Article pubs.acs.org/JAFC
Feruloyl Esterase from the Edible Mushroom Panus giganteus: A Potential Dietary Supplement Li Wang,† Zengqiang Ma,† Fang Du,† Hexiang Wang,*,† and Tzi Bun Ng*,§ †
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
§
ABSTRACT: A novel 61 kDa feruloyl esterase (FAE) was purified to homogeneity from freshly collected fruiting bodies of Panus giganteus. The isolation procedure involved chromatography on the ion exchangers DEAE-cellulose and Q-Sepharose, followed by size exclusion chromatography on Superdex 75, which produced a purified enzyme with a high specific activity (170.0 U/mg) which was 130-fold higher than that of crude extract. The purified FAE exhibited activity toward synthetic methyl esters and short-chain fatty acid nitrophenyl esters. The Km and Vmax for this enzyme on methyl ferulate were 0.36 mM and 18.97 U/ mg proteins, respectively. FAE activity was attained at a maximum at pH 4 and 40 °C, respectively. The FAE activity was inhibited by metal ions to various degrees. The purified FAE could bring about the release of ferulic acid from wheat bran and corn bran under the action of the single purified FAE, and the amount released from wheat bran rose to 51.9% (of the total amount) by the synergistic action of xylanase. KEYWORDS: cereal bran, feruloyl esterase, Panus giganteus, purification and characterization
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INTRODUCTION Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is the hydroxycinnamic acid found in greatest abundance and is widely distributed in plants and their byproducts. FA is best known for its antioxidant, anti-inflammatory, and antimicrobial activities as well as its activities in suppressing circulating and hepatic cholesterol levels and preventing coronary disease.1,2 Therefore, it has diverse applications in the food, medical, and cosmetic industries. Cereal bran is abundant in dietary fiber and is regarded as nutritionally valuable. The intake of cereal bran is associated with a cholesterol-lowering effect, as well as a decreased risk of colon cancer. These health-promoting effects arise from the antioxidant activity of phenolics present in cereal bran.3 In cereal grains, the major group of phenolics with antioxidative activity is represented by phenolic acids, particularly FAs, which are abundant in the cereal bran, present at levels varying from 0.66% (w/w, dry weight) in wheat bran, to 0.9% in rice endosperm cell wall, and to 3.1% in maize bran.4 Free FAs demonstrate antioxidant activity and exhibit a protective function in the human body.5,6 However, the majority of FAs in cereals are found in linkage with the cell walls through ester bonds, which are not degraded by enzymes in the human digestive tract. Two effective methods were reported to disrupt the cross-link and liberate FA from cereal brans.7 One of them is a chemical method that involves alkaline hydrolysis capable of completely liberating the bound FA quickly.8 However, there are many chemical residues in the hydrolysate, and it is difficult to purify FA, which restricts its application in the food and medical industries. Therefore, more and more attention has been paid to the enzymatic method, which is milder and more efficient, releasing FA from polysaccharides using feruloyl esterases (FAEs). © 2014 American Chemical Society
FAEs (EC 3.1.1.77) are carboxyl ester hydrolases that degrade the FA esters. They are extracellular, and the corresponding expression is inducible by byproducts in agricultural and food processing, such as wheat bran, sugar beet pulp, and maize bran, which contain feruloyl ester bonds.9 FAEs releasing FA from the crude hemicellulose preparation of wheat bran were first found in Streptomyces olivochromogenes cultures.10 FAEs are important in human health because they can de-esterify dietary fiber during digestion, which can release FAs and derivatives with health-promoting actions11 A new study found that feeding mice with cheeses with high FAE activity could enhance the hydrolysis of dietary hydroxycinnamates, releasing FA and thus improving the oxidative status and providing protection against oxidative stress-related disorders of animals.12 Studies revealed that FAEs possess many other biotechnological functions, such as production of fuel ethanol, use in the pulp and paper industries, as animal feed additives, and for synthesizing novel types of esters.4,9,13 Due to the biotechnological importance of FAEs, they have been studied from a diversity of bacterial and fungal species,14 but FAEs from edible food have rarely been reported, which restrict its application in the food industry. Panus giganteus is an edible and medicinal mushroom capable of fruiting during the summer in China. It is crispy, delicious, and nutritious and also exerts significant hepatoprotective effects.15 It is absolutely safe when used in the health protection and food industries. The present study aimed to isolate an FAE with novel properties Received: Revised: Accepted: Published: 7822
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been calibrated with molecular mass standards, was also employed to ascertain the molecular mass of the FAE from a calibration curve. Mass Spectrometry of the Purified FAE. The purified FAE band was excised from SDS−polyacrylamide gel, dried, and digested with trypsin. The resulting peptides were extracted and purified according to standard protocols. Then the peptides were analyzed by highperformance liquid chromatography combined with an LTQ Orbitrap mass spectrometer (HPLC-LTQ Orbitrap MS). The amino acid sequences were manually deduced and used for blast searches in public protein primary sequence databases (www.ncbi.nlm.nih.gov/BLAST. cgi).19 Substrate Specificities and Kinetics. Substrate preferences for the purified FAE against hydroxycinnamate ester derivatives were determined using the standard assay in the presence of the following esters at 50 μM concentration: methyl ferulate (MFA), methyl pcoumarate (MpCA), and methyl caffeate (MCA). Activities toward fatty acid nitrophenyl esters with different chain lengths, including pnitrophenyl acetate, butyrate, caproate, caprylate, palmitate, and stearate, were determined by following a published standard method. The Michaelis−Menten constants (Km) and maximal velocity (Vmax) of the P. giganteus FAE based on different MFA concentrations (50, 100, 200, 300, 500, and 900 μM) were determined using the Lineweaver−Burk plot. All determinations were conducted in triplicate at pH 4.0 and 40 °C. The reciprocals of the enzyme reaction velocities were plotted on the vertical axis, and the reciprocal of the substrate concentrations were plotted on the horizontal axis to generate the Lineweaver−Burk plot for calculation of Km and Vmax. Biochemical Properties. To determine the optimal temperature, the standard FAE activity assay using MFA as substrate was run at 20, 30, 40, 50, 60, 70, 80, and 90 °C. MOPS buffer was used as the assay buffer. In the thermostability assay, the enzyme solution was exposed for 120 min to various temperatures ranging from 20 to 80 °C before cooling to 25 °C. The activity remaining was then determined. Triplicate determinations were conducted. The pH optimum of the enzyme was measured using the standard FAE activity assay above, but in a series of McIlvaine buffers (mixtures of 0.1 M citric acid and 0.2 M sodium hydrogen phosphate) in the pH range from pH 2.0 to 8.0 instead of MOPS buffer (pH 5.0). Reaction conditions were the same as those described above for activities toward MFA. Effects of Metal Ions. For studying how metal ions affect the enzyme activity, the purified enzyme solution was exposed to metal ions, such as Pb2+, Al3+, Zn2+, Mg2+, Cu2+, K+, Mn2+, Ca2+, and Cd2+ ions at 5 mM concentration, at 4 °C for 2 h before the standard FAE assay using MFA as substrate was performed. The control sample was maintained in the absence of metal ions. Enzymatic Degradation of Different Cereal Brans. Enzymatic hydrolyses were performed in 100 mM MOPS buffer (pH 5) in an incubator at 37 °C with shaking. Wheat bran (100 mg) and corn bran (100 mg) were incubated with 0.02 U/mg FAE, in the absence and also in the presence of xylanase (0.6 U/mg) in a final volume of 1.0 mL. At the end of the incubation, the reaction mixture was boiled for 3 min to terminate the reaction. After cooling and centrifugation (13000 g, 5 min), the FA released into the supernatant was subjected to HPLC analysis by using a Hypersil ODS C18 column. E-FAERU (a commercial FAE from rumen microorganism) was used as a positive control. The dosage and the reaction conditions of this enzyme were identical to those for FAE from P. giganteus. Alkali Hydrolysis of the Substrates. To determine the total alkali-extractable FA of the substrate, 1.5 mL of 2 M NaOH was added to 100 mg of substrate. Incubation was carried out at 37 °C for 24 h. The supernatant obtained following centrifugation was neutralized using 6 M HCl. Determination of the FA content measured using HPLC was then carried out as described above.
from the fresh fruiting bodies of P. giganteus and to compare its properties with those of other fungal FAEs.
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MATERIALS AND METHODS
Materials. Wheat bran and corn bran were processed as described by Wang and Ou.16 Fresh fruiting bodies of the mushroom P. giganteus were purchased from a local market. DEAE-cellulose and Q-Sepharose were obtained from Sigma Chemical Co., St. Louis, MO, USA. A Hypersil ODS C18 column was from Agilent Technologies Co., Santa Clara, CA, USA. Superdex 75 HR 10/30 was from GE Healthcare. pNitrophenyl stearate, palmitate, caproate, butyrate, and acetate were purchased from Sigma. Methyl p-coumarate (MpCA), caffeate (CA), ferulate (MFA), and ferulic acid (FA) were obtained from Apin Chemicals Ltd., Abingdon, UK. Xylanase was obtained from Sigma. EFAERU (a commercial FAE) was purchased from Megazyme, Wicklow, Ireland. Purification Scheme. Fresh fruiting bodies of the mushroom P. giganteus (800 g) were homogenized in distilled water (2 mL/g) using a Waring blender followed by centrifugation (8000g, 15 min). TrisHCl buffer (pH 8.2) was added to the resulting supernatant until the concentration of Tris-HCl buffer was 10 mM. The supernatant was then applied on a DEAE-cellulose column (5 cm × 20 cm) previously equilibrated with 10 mM Tris-HCl buffer (pH 8.2). Following elution of unadsorbed proteins in fraction D1, adsorbed proteins were desorbed sequentially with 50, 150, and 300 mM NaCl in the same buffer, to produce fractions D2, D3, and D4, respectively. Fraction D3, which exhibited FAE activity, was dialyzed prior to ion exchange chromatography on a 3 cm × 20 cm column of Q-Sepharose in 10 mM Tris-HCl buffer (pH 7.2). Following elution of unadsorbed proteins in fraction D3Q1, adsorbed proteins were desorbed from the column with a linear 0− 1 M NaCl concentration gradient in the same buffer. The active fraction (D3Q2) was dialyzed and then resolved by ion exchange chromatography on a 1 cm × 10 cm DEAE-cellulose column in 10 mM sodium phosphate buffer (pH 6.8). Following removal of the unadsorbed proteins from the DEAEcellulose column in fraction D3Q2D1, adsorbed proteins were desorbed with 1 M NaCl in the same buffer. Fraction D3Q2D1 with activity was further purified on a gel filtration Superdex G-75 HR10/30 column by fast protein liquid chromatography (FPLC) using an AKTA purifier and was eluted with 0.2 M NH4HCO3 buffer (pH 8.5). The second adsorbance peak (D3Q2D1S2) from the Superdex G-75 column represented the purified FAE. Assay for Esterolytic Activity. p-Nitrophenyl butanoate (pNPB) was used as substrate. The test sample (5 μL) was mixed with 195 μL of 50 mM sodium phosphate buffer (pH 6.0) and 5 μL of 50 mM pNPB in isopropanol. The increase of absorbance was monitored at 37 °C at 415 nm for 20 min. One unit of enzyme activity was defined as 1 μmol of p-nitrophenol released within 1 min under the assay conditions. FAE activity was determined with a modified assay employing MFA as substrate.17 The assay was conducted at 30 °C for 10 min in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 5.0) containing 50 μM MFA, and then the decrease of the MFA was determined at 340 nm. One unit (U) of enzymatic activity was defined as the amount of enzyme that catalyzes the release of 1 μmol min−1 FA under the assay conditions. All determinations were performed in triplicate. Release of the FA was also detected by HPLC on a Hypersil ODS C18 column. Detection was achieved at 320 nm on the basis of calibration curves prepared using standard FA and MFA. Elution was conducted with 45/55% (v/v) methanol/sodium formate (10 mM) at a flow rate 0.3 mL min−1. Molecular Mass Determination. SDS-PAGE was carried out using a 12% resolving gel and a 5% stacking gel.18 Staining of the gel with Coomassie brilliant blue was performed at the end of electrophoresis. The electrophoretic mobility of the FAE was compared with those of molecular mass standards, and the molecular mass of the FAE was determined from a calibration curve. Gel filtration by FPLC on a Superdex G-75 column, which had previously
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RESULTS Purification of FAE. The FAE from P. giganteus was purified to homogeneity by employing three successive ion 7823
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exchange chromatography steps and a gel filtration step, resulting in 10.1% recovery of activity (Table 1).
peptides, VEMNAAPGVDL and GTAAGDNVV, showed some homology with FAEs from other microorganisms (Table 2).
Table 1. Yields and FAE Activities at Each Purification Step (from 800 g of Panus giganteus Fresh Fruiting Bodies)
Table 2. Comparison of the Internal Amino Acid Sequences of Panus giganteus FAE with Those of Other Microbial FAEs
purification step crude extract DEAEcellulose Q-Sepharose DEAEcellulose Superdex 75 on FPLC
yield (mg)
specific activity (U/mg)
total activity (U)
accession no. recovery (%)
microorganism
purification fold
15680 765.5
1.3 17.4
20384 13320
100 65.3
1 13.4
163.4 28.3
35.7 93.7
5833 2652
28.6 13.0
27.5 72.1
12.1
170.0
2057
10.1
130.8
Peptide 1 Panus giganteus Streptomyces hygroscopicus Janibacter sp. Colletotrichum f ioriniae Colletotrichum gloeosporioides Talaromyces stipitatus Fusarium f ujikuroi Fusarium oxysporum Peptide 2 Panus giganteus Postia placenta Mycobacterium chubuense Actinomyces sp. Gaeumannomyces graminis Aspergillus kawachii Botrytis cinerea Streptomyces afghaniensis
this study WP014670826.1 WP009774585.1 XP007600389.1 EQB57686.1 XP002341414.1 CCT67738.1 ENH67953.1
Upon chromatography on the ion exchanger DEAE-cellulose, the P. giganteus extract was separated into an unadsorbed fraction D1 and three adsorbed fractions, D2, D3, and D4, eluted at 50, 150, and 300 mM NaCl, respectively. Fraction D3, which contained FAE activity, was then fractionated into two peaks, D3Q1 and D3Q2, on a Q-Sepharose column. The unadsorbed fraction (D3Q1) was devoid of FAE activity. High FAE activity was found in the adsorbed fraction (D3Q2). Subsequently, D3Q2 was resolved on DEAE-cellulose into an unadsorbed fraction, D3Q2D1, and a large adsorbed fraction, D3Q2D2. D3Q2D1 was then fractionated into two peaks of about the same size after gel filtration on Superdex 75. FAE activity was confined to the second fraction, D3Q2D1S2. Determination of Molecular Mass and Internal Amino Acid Sequence. Fraction D3Q2D1S2 appeared as a single 61 kDa band as determined by SDS-PAGE and as a single 61 kDa peak upon rechromatography on Superdex 75 (data not shown). The data indicate that the purified FAE is a monomeric enzyme (Figure 1). Several internal sequences of the purified FAE were elucidated. Database search using BLAST indicated that two
peptide
this study XP002476624.1 YP006442683.1 WP021612423.1 EJT76105.1 GAA86475.1 CCD54784.1 WP020275788.1
VEMNAAPGVDL MNPGVDLSPAA SCMNPGVDK AVMAARGVDL VEMFDAADGVE FRLFLAPGVDH LRLFLAPGVDH LRLFLAPGVDH GTAAGDNVV GTQAGDNVV GQAAGDNVV AMAAGDDVV GTAASNNIV GTDTGDNLV HNGRGDNVV STADGGNVV
Substrate Specificities and Kinetics. The purified FAE was allowed to react with hydroxycinnamic acid methyl esters and fatty acid nitrophenyl esters with different chain lengths. The relative activities were calculated and are shown in Table 3. Table 3. Activities (Mean ± SD, n = 3) of the Purified Panus giganteus FAE on Different Substrates substrate methyl ferulate methyl caffeate methyl coumarate p-nitrophenyl acetate p-nitrophenyl butyrate p-nitrophenyl caproate p-nitrophenyl palmitate p-nitrophenyl stearate a
relative activity (%) 100.00 65.32 ± 53.17 ± 62.44 ± 38.30 ± 15.09 ± nda nd
2.46 2.84 4.01 3.58 2.00
nd, not detected.
The enzyme expressed the highest activity toward methyl ferulate, but it was about 35% less active on methyl caffeate and 47% less active on methyl coumarate. The FAE enzyme also showed activity toward short-chain fatty acid nitrophenyl esters: 62.44 ± 4.01% active on p-nitrophenyl acetate, 38.30 ± 3.58% active on p-nitrophenyl butyrate, and 15.09 ± 2.00% active on p-nitrophenyl caproate. There was no activity toward the longchain substrates. The Km and Vmax values for the purified P. giganteus FAE with MFA as substrate were determined by using the Lineweaver− Burk plot to measure the decreasing level of MFA at pH 4.0 and 40 °C. The Km and Vmax for the enzyme were estimated to be 0.36 mM and 18.97 U mg−1 protein, respectively. Characteristics of P. giganteus FAE Activity. FAE had its optimal temperature at 40 °C. At 20 °C, 68% of the activity remained, and at 90 °C, 45% activity was left (Figure 2a). FAE activity was completely abolished when the enzyme was boiled for 10 min (data not shown). P. giganteus FAE was stable at
Figure 1. Molecular mass of Panus giganteus FAE by SDS-PAGE: left lane, molecular mass standards (from top downward, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa)); right lane, P. giganteus FAE. 7824
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Enzyme Activity on Cereal Brans. Wheat bran and corn bran were selected for their high contents of FAs. The total amount of FA was first determined after alkaline hydrolysis and by HPLC analysis. Wheat bran and corn bran contained about 2.16 and 13.85 mg/g FA, respectively (Table 5). Before the action of the enzymes, no free FAs could be detected. Only 1% of the total FA was released from wheat bran, whereas no free FA was released from corn bran when hydrolyzed with xylanase alone. Without the help of accessory enzymes, 8.1 and 0.3% of the total FA was released, respectively, from wheat bran and corn bran with purified FAE. To investigate the action of purified FAE in synergism with xylanase, they were added to the reaction of substrates at the same time, and it was found that the release amount of FA from wheat bran and corn bran was significantly increased (51.9 and 1% of the total FA, respectively) (Table 5). No free FA was released from corn bran, and 5.3% of the total FA was released from wheat bran when hydrolyzed with E-FAERU alone. Under the combined action of E-FAERU and xylanase, 43.3 and 1.1% of the total FA were released from wheat bran and corn bran, respectively (Table 5).
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DISCUSSION FAE was first detected in cultures of S. olivochromogenes;20 since then, many FAEs have been isolated from fungi but rarely from mushrooms. Recently it has been reported that FA was detected in the broth of mycelial culture of four kinds of edible mushrooms.21 A new FAE was isolated from the mycelial culture of the edible mushroom Pleurotus sapidus.22 In the present study, we have purified a novel FAE from P. giganteus with a chromatographic procedure involving DEAEcellulose, Q-Sepharose, DEAE-cellulose, and gel filtration on Superdex 75. The procedure employed in the present study was useful for isolating P. giganteus FAE. The chromatographic behavior of P. giganteus FAE on ionic exchangers was similar to that of Sporotrichum thermophile23 and Aspergillus niger FAE.24 They were adsorbed only on anionic exchangers, such as DEAE-cellulose and Q-Sepharose. The specific activity of purified FAE was 170.0 U/mg, which was significantly higher than that of FAEs from other fungi, most of which ranged from 0.001 to 36.7 U/mg.14 The purified FAE was a monomeric protein with a molecular mass of 61 kDa. Its molecular weight was similar to that of FAEs from the mushroom P. sapidus (55 kDa)22 and also close to those of other fungi, such as Fusarium oxysporum (62 kDa), A. niger (63 kDa), and Penicillium pinophilum (57 kDa). P. giganteus FAE was larger in molecular mass than FAEs reported from A. tubingensis (36 kDa) and F. oxysporum (27 kDa) and smaller than FAEs from A. niger (120 kDa) and A. awamori (112 kDa).14 Due to the lack of a significant homology to known microbial FAEs, the purified FAE was likely a novel enzyme. Previous research has also shown that the homology of FAE was low between species.25,26 For instance, P. sapidus FAE, the only FAE from macrofungi, also showed no significant homology to any of the published FAE sequences.22 The activity of the purified FAE on the synthetic methyl esters declined with decreasing degree of substitution: it preferred methyl ferulate over methyl caffeate and was least active on methyl coumarate. It also displayed activity toward various p-nitrophenyl esters with short chain lengths, and the activity decreased as the number of carbon atoms increased, which was similar to the esterases reported from mushrooms
Figure 2. Effects of pH and temperature on Panus giganteus FAE: (a) effects of temperature on activity of P. giganteus FAE; (b) thermostability of P. giganteus FAE; (c) effects of pH on activity of P. giganteus FAE. Results represent the mean ± SD (n = 3).
20−40 °C, and 70% residual activity was detected after incubation of the enzyme at 50 °C for 120 min (Figure 2b). The optimal pH value of the purified FAE was pH 4.0, and 60% of the activity remained at pH 2.0, whereas the enzyme activity declined sharply as the pH was increased. Negligible FAE activity was detected at pH 7.0, whereas no activity was discerned at pH 8.0 (Figure 2c). The sensitivity of P. giganteus FAE to several metal ions is shown in Table 4. The purified enzyme activity was inhibited to different degrees in the presence of metal ions: >60% inhibition in the presence of Mn2+, Ca2+, and Cd2+ ions; >40% inhibition by K+, Al3+ Zn2+, Mg2+, and Cu2+ ions; and 20% inhibition by Pb2+ ions. Table 4. Effects of Different Metal Ions (5 mM) on the Activity (Mean ± SD, n = 3) of Purified Panus giganteus FAE metal ion Pb2+ Al3+ Zn2+ Mg2+ Cu2+
residual activity (%)
metal ion
± ± ± ± ±
K+ Mn2+ Ca2+ Cd2+
84.36 60.27 55.30 54.79 52.62
3.06 2.33 3.49 2.16 3.11
residual activity (%) 43.04 38.38 32.60 31.53
± ± ± ±
2.73 2.97 3.03 2.44
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Table 5. Alkaline and Enzymatic Hydrolysis of Ferulic Acid from Cereal Bransa ferulic acid (μg/100 mg substrates) enzymatic extraction
a
substrate
alkaline extract
xylanase
pgFAE
pgFAE + xylanase
E-FAERU
E-FAERU + xylanase
wheat bran corn bran
216.3 ± 7.5 1385.5 ± 96.0
2.3 ± 0.2 nd
17.6 ± 0.7 4.7 ± 0.1
112.2 ± 2.9 13.7 ± 0.3
11.5 ± 0.4 nd
93.7 ± 1.8 15.5 ± 0.5
Data represent the mean ± SD (n = 3); nd, not detected. pgFAE, Panus giganteus FAE; E-FAERU, a commercial FAE as positive control.
species, such as S. crispa,27 A. vaginata var. vaginata, Tricholoma terreum,28 and Lycoperdon perlatum,29 and showed specificity toward short-chain fatty acids such as p-nitrophenyl butyrate and p-nitrophenyl acetate. Apparently, this specificity study suggests that the enzyme is an esterase and not a lipase. The present FAE manifested an optimal temperature at 40 °C, which was similar to those of FAEs reported from A. niger, F. oxysporum, and A. awamori,14 whereas it was lower than that of FAE from P. sapidus (50 °C).22 P. giganteus FAE was stable at 20−40 °C. This feature can be successfully employed in applications that require a moderate temperature, such as in the production of pharmaceuticals and in the extraction of free phenolic acids. The purified FAE displayed an acidic pH optimum at pH 4.0, which is in accordance with human gastrointestinal pH values. Owing to its high catalytic efficiency under acidic conditions, the purified FAE was more valuable than most other fungal FAEs in many biotechnological applications such as used as functional food and animal feed additives. The FAE in this study was very sensitive to metal ions. It had some similarities to other mushroom esterases. Esterase activity in the mushroom S. crispa could be inhibited by the addition of Zn2+ and K+ ions.27 Addition of Zn2+ and Fe2+ ions could inhibit the esterase activity in the mushroom Volvariella volvacea.30 Addition of Cd2+ and Mn2+ ions inhibited the esterase activity in the mushroom T. terreum.28 On the other hand, the addition of Ca2+, Fe2+, and Mn2+ ions produced stimulatory effects on esterase activity in the mushroom L. perlatum.29 K+ and Al3+ ions had no effect on esterase activity in the mushroom A. vaginata var. vaginata.28 The present study suggests that FA can be recovered from corn bran and wheat bran by using the purified FAE alone. The percent recovery of FA in wheat bran was higher than those of E-FAERU and FAEs reported in the literature. It has been shown that AnFae alone releases a maximum of 4% of the FA present in wheat bran.31 The amount of FA released from wheat bran was greatly enhanced (51.9% of total FA) by the synergistic action of xylanase, which was consistent with the published data. This could be because xylanolytic enzymes can degrade long-chain feruloylated xylooligosaccharides to shorterchain feruloylated xylooligosaccharides, which are more easily hydrolyzed,31 whereas the purified FAE could release FAs from corn bran to much less extent and to only 1% of the total amount even with the addition of xylanase. The small extent of hydrolysis in corn bran could be attributed to the number of substitutions on the heteroxylan backbone, the presence of extensively branched xylose, and the existence of a linkage between arabinose and xylose in the vicinity of the ferulic acid group.31 The abundance of ferulic dimers in corn bran restricts enzymatic degradation.32 The amount of FA released from corn bran by E-FAERU was slightly higher than that by the purified FAE in the presence of xylanase. However, the E-FAERU was from a rumen microorganism, which is anaerobic and difficult
to propagate, and hence its further development is limited and it is not easy to widen the field of application. In comparison, P. giganteus is a cultivated edible mushroom, which is easy to culture and absolutely safe, and so FAE from P. giganteus will have broader prospects of application.
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AUTHOR INFORMATION
Corresponding Authors
*(H.W.) Phone: +86 10 62732578. Fax: +86 10 62732578. Email:
[email protected]. *(T.B.N.) E-mail:
[email protected]. Funding
This work was financially supported by National Grants of China (Biomass dissociation and low-molecular fragment green monomerization and transformation, 2010CB732202). Notes
The authors declare no competing financial interest.
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REFERENCES
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Journal of Agricultural and Food Chemistry
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dx.doi.org/10.1021/jf405654u | J. Agric. Food Chem. 2014, 62, 7822−7827
