Worfd Journa/
of Microbio/ogy
& f%otectmofogy
11, 169-162
Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis J-A. Donaghy* and A.M. McKay Ferulic and p-coumaric acid can be separated from their corresponding aliphatic methy esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl and p-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester horn wheat bran was also efficiently separated and used as substrate for the enzyme assays. PeniciZZium exp~~nsum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture. Key
WOYU!S:
Capillary,
coumaroyl,
electrophoresis,
esterase, feruloyl.
Feruloyi and coumaroyl ester groups, bound to plant-cellwall polysaccharides in many plants, help maintain the structural integrity of the cell-wall matrix and are the most recalcitrant residues of plant-cell-wall polysaccharides (Akin 1986). Feruloyl and p-coumaroyl e&erase activities, resulting in the cleavage of ester cross-linkages and the subsequent release of free phenolic acid, have been reported in Sfrepfomyces oliuochromogenes (Faulds 81 Williamson 1991), Penicill&n pinophil~~ (Castanares ef al. 1992), ~eocull~~asf~c spp. (Borneman ef al. IWO), Schizo~h~l~~~ co~~~~e (MacKenzie & Bilous 1988) and Aspergillm spp. (Tenkanen ef al. 1991). The aliphatic (methyl and ethyl) esters of the phenolic acids and phenolic acid-carbohydrate esters from wheat bran are commonly used as enzyme substrates. The phenolic acid resulting from enzyme de-esterification is normally measured by GC (Borneman ef al. 1990) or by HPLC (MacKenzie & Bilous 1988; Castanares ef al, 1992). Capillary electrophoresis (CE) has been used to investigate the separation of phenolic carboxylic acids (Morin & Dreux 1993) using micellar electrokinetic capillary chromatography (MECC), which involves the addition of a surfactant to the electrophoresis buffer. in this study, we investigated the possible use of capillary zone electrophoreThe authors are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland‘ Newforge Lane, Belfast BT9 5PX, UK; fax: 232 668376. A.M. McKay is also affiliated with the Department of Food Science (Microbiology), The Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK, *Corresponding author. @ 1995 Rapid
Communications
of Oxford
Ltd
sis (CZE), a simpler form of CE, to separate and measure the release of free phenolic acid from the methyl esters of ferulic and p-coumaric acid and from a phenolic acid-carbohydrate substrate isolated from wheat bran.
Materials
and Methods
Microorganism and Cube Condifions F’erticilbn eqatisgm was routinely subcultured on potato/ dextrose/agar (PDA) slopes. Spores were harvested from 4-day agar slopes and suspended in 10 ml sterile distilled water to give approximately 4.5 x IO9 spores ml-‘. One-litre flasks, each containing 7’5 g presterilized (121’C, 15 min) supplemented substrate comprising (%, w/w): unprocessed wheat bran, 33; distilled water, 65; (NH4)$04, 1; yeast nitrogen base, 1; and KHzP04, 0.5, were each inoculated with 2.5 m1 spore suspension, sealed with a cotton wool plug, shaken briefly to disperse the spores through the medium and incubated statically at 26OC for i’ days. Enzyme
Edracfion
The flask contents were extracted by adding 400 ml of 0.2 M sodium acetate buffer, pH 5.6, containing 0.1% (w/v) Tween 80 and homogenizing in a Waring blender for 4 min. The resultant slurry was shaken for 2 h at 26*C at 1.50 rev rnin-l before cenkifugation
(2000
X g, 30 min)
followed
by a further
cenkifuga-
tion of the supematant (28,000 x g, 30 min). The second supematant was dialysed for 3 days against 0.1 M sodium acetate buffer, pH 5.6, containing 0.2% (w/v) EDTA and 0.02% (w/v) sodium azide, with periodic changes of dialysis buffer. in some cases a further cent~fugation step (5000 X g, 15 min) was necessary after dialysis. All centrifugation and dialysis steps were perfomed at 4°C and the resultant ‘crude enzyme’ was stored at - ZO'C.
~easuremenf Ferulic Acid Ester Preparation
A water-soluble, sugar-fen&c acid ester (FAX) was produced by the digestion of unprocessed wheat bran with commercial ceilulase (Ceiiuclast; Novo Nordansk, Denmark), according to MacKenzie & Bilous (1988). FeruloyVp-coumaroyl esterase activities were assayed by the analysis of the acid released from the methyl esters of ferulic and coumarjc acid (Apin Chemicals, UK) or from FAX as prepared above. The aiiphatic methyl esters were prepared as 50 rnM solutions by dissolving the esters in 2 ml HPLC-grade ethanol followed by dilution to 50 ml in sterile distilled water. Reaction mixtures (I mi), containing 0.1 ml of I M Tris/HCl buffer, pH 8.0, 0.5 ml crude enzyme supematant, 0.35 ml distilled water and 0.05 ml of the methyl
esters (50 mM), were incubated at 30’C for up to 30 min. Assay mixtures for the measurement of fen&c acid released from FAX used 0.2 ml of lo-fold concentrated (lyophilized) FAX solution in place of the synthetic esters and samples were incubated at 3O’C for up to 1 h. In all cases, the reactions were terminated by the addition of 0.1 ml 1 M HCl (to lower the pH to 2.0). Fen&c acid released was extracted three times with equal volumes of HPLC-grade diethyl ether, dried under vacuum (Jouan, France), dissolved in OS ml of a methanol: water (50 :SO), filtered (O.l+m pore Anotop syringe filters) and subjected to capillary zone electrophoresis analysis. One unit of activity is defined as the amount of enzyme releasing 1 pmol of ferulic acid per min at ~o*C. time
intervals
Detection of Phenolic Esters and their Corresponding Acids &y Capillury Zone Electrophoresis Phenolic acids, their methyl esters and FAX were detected using a capillary electrophoresis system (CES I; Dionex, UK) modified to incorporate a spectral array detector (Spectra Focus; Spectra Physics, UK). Analysis conditions were as folIows: an open, fused-s&a capillary column, column length 110 cm, inner diam. 75 pm, with forced air cooling; injection by gravity at 100 mm for 30 s (24.5 nl): 30 kV; 60 PA: borate buffer (50 mM, pH 7.2). Mixtures containing methyl feruiate, methyl coumarate and their corresponding acids were dissolved in methanol:water (5o:50) (I rnM each) and separated using an open capillary as above, with 50 rnM borate buffer, pH 8.5. A filtered @.I-pm pore) aqueous solution of FAX was analysed under similar conditions. Determination of Alkali-extractable Feru/ic Acid from FAX Three ml of 1 M NaOH (purged with nitrogen) were added to 1 ml of FAX, flushed with nitrogen and sealed. Tubes were incubated in the dark in a shaking incubator (150 rev min-‘) at !@C for 18 h, then centrifuged (3000 X g, IO min) and the supernatant acidified to pH 2.0 with HCl. Fen&c acid was extracted, dried and analysed as described. Triplicate samples were saponified in this manner.
Results
and Discussion
Capillary zone electrophoresis (CZE) was successfully used to measure feruloyl/~-coumaroyl esterase activity. Methyl fendate and ferulic acid had migration times of 10.2 and 15.3 min, respectively (Figure lb) using borate buffer, pH 7.2. Minor peaks, detected at 7.8 and 10.6 min, may have been due to the ‘crude’ enzyme preparation used in
o{ eskemseactiuify by capii~~
electrop~resjs
the assay and disappeared after boiling, as shown in the control (Figure la). The enzymatic product and synthetic fen&c acid gave similar absorbance maxima (318 nm) and minima (254 nm). Attempts to separate a mixture of the neutral esters methyl ferulate and methyl coumarate using borate buffer with a pH of 7.2 were unsuccessful. However, selectivity was increased by adjusting the buffer pH to 8.5. Under these conditions the methyl esters appeared as two distinct peaks (methyl coumarate at 11.6 min and methyl ferulate at 12.1 min). The respective acids were also separated at this higher pH (coumaric acid at 18.33 min and ferulic acid at 19.9 min) (Figure 2). Plant-cell-wall structures may contain both feruloyl and p-coumaroyl residues, hence CZE could be used to monitor either the enzymatic or chemical release of both phenolic moieties simultaneously. FAX, shown to contain 78 ,ug ferulic acid ml-l, as determined by saponification and measured using CZE, migrated as a single peak (10.9 min) using the open, fused-silica capillary with borate buffer at pH 8.5. The spectral scan of FAX shows an increase in absorbance from 320 nm into the visible region of the spectrum, indicative of the sugar moiety within this substrate, CZE has not previously been used to separate such phenolic esters, although phenolic carboxylic acids have been separated by MECC (Morin & Dreux 1993). The above results show similar migration times to that obtained using MECC without the need for the addition of a surfactant to the run buffer. The results obtained demonstrate that CZE is an alternative to conventional liquid chromatography techniques for the measurement of phenolic acid esterase activities. The combination of electrophoretic and electroosmotic principles, coupled with the small sample loads required (nl compared with ~1 in HPLC) allow high-resolution separations of phenolic acids, their aliphatic esters and sugar esters containing phenolic groups. Feruloyl and p-coumaroyl esterase activities were present in the ‘crude enzyme’ preparation of P,expunsum. Mean (& S.D.) activities of 0.096 2 0.005 and 0.074 2 0.006 U ml-l of extract, respectively, were recorded using the aliphatic esters as substrates. The ‘crude enzyme’ also released ferulic acid from the feruloyl-carbohydrate ester isolated from wheat bran (an activity of 0.033 * O.o03 U ml-l. In all cases, the product formed was proportional to the time of reaction and to the concentration of the enzyme. Although Donaghy & McKay (1994) reported the production of phenolic acid esterases from this organism using a screening assay, there has been no previous attempt to quantify the activity. A feruloyl/ p”coumaroyl esterase has been purified from P,pirqdd~m (Castanares ef 81. 1992), the only such esterase characterized from Penicillium to date. The activities recorded were similar to those reported for other microbial phenolic acid esterases (Christov & Prior 1993; Faulds & Williamson 1994). The 3fold higher activity with methyl ferulate as substrate than
World &md
of A4icmbiolagy t3 Biofechmlqy, 5’01 ~1, ~995
161
].A.
Donaghy
and A.M.
McKay Akocaknasfic MC-2 using methyl ferulate and Coastal Bermuda grass as substrates (Bomeman et al. IWO). Clearly, there is a need to purify such enzymes further and elucidate their specificities with regard to more chemically-defined substrates or enzymatic hydrolysates of various plant-cell walls. CE should allow easy separation of such substrates and serve as a convenient method for measuring phenolic acid esterase activities.
Acknowledgement b
i
The authors thank P. Kelly for excellent throughout this work.
technical assistance
References ain,
Figure
1986 Interactions of ruminal bacteria and fungi with forages. ]o~r& of Animal Science 63, 962-977. Bomeman, W.S., Hartley, R.D., Morrison, W.H., Akin, D.E. & Ljungdahl, L.G. 1990 Feruloyl and p-coumaroyl e&erase from anaerobic fungi in relation to plant cell wall degradation. Applied Microbiology and Biotechnology 33, 315-35 1. Castanares, A., McCrae, S.1. & Wood, T.M. 1992 Purification and properties of a feruloyl/p-coumaroyl esterase from the fungus Penicillium pinophilum. Ertzyme und Microbial Technology 14, 875-884. Christov, L.P. 61 Prior, B.A. I993 Esterases of xylan-degrading microorganisms: Production, properties, and significance. Enzyme and Microbial Technology 15, 46&475, Donaghy, J.A. & McKay, A.M. I994 Novel screening assay for the detection of phenolic acid esterases. World]ournal of Microbiology and Biotechnology 10, 41-44. Faulds, C.B. & Williamson, G. 1991 The purification and characterization of 4-hydroxy-%methoxycinnamic (ferulic) acid esterase from Skeptomyces olivochromogenes. Journal of General Microbiologyl37,2337-2345. Faulds, C.B. & Williamson, G. 1994 Purification and characterization of a ferulic acid esterase (FAE-111) from Asperg& niger: specificity for the phenolic moiety and binding to microcrystalline cellulose. Microbiology 140, 77+787. MacKenzie, R.C. & Bilous, D. 1988 Ferulic acid esterase activity from Schizophyllum commcme. Applied and Environme& Microbiology 54, 1170-1173, Morin, P. &I Dreux, M. 1993 Factors influencing the separation of ionic and non-ionic chemical natural compounds in plant extracts by capillary electrophoresis. Journal of Liquid Chromatography 16,3735-3755. Tenkanen, M., Schuseii, J., Puls, J. & Poutanen, K. 1991 Production, purification and characterization of an esterase liberating phenolic acids from lignocellulosics. ]ournul of Biotechnology 18, 6984.
with the feruloyl-carbohydrate ester is similar to results reported for the feruloyl esterase from the anaerobic fungus
(Received
in
Sepfember
1994)
1. Separation of methyl ferulate and ferulic acid by capillary zone electrophoresis and their detection by U.V. spectral scanning. (a) Spectral scan showing methyl ferulate incubated with boiled ‘crude enzyme’ from F’.expansum. (b) Spectral scan showing release of ferulic acid from methyl ferulate after a 10 min incubation with ‘crude enzyme’,
Figure
0.07so
o.o6oo
0.04Sl -J 0.0301
o.oL52
IL, 20&l
2s.00
2. Separation of methyl coumarate (l), methyl ferulate (2), coumaric acid (3) and ferulic acid (4) by capillary zone electrophoresis at pH 6.5 and detection by U.V. spectral scanning at 340 nm.
D.E. southern
revised
form
19
Sepfember
1994;
accepfed
27
of Microbio/ogy
& f%otectmofogy
11, 169-162
Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis J-A. Donaghy* and A.M. McKay Ferulic and p-coumaric acid can be separated from their corresponding aliphatic methy esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl and p-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester horn wheat bran was also efficiently separated and used as substrate for the enzyme assays. PeniciZZium exp~~nsum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture. Key
WOYU!S:
Capillary,
coumaroyl,
electrophoresis,
esterase, feruloyl.
Feruloyi and coumaroyl ester groups, bound to plant-cellwall polysaccharides in many plants, help maintain the structural integrity of the cell-wall matrix and are the most recalcitrant residues of plant-cell-wall polysaccharides (Akin 1986). Feruloyl and p-coumaroyl e&erase activities, resulting in the cleavage of ester cross-linkages and the subsequent release of free phenolic acid, have been reported in Sfrepfomyces oliuochromogenes (Faulds 81 Williamson 1991), Penicill&n pinophil~~ (Castanares ef al. 1992), ~eocull~~asf~c spp. (Borneman ef al. IWO), Schizo~h~l~~~ co~~~~e (MacKenzie & Bilous 1988) and Aspergillm spp. (Tenkanen ef al. 1991). The aliphatic (methyl and ethyl) esters of the phenolic acids and phenolic acid-carbohydrate esters from wheat bran are commonly used as enzyme substrates. The phenolic acid resulting from enzyme de-esterification is normally measured by GC (Borneman ef al. 1990) or by HPLC (MacKenzie & Bilous 1988; Castanares ef al, 1992). Capillary electrophoresis (CE) has been used to investigate the separation of phenolic carboxylic acids (Morin & Dreux 1993) using micellar electrokinetic capillary chromatography (MECC), which involves the addition of a surfactant to the electrophoresis buffer. in this study, we investigated the possible use of capillary zone electrophoreThe authors are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland‘ Newforge Lane, Belfast BT9 5PX, UK; fax: 232 668376. A.M. McKay is also affiliated with the Department of Food Science (Microbiology), The Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK, *Corresponding author. @ 1995 Rapid
Communications
of Oxford
Ltd
sis (CZE), a simpler form of CE, to separate and measure the release of free phenolic acid from the methyl esters of ferulic and p-coumaric acid and from a phenolic acid-carbohydrate substrate isolated from wheat bran.
Materials
and Methods
Microorganism and Cube Condifions F’erticilbn eqatisgm was routinely subcultured on potato/ dextrose/agar (PDA) slopes. Spores were harvested from 4-day agar slopes and suspended in 10 ml sterile distilled water to give approximately 4.5 x IO9 spores ml-‘. One-litre flasks, each containing 7’5 g presterilized (121’C, 15 min) supplemented substrate comprising (%, w/w): unprocessed wheat bran, 33; distilled water, 65; (NH4)$04, 1; yeast nitrogen base, 1; and KHzP04, 0.5, were each inoculated with 2.5 m1 spore suspension, sealed with a cotton wool plug, shaken briefly to disperse the spores through the medium and incubated statically at 26OC for i’ days. Enzyme
Edracfion
The flask contents were extracted by adding 400 ml of 0.2 M sodium acetate buffer, pH 5.6, containing 0.1% (w/v) Tween 80 and homogenizing in a Waring blender for 4 min. The resultant slurry was shaken for 2 h at 26*C at 1.50 rev rnin-l before cenkifugation
(2000
X g, 30 min)
followed
by a further
cenkifuga-
tion of the supematant (28,000 x g, 30 min). The second supematant was dialysed for 3 days against 0.1 M sodium acetate buffer, pH 5.6, containing 0.2% (w/v) EDTA and 0.02% (w/v) sodium azide, with periodic changes of dialysis buffer. in some cases a further cent~fugation step (5000 X g, 15 min) was necessary after dialysis. All centrifugation and dialysis steps were perfomed at 4°C and the resultant ‘crude enzyme’ was stored at - ZO'C.
~easuremenf Ferulic Acid Ester Preparation
A water-soluble, sugar-fen&c acid ester (FAX) was produced by the digestion of unprocessed wheat bran with commercial ceilulase (Ceiiuclast; Novo Nordansk, Denmark), according to MacKenzie & Bilous (1988). FeruloyVp-coumaroyl esterase activities were assayed by the analysis of the acid released from the methyl esters of ferulic and coumarjc acid (Apin Chemicals, UK) or from FAX as prepared above. The aiiphatic methyl esters were prepared as 50 rnM solutions by dissolving the esters in 2 ml HPLC-grade ethanol followed by dilution to 50 ml in sterile distilled water. Reaction mixtures (I mi), containing 0.1 ml of I M Tris/HCl buffer, pH 8.0, 0.5 ml crude enzyme supematant, 0.35 ml distilled water and 0.05 ml of the methyl
esters (50 mM), were incubated at 30’C for up to 30 min. Assay mixtures for the measurement of fen&c acid released from FAX used 0.2 ml of lo-fold concentrated (lyophilized) FAX solution in place of the synthetic esters and samples were incubated at 3O’C for up to 1 h. In all cases, the reactions were terminated by the addition of 0.1 ml 1 M HCl (to lower the pH to 2.0). Fen&c acid released was extracted three times with equal volumes of HPLC-grade diethyl ether, dried under vacuum (Jouan, France), dissolved in OS ml of a methanol: water (50 :SO), filtered (O.l+m pore Anotop syringe filters) and subjected to capillary zone electrophoresis analysis. One unit of activity is defined as the amount of enzyme releasing 1 pmol of ferulic acid per min at ~o*C. time
intervals
Detection of Phenolic Esters and their Corresponding Acids &y Capillury Zone Electrophoresis Phenolic acids, their methyl esters and FAX were detected using a capillary electrophoresis system (CES I; Dionex, UK) modified to incorporate a spectral array detector (Spectra Focus; Spectra Physics, UK). Analysis conditions were as folIows: an open, fused-s&a capillary column, column length 110 cm, inner diam. 75 pm, with forced air cooling; injection by gravity at 100 mm for 30 s (24.5 nl): 30 kV; 60 PA: borate buffer (50 mM, pH 7.2). Mixtures containing methyl feruiate, methyl coumarate and their corresponding acids were dissolved in methanol:water (5o:50) (I rnM each) and separated using an open capillary as above, with 50 rnM borate buffer, pH 8.5. A filtered @.I-pm pore) aqueous solution of FAX was analysed under similar conditions. Determination of Alkali-extractable Feru/ic Acid from FAX Three ml of 1 M NaOH (purged with nitrogen) were added to 1 ml of FAX, flushed with nitrogen and sealed. Tubes were incubated in the dark in a shaking incubator (150 rev min-‘) at !@C for 18 h, then centrifuged (3000 X g, IO min) and the supernatant acidified to pH 2.0 with HCl. Fen&c acid was extracted, dried and analysed as described. Triplicate samples were saponified in this manner.
Results
and Discussion
Capillary zone electrophoresis (CZE) was successfully used to measure feruloyl/~-coumaroyl esterase activity. Methyl fendate and ferulic acid had migration times of 10.2 and 15.3 min, respectively (Figure lb) using borate buffer, pH 7.2. Minor peaks, detected at 7.8 and 10.6 min, may have been due to the ‘crude’ enzyme preparation used in
o{ eskemseactiuify by capii~~
electrop~resjs
the assay and disappeared after boiling, as shown in the control (Figure la). The enzymatic product and synthetic fen&c acid gave similar absorbance maxima (318 nm) and minima (254 nm). Attempts to separate a mixture of the neutral esters methyl ferulate and methyl coumarate using borate buffer with a pH of 7.2 were unsuccessful. However, selectivity was increased by adjusting the buffer pH to 8.5. Under these conditions the methyl esters appeared as two distinct peaks (methyl coumarate at 11.6 min and methyl ferulate at 12.1 min). The respective acids were also separated at this higher pH (coumaric acid at 18.33 min and ferulic acid at 19.9 min) (Figure 2). Plant-cell-wall structures may contain both feruloyl and p-coumaroyl residues, hence CZE could be used to monitor either the enzymatic or chemical release of both phenolic moieties simultaneously. FAX, shown to contain 78 ,ug ferulic acid ml-l, as determined by saponification and measured using CZE, migrated as a single peak (10.9 min) using the open, fused-silica capillary with borate buffer at pH 8.5. The spectral scan of FAX shows an increase in absorbance from 320 nm into the visible region of the spectrum, indicative of the sugar moiety within this substrate, CZE has not previously been used to separate such phenolic esters, although phenolic carboxylic acids have been separated by MECC (Morin & Dreux 1993). The above results show similar migration times to that obtained using MECC without the need for the addition of a surfactant to the run buffer. The results obtained demonstrate that CZE is an alternative to conventional liquid chromatography techniques for the measurement of phenolic acid esterase activities. The combination of electrophoretic and electroosmotic principles, coupled with the small sample loads required (nl compared with ~1 in HPLC) allow high-resolution separations of phenolic acids, their aliphatic esters and sugar esters containing phenolic groups. Feruloyl and p-coumaroyl esterase activities were present in the ‘crude enzyme’ preparation of P,expunsum. Mean (& S.D.) activities of 0.096 2 0.005 and 0.074 2 0.006 U ml-l of extract, respectively, were recorded using the aliphatic esters as substrates. The ‘crude enzyme’ also released ferulic acid from the feruloyl-carbohydrate ester isolated from wheat bran (an activity of 0.033 * O.o03 U ml-l. In all cases, the product formed was proportional to the time of reaction and to the concentration of the enzyme. Although Donaghy & McKay (1994) reported the production of phenolic acid esterases from this organism using a screening assay, there has been no previous attempt to quantify the activity. A feruloyl/ p”coumaroyl esterase has been purified from P,pirqdd~m (Castanares ef 81. 1992), the only such esterase characterized from Penicillium to date. The activities recorded were similar to those reported for other microbial phenolic acid esterases (Christov & Prior 1993; Faulds & Williamson 1994). The 3fold higher activity with methyl ferulate as substrate than
World &md
of A4icmbiolagy t3 Biofechmlqy, 5’01 ~1, ~995
161
].A.
Donaghy
and A.M.
McKay Akocaknasfic MC-2 using methyl ferulate and Coastal Bermuda grass as substrates (Bomeman et al. IWO). Clearly, there is a need to purify such enzymes further and elucidate their specificities with regard to more chemically-defined substrates or enzymatic hydrolysates of various plant-cell walls. CE should allow easy separation of such substrates and serve as a convenient method for measuring phenolic acid esterase activities.
Acknowledgement b
i
The authors thank P. Kelly for excellent throughout this work.
technical assistance
References ain,
Figure
1986 Interactions of ruminal bacteria and fungi with forages. ]o~r& of Animal Science 63, 962-977. Bomeman, W.S., Hartley, R.D., Morrison, W.H., Akin, D.E. & Ljungdahl, L.G. 1990 Feruloyl and p-coumaroyl e&erase from anaerobic fungi in relation to plant cell wall degradation. Applied Microbiology and Biotechnology 33, 315-35 1. Castanares, A., McCrae, S.1. & Wood, T.M. 1992 Purification and properties of a feruloyl/p-coumaroyl esterase from the fungus Penicillium pinophilum. Ertzyme und Microbial Technology 14, 875-884. Christov, L.P. 61 Prior, B.A. I993 Esterases of xylan-degrading microorganisms: Production, properties, and significance. Enzyme and Microbial Technology 15, 46&475, Donaghy, J.A. & McKay, A.M. I994 Novel screening assay for the detection of phenolic acid esterases. World]ournal of Microbiology and Biotechnology 10, 41-44. Faulds, C.B. & Williamson, G. 1991 The purification and characterization of 4-hydroxy-%methoxycinnamic (ferulic) acid esterase from Skeptomyces olivochromogenes. Journal of General Microbiologyl37,2337-2345. Faulds, C.B. & Williamson, G. 1994 Purification and characterization of a ferulic acid esterase (FAE-111) from Asperg& niger: specificity for the phenolic moiety and binding to microcrystalline cellulose. Microbiology 140, 77+787. MacKenzie, R.C. & Bilous, D. 1988 Ferulic acid esterase activity from Schizophyllum commcme. Applied and Environme& Microbiology 54, 1170-1173, Morin, P. &I Dreux, M. 1993 Factors influencing the separation of ionic and non-ionic chemical natural compounds in plant extracts by capillary electrophoresis. Journal of Liquid Chromatography 16,3735-3755. Tenkanen, M., Schuseii, J., Puls, J. & Poutanen, K. 1991 Production, purification and characterization of an esterase liberating phenolic acids from lignocellulosics. ]ournul of Biotechnology 18, 6984.
with the feruloyl-carbohydrate ester is similar to results reported for the feruloyl esterase from the anaerobic fungus
(Received
in
Sepfember
1994)
1. Separation of methyl ferulate and ferulic acid by capillary zone electrophoresis and their detection by U.V. spectral scanning. (a) Spectral scan showing methyl ferulate incubated with boiled ‘crude enzyme’ from F’.expansum. (b) Spectral scan showing release of ferulic acid from methyl ferulate after a 10 min incubation with ‘crude enzyme’,
Figure
0.07so
o.o6oo
0.04Sl -J 0.0301
o.oL52
IL, 20&l
2s.00
2. Separation of methyl coumarate (l), methyl ferulate (2), coumaric acid (3) and ferulic acid (4) by capillary zone electrophoresis at pH 6.5 and detection by U.V. spectral scanning at 340 nm.
D.E. southern
revised
form
19
Sepfember
1994;
accepfed
27
