Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

Bioresource Technology 102 (2011) 8117–8122

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant Yu-Chun Huang a, Yu-Fen Chen a, Cheng-Yu Chen a, Wei-Lin Chen a, Yong-Ping Ciou a, Wen-Hsiung Liu b, Chao-Hsun Yang a,⇑ a b

Department of Cosmetic Science, Providence University, Taichung 43301, Taiwan Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan

a r t i c l e

i n f o

Article history: Received 19 April 2011 Received in revised form 20 May 2011 Accepted 22 May 2011 Available online 30 May 2011 Keywords: Yarrowia lipolytica Thermobifida fusca Ferulic acid Esterase Fed-batch fermentor

a b s t r a c t A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12 h and then with the AXE esterase for an additional 12 h. Ferulic acid accumulated to 396 lM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Plant cell walls are the major reservoir of fixed carbon in nature. They contain three major polymers: cellulose (30–45%), hemicellulose (25–45%) and lignin (15–30%) (Betts et al., 1992). Ferulic acid (FA, 4-hydroxy-3-methoxycinnamic acid) is present at relatively high concentrations in the cell walls of several plants, including monocots and dicots (Hartley and Ford, 1989; Hartley and Harris, 1981). FA is found covalently linked to polysaccharides by ester bonds (Mueller-Harvey et al., 1986) and is a component of lignin (Scalbert et al., 1985). FA is best known for its antioxidant and anti-inflammatory properties (Castelluccio et al., 1995; Chawla et al., 1987). FA can also be biotransformed into vanillin, one of the most widely used aromatic molecules in the food, pharmaceutical and cosmetic fields (Clark, 1990). FA also can be bioconverted to other valuable molecules such as polymers, epoxides, alkylbenzenes, protocatechuic acid-related catechols, guaiacol and catechol (Rosazza et al., 1995). Ferulic acid esterases (FAEs, also known as feruloyl esterases, cinnamic acid esterases, or cinnamoyl esterases; EC 3.1.1.73) represent a diverse group of esterases that can release FA from plant cell wall constituents to which it is bound. FAEs are key enzymes ⇑ Corresponding author. Tel./fax: +886 4 26311167. E-mail address: [email protected] (C.-H. Yang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.05.062

for cell wall hydrolysis and are increasingly used for the extraction of phenolic acids from agricultural crops (Benoit et al., 2006; Wong, 2006; Faulds et al., 2006; Koseki et al., 2009). To produce enzymes for enzymatic degradation of renewable lignocelluloses, the extracellular lignocellulolytic enzymeproducing thermophilic actinomycete, Thermobifida fusca NTU22, was isolated from compost soils collected in Taiwan (Liu and Yang, 2002). Interestingly, it has been reported that T. fusca can produce intracellular and extracellular esterases on oat-spelt xylan (Bachmann et al., 1991). One of the extracellular esterases, AXE, was purified and found to be a ferulic acid esterase. Some of the properties of the purified enzyme were investigated. The optimum pH and temperature for the purified enzyme were 8.0 and 80 °C, respectively (Yang and Liu, 2008). The axe gene encoding the acetylxylan esterase from T. fusca NTU22 was cloned, sequenced and expressed in Escherichia coli (Accession No. HM 193859) (Huang et al., 2010). Several thermostable enzyme genes from thermophilic microorganisms have been cloned and expressed in mesophilic microorganisms to improve economy by reducing the energy needed for cultivation (Zamost et al., 1991; Cheng et al., 2005). Recently, the axe gene was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZaA, allowing constitutive expression and secretion of the esterase. Recombinant expression resulted in extracellular esterase production as high as 0.526 U/ml in Hinton flask culture broth. The esterase activity from

8118

Y.-C. Huang et al. / Bioresource Technology 102 (2011) 8117–8122

P. pastoris was higher than that from an E. coli host (Yang et al., 2010c), but remained lower than that produced by the original strain, T. fusca NTU22. Yarrowia lipolytica is widely used in the industrial production of citric acid, peach flavor and single cell protein (Fickers et al., 2005). It is one of the most promising nonconventional and generally regarded as safe (GRAS) yeasts available as a host for heterologous protein production (Madzak et al., 2000). A large number of molecular tools are available for heterologous protein expression in this yeast (Madzak et al., 2004), and it has a high secreting capacity (Barth and Gaillardin, 1997). This study was conducted to constitutively express the esterase gene (axe) from the thermophilic actinomycete T. fusca NTU22 in Y. lipolytica P01g. Fermentative esterase production and applications of the enzyme for the bio-production of ferulic acid from lignocellulolytic agricultural biomass were also studied. The interaction between T. fusca esterase and xylanase for FA production by hydrolysis of agricultural biomass was investigated. 2. Methods 2.1. Microorganisms and vectors The thermophilic actinomycete T. fusca NTU22, which was isolated from compost soils collected in Taiwan, was used in this study (Liu and Yang, 2002). Y. lipolytica P01g (MatA, leu2-270, ura3-302::URA3, xpr2-332, axp-2) and pYLSC1 (Fig. 1) were purchased from Yeastern Biotech Co., Ltd (Taipei, Taiwan). Plasmid propagation for the expression work was performed in E. coli TOP10 F’ (Invitrogen, San Diego, CA, USA). The plasmid pER405-6 was constructed from pGEMÒ-T Easy Vector and an 800-bp insert fragment containing the axe gene encoding AXE (Huang et al., 2010). 2.2. Materials Yeast extract, yeast nitrogen base without amino acids, peptone, tryptone and agar were purchased from BD (Sparks, MD, USA). Restriction endonucleases and a T4 DNA ligation kit were purchased from Roche (Mannheim, Germany). For PCR, Vio Twin Pack Kits (comprising VioTag DNA polymerase, PCR buffer and deoxynucleotides) were obtained from Viogene (Sunnyvale, CA, USA). Ni-Sepharose™ High Performance was supplied by GE Healthcare (Little Chalfont, UK). Endo-b-N-Acetylglucosaminidase

H was purchased from New England Biolabs Inc. (Beverly, MA, USA). The protein assay kit and SDS–PAGE molecular weight standards were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Ferulic acid, inorganic salts and all other chemicals were purchased from Sigma (St. Louis, MO, USA). 2.3. Construction of the esterase expression plasmid The T. fusca NTU22 axe esterase gene was amplified with primers 50 -CACGGCCGTTCTGGCCGCGGCGAATCCGTATGAAC-30 (SfiI site is underlined) and 50 -GGAGGTACCTCAATGATGATGATGATGATGAA ATGGACAGGTGCTGCG-30 (KpnI site is underlined) using plasmid pER405-6 as the template. The amplification was performed using a DNA thermal cycler (ABI 2720 Thermal Cycler, MA, USA) with the following thermal profile: 95 °C for 2 min, followed by 30 cycles of 95 °C for 20 s, 67 °C for 20 s and 72 °C for 30 s, and a final extension at 72 °C for 2 min. An 800-bp PCR product was recovered from the agarose gel and cloned into SfiI- and KpnI-digested pYLSC1. After transformation into E. coli TOP100 , one recombinant plasmid designated as pYLSC1-axe was selected on LB agar plates (5 g/l yeast extract, 10 g/l tryptone, 10 g/l NaCl, 15 g/l. agar; pH adjusted to 7.5) containing 100 lg/ml ampicillin. The insert orientation was verified by restriction analysis and sequencing as described above (Yang et al., 2010a). 2.4. Transformation and screening of Y. lipolytica The recombinant plasmid pYLSC1-axe was linearized with NotI and transformed into Y. lipolytica P01g by the method of Xuan et al. (1988). The transformants were selected at 28 °C on YNB agar plates (20 g/l glucose, 6.7 g/l yeast nitrogen base without amino acid) for 2–4 days. The transformants were tested for esterase activity by replicating the colonies onto YNB agar plates. After incubation at 28 °C for 48 h, the plates were stained with p-nitrophenyl acetate solution. Colonies with a yellow zone resulting from hydrolysis of p-nitrophenyl acetate were selected for further analysis; integration of the construct into the Y. lipolytica P01g genome was verified by genomic PCR. 2.5. Biomass and esterase activity assay Biomass production by the Y. lipolytica transformant was evaluated by optical density at 600 nm (OD600 value). To determine esterase activity, the p-nitrophenyl acetate was used as the

Fig. 1. The genetic map of pYLSC1.

Y.-C. Huang et al. / Bioresource Technology 102 (2011) 8117–8122

substrate. Esterase activity was determined by measuring the pnitrophenol released (Kademi et al., 1999). One unit of enzymatic activity is defined as the amount of the enzyme releasing 1 mmol p-nitrophenol per min at 25 °C. 2.6. Expression of esterase in Hinton flask The high-esterase-activity transformant was incubated in 50 ml YPD medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose, pH 4.0) in 500 ml Hinton flasks and shaken (200 rpm) at 28 °C. After several days of culture, the culture broth was centrifuged at 10,000  g for 30 min at 4 °C and the supernatant was tested for esterase activity. 2.7. Cultivation in fermentor The Y. lipolytica transformant was cultivated in a 5 l fermentor (Biostat B, B. Braun, Melsungen, Germany). A 500 ml Hinton flask containing 100 ml YPD medium inoculated with a pellet of Y. lipolytica transformant scraped from a maintenance agar plate was cultured at 28 °C and shaken (200 rpm) for 24 h; this was used as the seed culture. The 5 l fermentor was loaded with 3 l of YPD medium (supplemented after sterilization). Cultivation was performed at 28 °C, 2.0 vvm aeration and 500 rpm agitation speed. The fermentation process was divided into two phases. In the first phase, glucose was the sole carbon source for biomass accumulation. When the initial glucose in the medium was exhausted, a glucose fed-batch phase was initiated by feeding glucose.

8119

column (Agilent Technologies Industries Co., Ltd., USA) and a UV detector at 322 nm. A gradient mobile phase with 40%/60% (v/v) acetic acid/methanol from 0 to 5.5 min, 60%/40% (v/v) acetic acid/methanol from 7.5 to 10 min and then 100% acetic acid after 10 min was used to elute at a flow rate of 1.0 ml/min. Ferulic acid was used as standard. 2.11. Statistical analysis All analytic measurements were performed at least in triplicate. Data are expressed as mean ± SD. 3. Results and discussion 3.1. Amplification and construction of the axe gene in Y. lipolytica The axe coding sequence was cloned into the pYLSC1 vector using SfiI/KpnI restriction sites as described in the methods, which theoretically placed the axe coding sequence in frame. Following sequence verification, the construct denoted as pYLSC1-axe was NotI-linearized and transformeds into Y. lipolytica P01g. Many transformants were selected on YNB plates. Genomic PCR revealed that most of the transformants contained a genomically integrated axe coding sequence. Among these transformants, the one (pYLSC1-axe) that displayed the highest esterase activity was selected for further experiments. 3.2. Expression of the axe gene in Y. lipolytica

2.8. Enzyme purification All purification procedures were performed at 4 °C in 20 mM Tris–HCl buffer (pH 8.0) unless otherwise stated. After 72 h cultivation of the Y. lipolytica transformant (pYLSC1-axe) in a 5 l fermentor, the fermentation broth was centrifuged at 10,000  g for 30 min to remove cells. The supernatant was concentrated by ultrafiltration (Pellicon XL, Biomax 10K, Millipore). The concentrated solution was then applied to a Ni-Sepharose™ High Performance column (1.13 cm  8 cm) pre-equilibrated with Tris–HCl buffer. After washing with wash buffer (20 mM Tris–HCl, 500 mM sodium chloride, 40 mM imidazole; pH 8.0) to remove unbound protein, the enzyme was eluted with elution buffer (20 mM Tris–HCl, 500 mM sodium chloride, 500 mM imidazole; pH 8.0). The enzymatically active elution fractions were pooled and used as purified enzyme. The purified enzyme was denatured for 10 min at 100 °C followed by deglycosylation for 1 h at 37 °C with endo-b-N-acetylglucosaminidase H. All manipulations followed the manufacturer’s instructions. 2.9. Hydrolysis of agricultural biomass For enzymatic hydrolysis of agricultural biomass for FA production, the reaction mixture contained 200 mg agricultural biomass and a suitable concentration of enzyme in Tris buffer (50 mM, pH 8.0) in a final volume of 10.0 ml. The mixture was incubated with gentle shaking for 24 h at 45 °C. The reaction was stopped by placing the mixture in boiling water for 3 min. After centrifugation (10,000  g for 15 min), the FA content of the supernatant was determined. 2.10. Detection of FA The hydrolytic products of esterase from agricultural biomass were analyzed by high-performance liquid chromatography (Agilent 1100 series, Agilent Technologies Industries Co. Ltd., USA) using a pre-packed 4.6 mm  150 mm (5 lm) Zorbax XDB-C18

The culture conditions for constitutive expression of the esterase were investigated in a 500 ml Hinton flask loaded with 50 ml YPD broth at 28 °C. Transformant (pYLSC1-axe) grew logarithmically from 12 to 36 h and then entered a stationary phase. The biomass reached 43.2 of the OD600 value after 48 h of incubation. The extracellular esterase rapidly accumulated in parallel with the increase in biomass. The maximum activity (70.94 U/ml) in the culture broth was observed after 48 h of incubation. Little esterase activity was detected in the culture broth of the control strain, Y. lipolytica (pYLSC1), under the same culture conditions (data not shown). Recently, the P. pastoris and Y. lipolytica was attempted to be used as hosts for heterologous expression of thermostable enzymes. However, P. pastoris displayed low transformation efficiency and a high frequency of negative transformants (Madzak et al., 2004). In this study, the esterase gene from T. fusca NTU22 was successfully expressed in Y. lipolytica. The maximum accumulation of esterase activity (70.94 U/ml) in the culture broth of Y. lipolytica transformant (pYLSC1-axe) was about 140 times higher than that accumulated in the culture broth of P. pastoris transformant (pGAPZ-axe) (0.526 U/ml). Yang et al. heterologously expressed the amylase gene from T. fusca in P. pastoris, Y. lipolytica and E. coli (Yang et al., 2010a,b; Yang and Liu, 2007). The higher expression in Y. lipolytica is in accord with our result in this study. 3.3. Purification and properties of esterase from the Y. lipolytica transformant The purification of AXE was performed as described in the methods section. The results of the total purification are summarized in Table 1. The purified enzyme exhibited 3.65% of the total initial activity and a 1.73-fold increase in specific activity compared with the crude esterase solution. The amount of extracellular protein in the culture broth of the Y. lipolytica transformant was less than that in the cell-free extract of an E. coli transformant. Extracellular accumulation of the enzyme will facilitate its

8120

Y.-C. Huang et al. / Bioresource Technology 102 (2011) 8117–8122

Table 1 Summary of the purification of esterase from Y. lipolytica transformant (pYLSC1-axe). Step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Purification (fold)

Yield (%)

Crude enzyme Ultrafiltration concentration His-tag affinity purification

56,752.6 11,270.9 2072.5

249.6 193.1 5.274

227.3 58.3 392.9

1 0.26 1.73

100 19.86 3.65

As shown in Fig. 2, the purified enzyme ran as an apparent single protein band by SDS–PAGE (10% gel). The subunit size of the single protein band was estimated from its mobility relative to standard proteins to be 28 kDa. The optimal pH and temperature of the esterase from Y. lipolytica transformant (pYLSC1-axe) were 8.0 and 60 °C, respectively. About 70% of the original esterase activity remained after heat treatment at 60 °C for 3 h. These parameters are similar to the enzyme from T. fusca NTU22. The enzyme purified from Y. lipolytica transformant was stable over a pH range from 6.0 to 10.0 at 4 °C for 24 h. 3.4. Production of transformant esterase in fed-batch fermentor The culture conditions for the production of esterase were investigated in a 5 l fermentor. A typical growth curve and enzyme production levels by the Y. lipolytica transformant in a 5 l fermentor are shown in Fig. 3. The cultivation was initiated in a 20 g/l glucose batch culture for about 36 h. A fed-batch phase was then initiated by feeding glucose (60 g) to accumulate biomass. As shown in Fig. 3, rapid consumption of glucose paralleled an increase in biomass (OD600) during the growth phase of the culture. The appearance of esterase activity in the culture broth became significant after 30 h of cultivation. The maximum esterase activity was about 41.11 U/ml in the culture broth after 72 h of cultivation. Fig. 2. SDS–PAGE of the purified esterase from Y. lipolytica transformant (pYLSC1axe). Lane M: Molecular weight marker; Lane 1: Purified esterase from Y. lipolytica transformant (pYLSC1-axe).

3.5. Bio-production of FA from agricultural biomass

application in an industrial process immediately, without complicated purification procedures. Some of the esterase activity was lost in the ultrafiltration process, resulting in a decrease in the apparent activity.

The enzymatic hydrolysis conditions for production of FA were investigated. When using corncob as an agricultural-biomass substrate, 2% was hydrolyzed by 1 U crude esterase solution/mg substrate after 24 h at 45 °C and pH 8.0. As shown in Fig. 4, FA accumulated rapidly during the initial 4 h. After 4 h of incubation,

Fig. 3. Time course for production of esterase in a 5 l fermentor by Y. lipolytica transformant (pYLSC1-axe). The operation conditions were as follows: working volume, 3 l; inoculum size, 5%; agitation speed, 500 rpm; aeration rate, 2 vvm; temperature, 28 °C. Symbols: (d) esterase, (h)OD 600, (4)dissolved oxygen, (–)pH value, (})glucose.

8121

Y.-C. Huang et al. / Bioresource Technology 102 (2011) 8117–8122

400

Ferulic acid ( µM)

300

200

100

0 0

4

8

12

16

20

24

Time (h) Fig. 4. Time course of ferulic acid accumulation from corncob. The reaction mixture contained 200 mg agricultural biomass and 1 U esterase/mg substrate or 0.1 U xylanase/ mg substrate in Tris buffer (50 mM, pH 8.0) in a final volume of 10 ml, which was gently shaken and incubated for 24 h. Symbols: (d) xylanase for first 12 h and then esterase for further 12 h, (h) esterase, (4) joint action of esterase and xylanase, (s)xylanase.

the FA continued accumulating slowly. After 12 h incubation, 328 lM FA had accumulated in the broth. The FA releasing yield was 39.7%. A similar FA accumulation pattern was observed for enzymatic hydrolysis of all other tested agricultural biomass substrates. The highest FA accumulation was found in medium containing corncob (Table 2). Rice bran and bagasse were poor sources, with FA production of only 9.5 lM and 5.7 lM, respectively. Similar results were obtained when monitoring the production of hemicellulose or xylan. 3.6. Co-operative enzymatic production of FA from agricultural biomass The AXE esterase proved to be an active biocatalyst for the production of FA from corncob. Xylanase may be used synergistically with esterase to enhance hydrolysis of lignocellulolytic substrates. As shown in Table 2 and Fig. 4, xylanase alone caused only a slight release of FA. The addition of T. fusca xylanase (Tfx) to AXE esterase did not improve the release of FA from corncob. However, when the corncob was incubated first with Tfx (0.1 U/mg corncob) at 45 °C and pH 8.0 with gentle shaking for 12 h, then with added esterase (1.0 U/mg corncob) for an additional 12 h at 50 °C, the

Table 2 Production of ferulic acid from various agricultural biomass sources using T. fusca enzymes. Substrate

Xylan Rice bran Wheat bran Bagasse Corncob

Ferulic acid (lM) Xylanase

Esterase

Xylanase + Esterase

7.5 9.0 9.7 9.5 10.2

9.2 9.5 88.8 5.7 328.0

7.0 8.5 55.2 3.7 265.0

The reaction mixture contained 200 mg agricultural biomass and 1 U esterase/mg substrate or 0.1 U xylanase/mg substrate in Tris buffer (50 mM, pH 8.0) in a final volume of 10 ml, which was gently shaken and incubated for 12 h.

accumulation of FA was higher than with esterase alone or with both esterase and Tfx for 24 h. Factors limiting efficient enzymatic extraction of FA from various biomass sources have been discussed (Faulds et al., 2006; Shin et al., 2006; Panagiotou et al., 2007; Barberousse et al., 2009; Xiros et al., 2009). It was reported that the source of xylanase is important, as is the type of FAE used in conjunction with the xylanase. For example, using arabinoxylan derived from brewers’ spent grain, family 10 xylanases in conjunction with Aspergillus niger FAE were more effective in releasing the 5, 50 form of diferulic acid, whereas family 11 xylanases preferentially released FA (Faulds et al., 2006). Tfx from T. fusca is a family 11 xylanase (Irwin et al., 1994). Xylanase treatment of lignocelluloses led to the release of ferulated sugar, which could be detected by measuring the absorbance of the solution at 375 nm. Treatment of this solution with esterase produced a shift in the absorbance from 375 to 345 nm, the latter peak characteristic for FA (Abokitse et al., 2010).

4. Conclusions In this process, we used lignocellulolytic agricultural waste as substrate to produce ferulic acid. The axe esterase gene from the thermophilic T. fusca NTU22 was successfully expressed in a Y. lipolytica expression system to improve economy by reducing the energy needed for cultivation. The esterase accumulated in the fermentation broth was about 140 times than that accumulated in the culture broth of P. pastoris. After concentration by ultrafiltration, the esterase solution can be used directly for industrial applications. As a result, the cost of the enzyme preparation may be reduced. The esterase, AXE, proved to be an active biocatalyst for the production of FA from corncob. Additional information about the interaction between T. fusca esterase and xylanase for FA production was obtained in the corncob-hydrolysis experiments. These findings may be helpful to understand the catalysis of lignocellulolytic substrates by complex hydrolytic enzyme systems.

8122

Y.-C. Huang et al. / Bioresource Technology 102 (2011) 8117–8122

Acknowledgements Financial support for this study from the National Science Council of the Republic of China (NSC98-2320-B-126-001-MY3) is gratefully acknowledged. References Abokitse, K., Wu, M., Bergeron, H., Grosse, S., Lau, P.C.K., 2010. Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran. Appl. Microbiol. Biotechnol. 87, 195–203. Bachmann, S.L., McCarthy, A.J., 1991. Purification and co-operative activity of enzymes constituting the xylan-degrading system of Thermomonospora fusca. Appl. Environ. Microbiol. 57, 2121–2130. Barberousse, H., Kamoun, A., Chaabouni, M., Giet, J.M., Roiseux, O., Paquot, M., Deroanne, C., Blecker, C., 2009. Optimization of enzymatic extraction of ferulic acid from wheat bran, using response surface methodology, and characterization of the resulting fractions. J. Sci. Food Agric. 89, 1634–1641. Barth, G., Gaillardin, C., 1997. Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol. Rev. 19, 219–237. Benoit, I., Navarro, D., Marnet, N., Rakotomanomana, N., Leasage-Messen, L., Sigoilot, J.C., Asther, M., Asther, M., 2006. Feruloyl esterases as a tool for the release of phenolic compounds. Carbohydr. Res. 341, 1820–1827. Betts, W.B., Dart, R.K., Ball, A.S., Pedlar, S.L., 1992. Biosynthesis and structure of lignocellulose. In: Betts, W.B. (Ed.), Biodegradation natural and synthetic materials. Springer-Verlag, London, pp. 139–156. Castelluccio, C., Paganga, G., Melikian, N., Bolwell, G.P., Pridham, J., Sampson, J., RiceEvans, C., 1995. Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Lett. 368, 188–192. Chawla, A.S., Singh, M., Murthy, M.S., Gupta, M.P., Singh, H., 1987. Antiinflammatory action of ferulic acid and its esters in carrageen induced rat paw edema model. Indian J. Exper. Biol. 25, 187–189. Cheng, Y.F., Yang, C.H., Liu, W.H., 2005. Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme Microb. Technol. 37, 541–546. Clark, G.S., 1990. Vanillin. Perfum. Flavo. 15, 45–54. Faulds, C.B., Mandalari, G., Lo-Curto, R.B., Bisignano, G., Christakopoulos, P., Waldron, K.W., 2006. Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acid from cereal arabinoxylan. Appl. Microbiol. Biotechnol. 71, 622–629. Fickers, P., Benetti, P.H., Wache, Y., Marty, A., 2005. Hydrophobic substrate utilization by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res. 5, 527–543. Hartley, R.D., Ford, C.W., 1989. Phenolic constituents of plant cell walls and wall biodegradability. In: Lewis, N.G., Paice, M.G. (Eds.), Plant cell wall polymers: Biogenesis and Biodegradation. ACS Series 399. ACS, Washington, pp. 137–145. Hartley, R.D., Harris, P.J., 1981. Phenolic constituents of the cell walls of dicotyledons. Biochem. Syst. Ecol. 9, 189–203. Huang, Y.C., Chen, G.H., Chen, Y.F., Chen, W.L., Yang, C.H., 2010. Heterologous expression of thermostable acetylxylan esterase gene from Thermobifida fusca and its synergistic action with xylanase for the production of xylooligosaccharides. Biochem. Biophys. Res. Commun. 400, 718–723. Irwin, D., Jung, E.D., Wilson, D.B., 1994. Characterization and sequence of a Thermomonospora fusca xylanase. Appl. Environ. Microbiol. 60, 763–770.

Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., Baratti, J.C., 1999. A thermostable esterase activity from newly isolated moderate thermophilic bacterial strains. Enzyme Microb. Technol. 24, 332–338. Koseki, T., Fushinobu, S., Ardiansyah, S.H., Komai, M., 2009. Occurrences, properties, and applications of feruloyl esterases. Appl. Microbiol. Biotechnol. 84, 803–810. Liu, W.H., Yang, C.H., 2002. The isolation and identification of a lignocellulolytic and thermophilic actinomycete. Food Sci. Agric. Chem. 4, 89–94. Madzak, C., Gaillardin, C., Beckerich, J.M., 2004. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica. J. Biotechnol. 109, 63–81. Madzak, C., Treton, B., Blanchin-Roland, S., 2000. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J. Mol. Microbiol. Biotechnol. 2, 207–216. Mueller-Harvey, I., Hartley, R.D., Harris, P.J., Curzon, E.H., 1986. Linkage of pcoumaroyl and feruloyl groups to cell wall polysaccharides of barley straw. Carbohydr. Res. 148, 71–85. Panagiotou, G., Olavarria, R., Olsson, L., 2007. Penicillium brasilianum as an enzyme factory: the essential role of feruloyl esterases for the hydrolysis of the plant cell wall. J. Biotechnol. 130, 219–228. Rosazza, J., Huang, Z., Dostal, L., Volm, T., Rosseau, B., 1995. Biocatalytic transformation of ferulic acid, an abundant aromatic natural product. J. Ind. Microbiol. 15, 457–471. Scalbert, A., Monties, B., Lallemand, J.Y., Guittet, E.R., Rolando, C., 1985. Ether linkage between phenolic acids and lignin fractions of wheat straw. Phytochemistry 24, 1359–1362. Shin, H.D., McClendon, S., Le, T., Taylor, F., Chen, R.R., 2006. A complete enzymatic recovery of ferulic acid from corn residues with extracellular enzymes from Neosartorya spinosa NRRL 185. Biotechnol. Bioeng. 95, 1108–1115. Wong, D.W.S., 2006. Feruloyl esterase: a key enzyme in biomass degradation. Appl. Biochem. Biotechnol. 133, 87–112. Xiros, C., Moukouli, M., Topakas, E., Christakopoulous, P., 2009. Factors affecting ferulic acid release from Brewer’s spent grain by Fusarium oxysporum enzymatic system. Bioresour. Technol. 100, 5917–5921. Xuan, J.W., Fournier, P., Gaillardin, C., 1988. Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration. Curr. Genet. 14, 15–21. Yang, C.H., Huang, Y.C., Chen, C.Y., Wen, C.Y., 2010a. Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation. J. Ind. Microbiol. Biotechnol. 37, 953–960. Yang, C.H., Huang, Y.C., Chen, C.Y., Wen, C.Y., 2010b. Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis. J. Ind. Microbiol. Biotechnol. 37, 401– 406. Yang, C.H., Lin, K.I., Chen, G.H., Chen, Y.F., Chen, C.Y., Chen, W.L., Huang, Y.C., 2010c. Constitutive expression of Thermobifida fusca thermostable acetylxylan esterase gene in Pichia pastoris. Int. J. Mol. Sci. 11, 5143–5151. Yang, C.H., Liu, W.H., 2007. Cloning and characterization of a maltotriose-producing a-amylase gene from Thermobifida fusca. J. Ind. Microbiol. Biotechnol. 34, 325– 330. Yang, C.H., Liu, W.H., 2008. Purification and properties of an acetylxylan esterase from Thermobifida fusca. Enzyme Microb. Technol. 42, 181–186. Zamost, B.L., Nielsen, H.K., Starnes, R.L., 1991. Thermostable enzymes for industrial application. J. Ind. Microbiol Biotechnol. 8, 71–82.