Effects of crude feruloyl and acetyl esterase solutions of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 isolated from Holstein steers on hydrolysis of Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks

Animal Feed Science and Technology 154 (2009) 218–227

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Effects of crude feruloyl and acetyl esterase solutions of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 isolated from Holstein steers on hydrolysis of Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks H.J. Yang a,b,∗, Q. Yue b,1, Y.C. Cao b, D.F. Zhang b, J.Q. Wang c a

Department of Animal Nutrition & Feed Science, College of Animal Science and Technology, China Agricultural University (CAU), Beijing 100193, PR China b Department of Animal Biology & Physiology, College of Biological Sciences, China Agricultural University (CAU), Beijing 100193, PR China c State key Laboratory of Animal Nutrition, Institute of Animal Science, China Academy of Agricultural Sciences (CAAS), Beijing 100193, PR China

a r t i c l e

i n f o

Article history: Received 1 April 2009 Received in revised form 14 September 2009 Accepted 15 September 2009

Keywords: Rumen Anaerobic fungi Ferulic acids esterase Acetyl esterase Plant cell wall Agricultural by-products

a b s t r a c t Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 were isolated from rumen fluids of Holstein steers using a modification of the roll-tube technique of Hungate (1969). After a 9-day incubation for YQ1, and a 4-day incubation for YQ3, with optimal carbon and N supply in cultures, crude enzyme solutions of both fungi were prepared by centrifuging cultures for 10 min at 1000 × g at 4 ◦ C. Enzymological characteristics of ferulic acid esterase and acetyl esterase were measured in the crude enzyme solution. The Michaelis constants (Km ) and maximum velocities (Vmax ) of ferulic acid esterase against methyl ferulate at pH 6.0 and 39 ◦ C were 30 ␮M and 8.05 mU for YQ1 and 129 ␮M and 3.15 mU for YQ3, respectively. With substrate of pnitrophenyl acetate, the Km and Vmax of AE at pH 6.0 and 39 ◦ C were 0.21 mM and 336 mU for YQ1 and 5.26 mM and 854 mU for YQ3. Enzymatic release of reducing sugar, ferulic acid, and p-coumaric acid from complex fibre-rich feedstuffs was evaluated, and YQ3 released more reducing sugars from wheat straw, but less from the

Abbreviations: ADF, acid detergent fibre; AE, acetyl esterase; CES, crude enzyme solution; CS, corn stalks; CW, Chinese wildrye grass hay; DM, dry matter; FAE, ferulic acid esterase; Km , Michaelis constants; MB, maize bran; NDF, neutral detergent fibre; WB, wheat bran; WS, wheat straw; Vmax , maximum velocities. ∗ Corresponding author at: Department of Animal Nutrition & Feed Science, College of Animal Science and Technology, China Agricultural University, Beijing 100193, PR China. Tel.: +86 10 6273 2874; fax: +86 10 6281 4346. E-mail address: yang [email protected] (H.J. Yang). 1 The author who equally contributed to this work. 0377-8401/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2009.09.006

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other plant materials especially maize bran and corn stalks. YQ3 liberated much more hydroxycinnamic acids from corn stalks, and YQ1 liberated much more hydroxycinnamic acids from maize bran. Anaerobic rumen fungi showed species diversity of enzymological characteristics of ferulic acid esterase and acetyl ester esterase, and exhibited a strong capability to release ferulic acid and p-coumaric acid from complex fibre-rich feedstuffs. Therefore anaerobic fungi, especially their FAE, should not be neglected in studies on biodegradation of plant cell walls in the rumen as well as on industrial processes developed to release ferulic acid and p-coumaric acid from agricultural by-products with potential commercial industrial applications, such as precursors of natural vanillin, natural antioxidants, food preservative agents, anti-inflammatory agents and photoprotectants. © 2009 Elsevier B.V. All rights reserved.

1. Introduction There are many cross-linkages between lignin polymers and polysaccharides in the plant cell wall through phenolic acids, predominantly ferulic and p-coumaric acids, that provide cell wall integrity and resist enzymatic attack by microorganisms (Ralph et al., 1992; Williamson et al., 1998; Reid, 2000; Trzcinska et al., 2005). Phenolic acids exist particularly in graminaceous plants where approximately 0.45 of the cell walls are arabinoxylans to which ferulic acid is esterified (Panagiotou et al., 2007). In China, agricultural by-products including wheat bran, maize bran, wheat straw and corn stalks as well as Chinese wildrye grass hay, have extensively been included in ruminant diets, but their digestibility is quite low due to the formation of cross-linkages between ferulic acid and arabinoxylans in cell walls. Therefore, besides main-chain degrading enzymes such as cellulases and xylanase, side-chain degrading enzymes, such as ferulic acid esterase (EC 3.1.1.73, FAE) and acetyl esterase (EC 3.1.1.6, AE), might be key lignin-degrading esterases to break the cross-linkages in plant cell walls. Fungi have been regarded as the primary colonizers and degraders of plant fragments (Hebraud and Fevre, 1988; Gordon and Phillips, 1998) and their capacity to degrade lignin containing tissues is believed to be even higher than that of bacteria in the rumen (Akin and Borneman, 1990). Since Orpin (1975) first confirmed the presence of fungi in the rumen, they have received attention because of their potential to degrade fibre. In the literature, the cellulase of Neocallimastix frontalis had a higher rate of degradation of crystalline cellulose than those of the aerobic fungus Trichoderma reesei and Penicillium pinophilum that are used commercially (Wood and McCrae, 1986; Wilson and Wood, 1992). Comparatively high attention that has been paid to main-chain degrading enzymes of rumen fungi (Hodrová et al., 1998; Tripathi et al., 2007; Atanasova-Pancevska and Kungulovski, 2008) contrasts to the little reported on rumen fungal side-chain degrading enzymes that can remove the crosslinkages of plant cell walls, and could possibly improve the action of main-chain degrading enzymes. Although isolation and characterization of the FAE of Neocallimastix sp. MC-2, Piromyces sp. MC-1, Orpinomyces sp. PC-2 and Orpinomyces sp. PC-3 from rumen of cattle and effects of organic acids on FAE production have been investigated, little information exists about their hydrolyzation of complex fibre-rich feedstuffs, except coastal Bermuda grass (Borneman et al., 1990, 1991, 1992; Paul et al., 2003; Barahona et al., 2006). The objective of this work was to measure the enzymological characteristic of FAE and AE of two anaerobic rumen fungi isolates, and to evaluate their capability to release phenolic acids in Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks. 2. Materials and methods 2.1. Microorganism The anaerobic rumen fungi Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 were isolated from the rumen fluid of Holstein steers. The steers aged 22 ± 0.2 months with body weight of 641 ± 27.5 kg

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Table 1 The optimal combination of carbon and N source (g/L) in anaerobic cultures for growth of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 to produce feruloyl esterase in vitro.

Wildrye grass hay, chopped Corn stalks, chopped Glucose Tryptone (NH4 )2 SO4 Yeast extract a

Neocallimastix sp. YQ1

Anaeromyces sp. YQ3

0.08 NAa 1.0 1.6 0.5 NAa

NAa 0.08 1.0 1.7 NAa 1.4a

NA = not added in cultures.

were kept at the experimental farm of the China Agriculture University. They were fed at 06:30, 12:30 and 17:30 h and were offered each time 6.5 kg dry matter (DM) of a standard diet composed of (on a DM basis) 500 g CW, 325 g maize grain, 55 g WB, 44 g soybean meal, 51 g cottonseed meal, 5.0 g Ca(HCO3 )2 , 5.0 g NaHCO3 , 5.0 g limestone, 5.0 g iodined salt and 5.0 g commercial vitamin and trace mineral premix. The roll-tube technique of Hungate (1969) was used to isolate the fungi. Salt solution I contained (per liter) NaCl, 6.0 g (NH4 )2 SO4 , 3.0 g; KH2 PO4 , 3.0 g; CaCl2 ·2H2 O, 0.4 g; MgSO4 ·2H2 O, 0.6 g. Salt solution II contained 4.0 g K2 HPO4 . The basal culture medium consisted of (per liter) yeast extract (Sinopharm Chemical Reagent Beijing Co., Ltd, Beijing), 1.0 g; tryptone (Beijing Abxing Biological Technology Co., Ltd, Beijing), 1.0 g; glucose, 1.0 g; NaHCO3 , 7.0 g; solution I, 165 mL; solution II, 165 mL; rumen fluid which was centrifuged at 8000 × g at 4 ◦ C for 10 min, 170 mL; resazurin (1.0 g/L), 1 mL; l-cysteine hydrochloride, 1.7 g; and distilled water to a volume of 1000 mL. In the liquid basal culture, air dry 2.0 mm chopped wheat straw was weighed and added to the basal culture in each tube with a final substrate concentration of 8.0 g/L; in the solid basal culture, agar was added to the basal culture to a concentration of 15 g/L. Inocula (0.1 mL) were added to the solid basal culture (9 mL) supplemented with 1600 IU/mL Penicillin and 2000 IU/mL Streptomycin, and then incubated anaerobically at 39 ◦ C until fungal colonies appeared. These colonies were subcultured in the liquid basal culture (9 mL) supplemented with 1600 IU/mL Penicillin and 2000 IU/mL Streptomycin. The fungi were identified according to morphological characteristics of fungal isolates as well as their partial 18S rDNA sequences. The sequences of the partial 18S rDNA were assigned the GenBank accession numbers FJ687479 for YQ1 and FJ687481 for YQ3. The fungi were incubated anaerobically at 39 ◦ C and transferred every 4 days. 2.2. Preparation of crude enzyme Based on the results of a preliminary unpublished study, the optimal combinations (see Table 1) of carbon and N sources to produce FAE were used as liquid medium components to incubate the fungi. FAE activity reached the highest level after 9 days of incubation for YQ1 and 4 days of incubation for YQ3, respectively. Cultures were then centrifuged for 10 min at 1000 × g at 4 ◦ C (Koleva et al., 2008), and the supernatants were used as the crude enzyme solutions (CES) of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 to analyze for activity of FAE, AE, xylanse, carboxymethyl cellulase (EC 3.2.1.4) and avicelase (EC 3.2.1.91), enzymological characteristics of FAE and AE and hydrolysis of different substrates, immediately. 2.3. Plant cell wall glycoside hydrolase and esterase assays According to Yue et al. (2009), crude enzyme solutions of both fungi were diluted to ensure absorption between 0.7 and 1.0 at the start of measurement in order to reduce the influence of pigments on FAE measurement. 100 ␮L suitably diluted enzyme solution was incubated with 200 ␮L of 100 ␮M methyl ferulate in 100 mM 3-(N-morpholino) propane sulfonic buffer (pH 6.0) at 39 ◦ C for 30 min, and the absorbance was estimated by microplate reader 680XR (Bio-rad, Hercules, CA, USA) at 340 nm (recommended by Biocatalysts Co. Ltd., Cardiff, Wales, UK). Enzyme activity was calculated using stan-

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dard curves of ferulic acid and methyl ferulate according to the equation provided by Biocatalysts Co as: activity (U) = [(OD0

min

− OD30 min ) − (ODblank 0 min − ODblank 30 min )]

× Vreaction × dilution/[(εMFA × l − εFA × l) × Vsample ]

(1)

where OD0 min and OD30 min are absorbance of reaction system at 0 min and 30 min respectively, ODblank 0 min and ODblank 30 min are absorbance of substrate-containing at 0 min and 30 min respectively, Vreaction is reaction system volume, Vsample is volume of sample, εMFA and εFA are extinction coefficient of methyl ferulate and ferulic acid respectively, and l is pathlength. The activity of xylanase, carboxymethyl cellulase, avicelase and AE were determined at 39 ◦ C and pH 7.0. xylan, carboxymethyl cellulose and avicael were used for measurement of xylanase, carboxymethyl cellulase and avicelase, respectively. Production of reducing sugars (RS) was quantified by the dinitrosalicylic acid method as described by Colombatto et al. (2003) and determined colorimetrically at 540 nm. p-Nitrophenyl acetate was the substrate of AE, and production of p-nitrophenol was measured at 415 nm. One unit of enzyme activity was defined as the amount of enzyme releasing 1.0 ␮mol of ferulic acid or reducing sugar or p-nitrophenol per minute per millilitre under the conditions described above. 2.4. pH and temperature optima, cation stability and kinetic constants of FAE and AE The optimal pH was determined by measuring the FAE and AE activity as described in Section 2.3 except that the buffer was replaced by the following buffers: 50 mM citrate buffer (pH 3.0–5.0), 50 mM phosphate buffer (pH 6.0–8.0), and 50 mM glycine–NaOH buffer (pH 9.0–10.0). The optimal temperature was studied by measuring the enzyme activity at various temperatures (20–60 ◦ C). Cation stability was studied by inclusion in the buffer of 0.667 mM cations such as Fe2+ , Co2+ , Mg2+ , Zn2+ , Mn2+ , K+ , Cu2+ and Ca2+ were used to study their effects on FAE and AE activity. The Michaelis constants (Km ) and maximum velocities (Vmax ) were estimated by using varying concentrations of the substrate (methyl ferulate 16.7–100 ␮M for FAE and p-nitrophenyl acetate 0.1–0.9 mM for AE). The experiment was in triplicate. 2.5. Hydrolysis of Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks The hydrolysis procedure was according to Xie (2007). About 10 mg of Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks were incubated in triplicate with 200 ␮L crude enzyme solution at 39 ◦ C for 24 h or 2 mL 1.0 M NaOH at 100 ◦ C for 2 h. The release of reducing sugar was measured with a microplate reader 680XR (Bio-rad, Hercules, CA, USA), and that of ferulic acid and p-coumaric acid with HPLC (515 HPLC; Waters, MA, USA). The experiment was in duplicate. 2.6. Chemical analyses According to AOAC (1999, ID 930.5), samples of the feedstuffs were dried at 105 ◦ C for 4 h and equilibrated in a desiccator to determine DM. Crude protein of air dried sample was determined using a Kjeldahl method (AOAC, 1999, ID 984.13) with a Kjelfoss apparatus (KjeltecTM, Hollgard, Denmark). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined as described by Van Soest et al. (1991). Alpha amylase was not used but sodium sulphite was added to each sample separately for NDF determination. Both NDF and ADF were expressed inclusive of residual ash. The concentration of reducing sugar was measured by the dinitrosalicylic acid method and determined colorimetrically at 540 nm. Release of ferulic acid and p-coumaric acid by crude enzyme solution or 1.0 mol/L NaOH were done by HPLC (515 HPLC, Waters) on a Symmetry RP C18 column (4.6 mm × 25 cm; Waters). Elution was conducted with a linear gradient of 0–0.5 acetonitrile in 50 mM of sodium acetate buffer (pH 4.0) for 20 min at a flow rate of 1.0 mL/min. Peak detection was at 320 nm. Pure standards of ferulic acid and p-coumaric acid were used for calibration.

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Table 2 Chemical analysis (g/kg DM) of Chinese wildrye grass hay (CW), wheat bran (WB), maize bran (MB), wheat straw (WS) and corn stalk (CS).

DM CP NDF ADF Ash Ferulic acid p-Coumaric acid

CW

WB

MB

WS

CS

SEM

896.0b 145.1b 683.9c 383.5b 65.3a 5.9ab 5.2bc

876.9d 214.7a 435.2d 185.5c 61.1c 2.5c 2.7bc

885.0c 119.0c 215.0e 62.0d 27.0d 6.9a 1.0c

910.0a 26.0d 815.0a 591.0a 64.0b 5.0b 8.7b

871.9e 69.4e 698.7b 402.1b 64.3b 4.7b 20.4a

0.16 0.62 3.25 10.45 0.17 0.48 1.82

Least square means (n = 3) in same rows without common superscripts differ (P<0.05).

2.7. Statistical analyses Chemical composition, release of reducing sugar, ferulic acid and p-coumaric acid for all feedstuffs were analysed to compare differences among Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks as well as differences between crude enzyme solutions of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 using the GLM procedure of SAS (1999) with a Tukey/Kramer multiple comparison testas: Y =  + Ri + Fj + Sk + (F × S)jk + εijk

(2)

where Y is the dependent variable under examination,  is the overall mean, Ri is the replicate effect (i = 3), Fj is the feed effect (j = 5, CW, WB, MB, WS and CS), Sk is the crude enzyme solution effect of fungi (k = 2), and εijk is the error term. Least square means, standard errors of least square means and significance level (P value) were calculated using the LSMEANS statement of SAS (1999).

3. Results 3.1. Nutrients, ferulic acid and p-coumaric acid composition of fibre-rich feedstuffs The contents of crude protein were low (Table 2), especially in WS and CS, and significantly differed (P<0.05) among feeds. The highest NDF and ADF contents were in WS whereas the lowest was in MB. The forages contained more than 680 g/kg DM of NDF and 380 g/kg DM of ADF which were much higher than in WB and MB. The content of ferulic acid ranged from 2.5 g/kg DM in WB to 6.9 g/kg DM in MB which also had the lowest (P<0.05) proportion of p-coumaric acid. The highest content of p-coumaric acid was found in CS, which was up to 20.4 g/kg DM.

3.2. The pH and temperature optima of FAE and AE for both fungal isolates FAE and AE activities were dramatically influenced by the pH values of reaction system (Fig. 1). pH optima of FAE activity was 7.0 for YQ1 and 4.0 for YQ3, while that of AE was 8.0 for both fungi. Temperature optima of FAE were 40 ◦ C for both fungi, and there was no FAE activity when the temperature of reaction system exceeded 60 ◦ C, while optimal temperatures for AE activity were 50 ◦ C for YQ1 and 60 ◦ C for YQ3 (Fig. 2). FAE activities of the two fungi, and AE activities of YQ3 were markedly inhibited by these cations, except Fe2+ which had a stimulatory effect on FAE activity of YQ3.In contrast, most of cations tested were stimulators for AE of YQ1 except Fe2+ and Cu2+ . Moreover, the Km and Vmax of FAE against methyl ferulate were 30 ␮M and 8.05 mU for YQ1 and 129 ␮M and 3.15 mU for YQ3, respectively. With substrate of p-nitrophenyl acetate, the Km and Vmax of AE were 0.21 mM and 336 mU for YQ1 and 5.26 mM and 854 mU for YQ3.

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Fig. 1. Effects of pH on ferulic acid esterase (FAE, rhombus) and acetyl esterase (AE, squares) activity of both Neocallimastix sp. YQ1 (closed) and Anaeromyces sp. YQ3 (open). To investigate the optimal pH and stability of the esterase activity, three buffers were used: 50 mM citrate buffer (pH 4.0–5.0), 50 mM phosphate buffer (pH 6.0–8.0), and 50 mM glycine–NaOH buffer (pH 9.0–10.0). Since relative activities of ferulic acid esterase or acetyl esterase were calculated in mU/mU according to one maximum mean activity under optimal temperature of incubation fluids, standard error bars are not added.

3.3. Effect of crude fugal enzyme solutions on the hydrolysis of fibre-rich feedstuffs FAE, AE, xylanase, carboxymethyl cellulase and avicelase activities of the crude enzyme solution of YQ1 were 5.71, 65, 136, 41.4 and 34.0 mU, while the values of YQ3 were 1.07, 14, 215, 13.9 and 10.0 mU, respectively. Releases of ferulic acid, p-coumaric acid and reducing sugars from CW, WB, MB, WS and CS are in Table 3, which illustrate the interactions between feed and CES for release of reducing sugars, ferulic acid and p-coumaric acid. In contrast to CES of YQ1, CES of YQ3 released more reducing sugars from WS, but less from the other plant materials, especially MB and CS. More than 100 g/kg DM of alkali-extractable ferulic acid was liberated from CW and WB by CES of YQ1 and from WB and CS by CES of YQ3. Meanwhile, a higher proportion of alkali-extractable p-coumaric acid was released from CW and MB by CES of both fungi, and CES of YQ3 exhibited higher potential to release p-coumaric acid from WS and CS than that of YQ1.

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Table 3 Release of ferulic acid, p-coumaric acid and reducing sugar from the Chinese wildrye grass hay (CW), wheat bran (WB), maize bran (MB), wheat straw (WS) and corn stalk (CS) after 24 h of enzymatic degradation by crude enzyme solutions of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3. CES

Feeds CW

SEM WB

MB

WS

CS

P Feed

CES

Feed × CES

Ferulic acid (g/kg DM)

YQ1 YQ3

0.72a 0.37b

0.90a 0.41b

0.52a 0.17b

0.36a 0.29b

0.03b 0.58a

0.046

<0.0001

0.0007

<0.0001

p-Coumaric acid (g/kg DM)

YQ1 YQ3

0.42 0.43

0.03 0.04

0.69 0.53

0.03b 0.35a

0.01b 1.57a

0.037

<0.0001

<0.0001

<0.0001

Reducing sugar (g/kg DM)

YQ1 YQ3

9.07 8.12

15.23 14.25

11.19a 2.77b

2.45b 5.36a

12.67a 2.42b

0.607

<0.0001

<0.0001

<0.0001

Least square means (n = 5) in the same column within crude enzyme solutions with different superscripts differ (P<0.05). CES = crude enzyme solutions.

Fig. 2. Effects of temperature on ferulic acid esterase (FAE, rhombus) and acetyl esterase (AE, squares) activity of both Neocallimastix sp. YQ1 (closed) and Anaeromyces sp. YQ3 (open). Since relative activities of ferulic acid esterase or acetyl esterase were calculated in mU/mU according to one maximum mean activity under the optimal temperature of incubation fluids, standard error bars are not added.

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4. Discussion The chemical compositions for most feedstuffs were close to tabulated values published by China Feedstuffs (Zhang, 2000), NRC (2001) and Feeding Standard of Beef Cattle (Feng et al., 2005). However, the alkali-extractable ferulic acid and p-coumaric acid tested were dramatically different from those in the literatures, such as higher values for wheat bran and lower values for maize bran (Bonnin et al., 2002; Benoit et al., 2006). These differences have been contributed by the different plant varieties, cultivation regions, climate, processing and chemical analytical methods. From the enzymological characteristics of FAE and AE, although the range of pH in the rumen is 6–7, not all esterases in our study produced by rumen fungi exhibited their highest activity in this pH range. This is in agreement with Blum et al. (1999) for the AE of Orpinomyces sp. Strain PC-2, but inconsistent with Borneman et al. (1992) for the FAE of Neocallimastix Strain Mc-2. Not all cations such as Fe2+ , Co2+ , Mg2+ , Zn2+ , Mn2+ , K+ , Cu2+ and Ca2+ were always found to inhibit FAE and AE activities of both fungi. Cu2+ in the literatures has had an inhibitory effect on FAE of the intestinal bacterium Lactobacillus acidophilus (Wang et al., 2004) and AE of Candida guilliermondii (Basaran and Hang, 2000) and Streptomyces sp. PC22 (Chungool et al., 2008). Chungool et al. (2008) found that Ca2+ , Co2+ , Mg2+ , and Mn2+ had no inhibitory, or an activation, effect at concentrations as high as 10 mM for Streptomyces sp. PC22, which contrasts to AE from Bacillus pumilus, in which Cu2+ , Zn2+ , Mn2+ , Co2+ , and Ca2+ had no effect at 2 mM, but markedly inhibited the enzyme activity at 10 mM (Degrassi et al., 1998). The presence of these cations should be paid more attention in process application because of their inhibitory influence on the activity. Some researchers have found that the crude enzyme solution, prepared from Aspergillus niger, Streptomyces avermitilis and Penicillium brasilianum, released hydroxycinnamic acids from wheat bran, brewer’s spent grain, eight complex or model substrates continually for up to 12 h and in some cases for up to 24 h (Faulds and Williamson, 1995; Bartolomé et al., 2003; Panagiotou et al., 2007). Therefore, we decided to determine the amount of ferulic acid and p-coumaric acid after 24 h incubation in vitro. Borneman et al. (1990) reported that monocentric isolates had higher esterase activities against methyl ferulate than polycentric ones, which was consistant with our results for YQ1 and YQ3. However, their capability of release ferulic acid from complex plant materials did not follow the same pattern. Although CES of YQ1 released more ferulic acid from most plant materials than CES of YQ3, the ferulic acid released by CES of YQ3 from corn stalks was almost 20 times that released by CES of YQ1. Furthermore, CES of YQ3 liberated much more hydroxycinnamic acids from corn stalks than P. brasilianum (Panagiotou et al., 2007) and CES of YQ1, which in turn resulted in higher liberation from maize bran. The liberation of ferulic acid and p-coumaric acid from wheat straw by the crude enzyme solutions of the two rumen fungi was higher than that of P. brasilianum (Panagiotou et al., 2007), and between the values for purified FAE of A. niger reported by Benoit et al. (2006). In our study, 138 and 71 g/kg of alkali-extractable ferulic acid were released from Chinese wildrye grass hay by CES of YQ1 and YQ3, respectively, while 92 and 94 g/kg of alkali-extractable p-coumaric acid were released by CES of YQ1 and YQ3, respectively. However, little information on Chinese wildrye grass hay could be found in the literature. These different hydrolysis results might be due to synergism occurred between ferulic acid esterase and other fibrolytic enzymes in the degradation of large feruloyl-polysaccharides (Borneman et al., 1992). Ferulic acid and p-coumaric acid are specific useful compounds which present potential commercial applications, such as precursors of natural vanillin (Bonnin et al., 2002), natural antioxidants, food preservative agents, anti-inflammatory agents and photoprotectants (Graf, 1992). Liberation of ferulic acid and p-coumaric acid from agricultural by-products through enzymatic methods has been increasingly researched, and most of these studies focused on the aerobic fungi (Faulds et al., 1995, 2004; Faulds and Williamson, 1995; Bartolomé et al., 1997, 2003; Kroon et al., 2000; Bonnin et al., 2002; Yu et al., 2002; Topakas et al., 2004; Ferreira et al., 2007). In our study, rumen fungi exhibited a strong capability to release ferulic acid and p-coumaric acid from fibre-rich feedstuffs. Therefore, anaerobic fungi, and especially their production of ferulic acid esterase, should not be neglected in studies on the biodegradation of plant cell walls in the rumen as well as in industrial processes developed to obtain ferulic acid and p-coumaric acid from agricultural by-products.

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5. Conclusions Enzymological characteristics of ferulic acid esterase in pH optima showed differences between Neocallimastix and Anaeromyces, and no such differences occurred in that of acetyl esterase. Both fungi had the highest ferulic acid esterase activity at 40 ◦ C, which might be a result of long term evolution of anaerobic fungi to the rumen environment. However, acetyl esterase activities of YQ1 and YQ3 at 40 ◦ C were only 54% and 34% of the activities at their optimal temperatures, respectively. Both fungi exhibited strong capability to release hydroxycinnamic acids from fibre-rich agricultural by-products (e.g., corn stalks and maize bran). 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