Schizophyllum commune produced an esterase which released ferulic acid from ... from a soluble ferulic acid-sugar ester that was isolated from wheat bran.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1988, p. 1170-1173 0099-2240/88/051170-04$02.00/0 Copyright © 1988, American Society for Microbiology
Ferulic Acid Esterase Activity from Schizophyllum
Vol. 54, No. 5
communet
C. ROGER MAcKENZIE* AND DORIS BILOUS
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KIA OR6, Canada Received 14 October 1987/Accepted 5 February 1988
Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate. There have been numerous reports of feruloylated and p-coumaroylated polymers in plant cell walls. Ferulic acid has been shown to be esterified to arabinoxylan in wheat cell walls (17). A compound identified as 2-0-[5-0-(trans-feruloyl)-p-L-arabinofuranosyl]-D-xylopyranose is released by treatment of wheat bran with Oxysporus "cellulase," a mixture of polysaccharide hydrolases (17). The same linkages have been identified in the cell walls of barley straw. In barley straw, trans-p-coumaric acid is also linked to arabinoxylans in the same way as trans-ferulic acid (13). The cell walls of spinach and sugar beet have been reported to contain ferulic acid and p-coumaric acid residues that are esterified with pectin (8, 9, 15). In spinach, the phenolic acids are esterified with galactopyranose and arabinopyranose residues. It is thought that these various polymers may be cross-linked by oxidative phenolic coupling and that the phenolic acid moieties play a role in cell wall growth and stabilization (8, 9). Ester linkages involving phenolic acids have also been identified in certain lignins (16). Little information is available on the enzymatic hydrolysis of these linkages. It has been reported that Streptomyces olivochromogenes produces an esterase that releases ferulic acid from wheat bran and that its synthesis is linked with the production of endoxylanase and xylan-debranching enzymes (12). In the present study we show that the fungus Schizophyllum commune produces a ferulic acid esterase which acts synergistically with xylanase in releasing ferulic acid from wheat bran.
Ferulic acid ester preparation. A soluble compound containing ferulic acid was produced by digestion of wheat bran with Celluclast (Novo Laboratories, Denmark). A suspension containing 50 g of wheat bran in 700 ml of distilled water was heated to 90°C and maintained at this temperature for 10 min to inactivate the ferulic acid esterase in wheat bran. The suspension was cooled, and 50 ml of Celluclast, 200 ml of 0.5 M morpholinepropanesulfonic acid (pH 6), and 2 ml of 10% NaN3 were added. This was followed by an 18-h incubation at 50°C. The suspension was then heated to 100°C and maintained at this temperature for 3 min, cooled, filtered, and centrifuged. The supernatant fraction was used as a ferulic acid esterase substrate. The composition of this fraction was analyzed by high-performance liquid chromatography as described previously (12). The main UV-adsorbing peak was collected, and the UV-adsorption spectrum was determined before and after the addition of 0.1 N NaOH. Enzyme assays. Ferulic acid esterase was assayed with wheat bran as the substrate as previously described (12). The assay was also carried out with the soluble ferulic acid substrate prepared as described above. The latter assays contained 0.4 ml of substrate and enzyme at an appropriate dilution in a final volume of 0.5 ml. Ferulic acid release was analyzed by reversed-phase high-performance liquid chromatography (12) and by high-performance liquid chromatography with a Polypore H column (Brownlee Laboratories) maintained at 65°C. The eluant for the latter column was 0.01 N H2SO4-CH3CN (9:1, vol/vol). Ferulic acid was also detected by thin-layer chromatography with Silica gel G plates. The solvent system was toluene-acetic acid (9:1, vol/vol). Plates were stained with iodine vapor. Endoglucanase, pnitrophenyllactopyranosidase, xylanase, acetyl xylan esterase, and arabinofuranosidase were assayed as described previously (12). p-Nitrophenyllactopyranoside is a substrate for exo-1,4-p-glucanase and 3-glucosidase (6). Enzyme fractionations. Culture supernatants were concentrated and dialyzed against 10 mM phosphate buffer (pH 6) in an Amicon ultrafiltration cell with a PM-10 membrane. Protein (20 mg) was applied to a 7.5-by 150-mm DEAE-TSK column (LKB, Sweden). The sample was eluted at a flow rate of 1 ml/min with a 0 to 1 M linear NaCl gradient in 10 mM phosphate buffer (pH 6).
MATERIALS AND METHODS Organism and growth conditions. One-liter cultures of S. commune ATCC 38548 were grown in 4-liter flasks with the medium of Desrochers et al. (7). Cultures were harvested after 14 days of incubation at 30°C on a rotary shaker at 150 cycles per min. The supernatant fractions were retained for enzyme assay and fractionation. Protein determinations. Protein concentrations were estimated by the method of Bradford (5) with gamma globulin as the standard. * Corresponding author. t Issued as National Research Council of Canada article no.
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SCHIZOPHYLLUM COMMUNE FERULIC ACID ESTERASE ACTIVITY
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TABLE 1. Maximum levels of various activities produced by S. commune during growth on cellulose, xylan, and wheat bran Carbon source
Protein (mg/ml)
Cellulose Xylan Wheat bran
1.0 0.21 0.17
a
b
Activity (U/mI)
Endogiucanase
pNPLase'
Xyianase
Acetyl xylan esterase
Arabinofuranosidase
Ferulic acid
37 1.7 5.6
0.014 0.065 0.005
145 7.1 18.2
16 1.5 6.7
0.048 0.43 0.077
0.028 0.002 0.007
a
esteraseb
pNPLase, p-Nitrophenyllactopyranosidase. Activity was measured with wheat bran as the substrate.
RESULTS With the exception of arabinofuranosidase, growth on Avicel resulted in the highest levels of all measured activities, whereas growth on xylan resulted in the lowest activities (Table 1). Extracellular endoglucanase and xylanase levels were approximately 20-fold higher in cellulose than in xylan cultures, whereas the levels of p-nitrophenyllactopyranosidase, acetyl xylan esterase, and ferulic acid esterase were 2-, 11-, and 14-fold higher, respectively. In contrast, the levels of arabinofuranosidase were sixfold higher in xylan cultures than in cellulose cultures. Growth on wheat bran resulted in activity levels for each enzyme that were intermediate between those in cellulose and xylan cultures. There was good reproducibility between identical cultures. Appreciable levels of enzyme activities were detected in cultures on day 3 of growth on cellulose (Fig. 1). Extracellular protein reached a maximum level at 12 days of growth and dropped considerably thereafter. Peak levels of p-nitrophenyllactopyranosidase and endoglucanase were reached at days 10 and 12, respectively, after which there was a slight decline. The enzymes associated with heteroxylan degradation (xylanase, acetyl xylan esterase, ferulic acid esterase, and arbinofuranosidase) rose steadily up to culture harvest at day 14. Fractionation of S. commune extracellular protein by TSK-DEAE chromatography revealed that ferulic acid esterase levels were dependent on the presence of xylanase. With wheat bran as the substrate, only very low levels (less than 0.05 U/ml) were detected in column fractions. However, appreciably higher levels were obtained when samples of fractions 19 through 21, known to be xylanase-rich fractions (2), were added to each fraction before assay with wheat bran (Fig. 2). Two peaks of activity were thereby detected in fractions 30 through 35. Conversely, when samples of fractions 30 through 35 were added to each of the fractions, ferulic acid esterase was detected only in fractions 19 through 21 (Fig. 2). The observed cooperativity between the xylanase and ferulic acid esterase fractions indicated that a soluble sugar-ferulic acid substrate must be formed by the action of xylanase before the esterase can function efficiently. The activity elution pattern obtained with the low-molecular-weight ferulic acid-carbohydrate complex as the substrate was similar to that obtained by the addition of xylanase to wheat bran assays. However, activity levels were approximately 10-fold higher with the low-molecular-weight substrate. The UV-absorption spectrum of this substrate was identical to that of 2-0-[5-O-(trans-feruloyl)-3-L-arabinofuranosyl]-D-xylopyranose (17). Addition of 0.1 N NaOH resulted in an immediate shift in the absorption maximum from 322 to 370 nm, followed by a slow shift to an absorption maximum of 347 nm over the next 90 min. These spectral changes are also characteristic of ferulic acid esters such as
2-0-[5-0-(trans-feruloyl)-p-L-arabinofuranosyl]-D-xylopyra-
nose. Treatment of the substrate with S. commune extracellular enzyme released a component that was identified as ferulic acid based on the high-performance liquid and thinlayer chromatography techniques employed here.
DISCUSSION Ferulic acid esterase production by S. commune paralleled that of most other enzymes associated with heteroxyIan degradation in that extracellular levels were highest in cellulose-grown cultures and increased throughout the 14day growth period. Of the enzymes thought to be involved in heteroxylan hydrolysis, only arabinofuranosidase did not follow this pattern. Ferulic acid esterase was first identified as a component of xylanolytic enzyme systems in extracellular enzyme preparations from Streptomyces olivochromogenes (12). This enzyme was produced as part of a complex mixture of activities that were capable of debranching heteroxylans and of hydrolyzing the ,-1,4-xylan backbone. The substrate specificities of the ferulic acid esterases produced by Streptomyces olivochromogenes and S. commune have not been studied. It is known, however, that the S. commune enzyme A
10.02
i 50 E
E
E
'3 0-
25
v
0
0.01
aou
-j X -J a.
oCF
z
c
B
Jo0 120
200r
0 0.05
o, I
a v na 0 c
10
100
0.025 X-X 0' wr0
4-
t0
,*
CII 8 6 Days
it -C
0
Jo
FIG. 1. Production of cellulolytic (A) and xylanolytic (B) enzymes by S. commune during growth on 1% Avicel. (A) Symbols:
protein (0), endoglucanase (A), p-nitrophenyllactopyranosidase (pNPLase) (0). (B) Symbols: xylanase (A), arabinofuranosidase (0), acetyl xylan esterase (0), ferulic acid esterase (A).
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APPL. ENVIRON. MICROBIOL.
MAcKENZIE AND BILOUS
-
0.2
I
N
E D _
0
0
0.1
cr OD
0.5= c0 z0 .2 0
a,
5-
50
0
Fraction number
), FIG. 2. TSK-DEAE chromatography of S. commune extracellular protein from cultures grown on 1% Avicel. Lines: protein ( NaCl (- - -). Symbols: ferulic acid esterase profile when equal portions of fractions 19 through 20 were pooled and 100 ,ul of the pooled material was added to each assay (0), ferulic acid esterase profile when equal portions of fractions 30 through 35 were pooled and 100 [L1 of the pooled material was added to each assay (0). Wheat bran was the substrate for esterase activity in each instance.
elutes in a region of the TSK-DEAE chromatogram that is devoid of general esterase activity as measured with pnitrophenyl acetate as the substrate (2). The present study has shown that ferulic acid esterase acts optimally on wheat bran only in the presence of xylanase. Cooperativity between enzymes involved in the liberation of plant cell wall components has previously been demonstrated with several other enzyme combinations. Greve et al. (11) reported that an cx-L-arabinofuranosidase from Ruminococcus albus significantly enhanced alfalfa cell wall hydrolysis by xylanase. Biely et al. (1, 3) observed that fungal xylanolytic enzyme systems contained esterase activity with considerable specificity for acetyl xylan and that these esterases acted synergistically with xylanases in the hydrolysis of acetylated xylans. More recently, Puls et al. (14) reported that two fungi, Agaricus bisporus and Pleurotus ostreatus, were good producers of a-4-0-methyl-D-glucuronidase and that this enzyme acted synergistically with xylanase in releasing xylose from xylans. The enhancement of ferulic acid esterase by xylanase suggests that the size or inaccessibility of the material bearing the ferulic acid residue is important in determining the rate or extent of reaction. Elucidation of the details of the size effect on ferulic acid esterase activity, as well as the related question of the effect of ferulic acid substituents on xylanase action, will require the availability of ferulate substrates with a range of sizes. There is evidence to suggest that the presence of phenolic acid linkages in plant cell walls retards the digestion of these materials in the rumen. It has been found that the amount of phenols released from plant cell walls by alkali treatment is correlated with the amount of forage digested by ruminants (10). This effect on digestion could at least partly be due to inhibition of ruminal bacteria by phenolic compounds (4). However, limited hydrolysis in the rumen of phenolic acid
ester linkages found in hemicelluloses (13, 17) and lignins (16) could also be a contributing factor. ACKNOWLEDGMENT We thank Henry Schneider for his encouragement and many helpful suggestions. LITERATURE CITED 1. Biely, P., C. R. MacKenzie, J. Puls, and H. Schneider. 1986. Cooperativity of esterases and xylanases in the enzymic degradation of acetyl xylan. Bio/Technology 4:731-733. 2. Biely, P., C. R. MacKenzie, H. Schneider, and J. Puls. 1987. The role of fungal acetyl xylan esterases in the degradation of acetyl xylan by fungal xylanases, p. 283-289. In J. F. Kennedy, G. 0. Phillips, and P. A. Williams (ed.), Wood and cellulosics. Ellis Harwood, Chichester, United Kingdom. 3. Biely, P., J. Puls, and H. Schneider. 1985. Acetyl xylan esterases in fungal cellulolytic systems. FEBS Lett. 186:80-84. 4. Borneman, W. S., D. E. Akin, and W. P. Van Eseltine. 1986. Effects of phenolic monomers on ruminal bacteria. Appl. Environ. Microbiol. 52:1331-1339. 5. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72:248-254. 6. Deshpande, M. V., K.-E. Eriksson, and L. G. Pettersson. 1984. An assay for the selective determination of exo-1,4-,-glucanases in a mixture of cellulolytic enzymes. Anal. Biochem. 138: 481-487. 7. Desrochers, M., L. Jurasek, and M. G. Paice. 1981. Production of cellulase, P-glucosidase, and xylanase by Schizophyllum commune grown on a cellulose-peptone medium. Dev. Ind. Microbiol. 22:679-684. 8. Fry, S. C. 1982. Phenolic components of the primary cell wall. Biochem. J. 203:493-504. 9. Fry, S. C. 1983. Feruloylated pectins from the primary cell wall: their structure and possible functions. Planta 157:111-123. 10. Graham, H., and P. Aman. 1984. A comparison between deg-
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radation in vitro and in sacco of constituents of untreated and ammonia-treated barley straw. Anim. Feed Sci. Technol. 10: 199-211. 11. Greve, L. C., J. M. Labavitch, and R. E. Hungate. 1984. a-L-Arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall. Appl. Environ. Microbiol. 47:1135-1140. 12. MacKenzie, C. R., D. Bilous, H. Schneider, and K. G. Johnson. 1987. Induction of cellulolytic and xylanolytic enzyme systems in Streptomyces spp. Appl. Environ. Microbiol. 53:2835-2839. 13. Mueller-Harvey, I., R. D. Hartley, P. J. Harris, and E. H. Curzon. 1986. Linkage of p-coumaroyl and feruloyl groups to
14. 15. 16. 17.
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cell wall polysaccharides of barley straw. Carbohydr. Res. 148:71-85. Puls, J., 0. Schmidt, and C. Granzow. 1987. a-Glucuronidase in two microbial xylanolytic systems. Enzyme Microb. Technol. 9:83-88. Rambouts, F. M., and J.-F. Thibault. 1986. Feruloylated pectin substances from sugar-beet pulp. Carbohydr. Res. 154:177-187. Shimada, M., T. Fukuzuka, and T. Higuchi. 1971. Ester linkages of p-coumaric acid in bamboo and grass lignins. Tappi 54:72-78. Smith, M. M., and R. D. Hartley. 1983. Occurrence and nature of ferulic acid substitution of cell-wall polysaccharides in graminaceous plants. Carbohydr. Res. 118:65-80.
