degradations were carried out with a ratio of enzyme/substrate (1/100). Ferulic acid is expressed as a percentage of initial amount of ferulic acid in the raw material. Values are means of replicated dosages after each degradation.

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dase activity and a very low arabinofuranosidase activity (Table 2) and did not increase the proportion of monomers present in AFMB, except for glucose (Table 4). On the contrary, feruloyl esterases from A. niger were very efficient since 95% of ferulic acid esterified to sugars were solubilized among which 90.3% were free (Table 5). Galactosidases from A. niger culture supernatant were also able to partly hydrolyse the oligosaccharides present in AFMB since the level of galactose monomers increased after enzymatic treatment (Table 4). Therefore, A. niger enzymes do not exhibit the appropriate combination of cell wall-degrading enzymes to solubilize extensively the heteroxylans from raw maize bran. However, feruloyl esterases of A. niger were very efficient to release free ferulic acid from the feruloylated oligosaccharides of pretreated maize bran which can be easily obtained by autoclaving maize bran.

co-regulated, and that sugar beet pulp mimics the conditions of the host–pathogen interaction, when it is used as carbon source. These genes exhibit a low level constitutive transcription producing enzymes that release cell wall polysaccharide fragments from sugar beet pulp. These fragments would further promote the synthesis of an intermediate molecule which would activate the transcription of the different genes coding for polysaccharide-degrading enzymes, including feruloyl esterases.

4. Discussion

[1] Ishii T. Structure and functions of feruloylated polysaccharides. Plant Sci 1997;127:111–27. [2] Colquhoun IJ, Ralet M-C, Thibault J-F, Faulds CB, Williamson G. Feruloylated oligosaccharides form cell wall polysaccharides from sugar beet pulp by NMR spectroscopy. Carbohydr Res 1994;263:243– 56. [3] Micard V, Renard CMGC, Colquhoun IJ, Thibault J-F. End-products of enzymic saccharification of beet pulp, with a special attention to feruloylated oligosaccharides. Carbohydr Polym 1997;32:283–92. [4] Micard V, Renard CMGC, Thibault J-F. Enzymatic saccharification of sugar-beet pulp. Enzyme Microb Technol 1996;19:162–70. [5] Faulds CB, Williamson G. Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger. Appl Microbiol Biotechnol 1995;43:1082–7. [6] Lesage-Meessen L, Delattre M, Haon M, Thibault J-F, Colonna Ceccaldi B, Brunerie P, et al. A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus. J Biotechnol 1996;50:107–13. [7] Saulnier L, Marot C, Elgorriaga M, Bonnin E, Thibault J-F. Thermal and enzymatic treatments for the release of free ferulic acid from maize bran. Carbohydr Polym 2001;45:269–75. [8] Bonnin E, Brunel M, Gouy Y, Lesage-Meessen L, Asther M, Thibault J-F. Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases. Enzyme Microb Technol 2001; 28:70–80. [9] Lahaye M, Thibault J-F. Purification of arabinanases and galactanases from Aspergillus niger. In: Third Workshop on Plant Polysaccharides, Structure, Function. Le Croisic, France, 17–18 Sept., 1990. [10] Lahaye M, Vigouroux J, Thibault J-F. Endo-␤-14-D-galactanase from Aspergillus niger var aculeatus. Purification and some properties. Carbohydr Polym 1991;15:431–44. [11] Saulnier L, Vigouroux J, Thibault J-F. Isolation and partial characterization of feruloylated oligosaccharides from maize bran. Carbohydr Res 1995;272:241–53. [12] Kroon PA, Conesa MTG, Colquhoun IJ, Williamson G. Process for the isolation of preparative quantities of 2-O-trans-feruloyl-␣L-arabinosyl-15-L-arabinofuranose from sugar beet. Carbohydr Res 1997;300:351–4. [13] Nelson N. A photometric adaptation of the Somogyi method for determination of glucose. J Biol Chem 1944;153:375–80. [14] Rouau X, Odier E. Production of extracellular enzyme by the white-rot fungus Dichomitus squalens in cellulose-containing liquid culture. Enzyme Microb Technol 1986;8:22–6.

This study was carried out in order to evaluate the possibility to use the cell wall polysaccharide-degrading enzymes produced by A. niger I-1472 in the release of ferulic acid from sugar beet pulp or maize bran. Our results clearly demonstrate that (i) the enzymes from A. niger I-1472 were able to release ferulic acid either from beet pulp or from autoclaved maize bran. These results have to be related with the large spectrum of enzymatic activities found when A. niger was grown on sugar beet pulp as carbon source [8]. Aspergillus species are efficient producers of cell wall-degrading enzymes. Synergy between enzymes was already demonstrated for the complete degradation of plant cell wall [21,22]. As ferulic acid is covalently linked to polysaccharides, this synergy was particularly important in the case of feruloyl esterases, as most of them were unable to release significant amount of ferulic acid when acting alone on cell wall [5,23,24]. The ester-linkage between ferulic acid and the sugar moiety is different according to the polysaccharide considered and feruloyl esterases exhibit different specificities [25,26]. The effect of different carbon sources as inducer has been studied. The results suggested that sugar beet pulp and oat splet xylan did not induce the same esterases and that the presence of feruloyl groups in the carbon source was not essential to induce feruloyl esterases [26,27]. In our study, feruloyl esterases are induced by using sugar beet pulp in the culture of A. niger I-1472. They were able to release ferulic acid from feruloylated arabinoxylan oligosaccharides, although the ester links of ferulic acid to sugar moiety in arabinoxylan or in pectins are different. Thus, feruloyl esterase specificity did not seem to be related to the type of ester linkage in the carbon source. This supports the hypothesis that the presence of ferulic ester groups was not necessary to induce feruloyl esterase. It is likely that the genes coding for polysaccharide-degrading enzymes are

Acknowledgments This work was supported by the European Commission (FAIR No. CT96 1099). References

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