J Korean Soc Appl Biol Chem (2015) 58(3):415–421 Online ISSN 2234-344X DOI 10.1007/s13765-015-0061-7 Print ISSN 1738-2203

ARTICLE

The ascomycete polymorpha produces an acetyl esterase that solubilises beech wood material to release water-soluble lignin fragments

Do Huu Nghi • Rene´ Ullrich • Franco Moritz • Le Mai Huong • Vu Dinh Giap • Do Huu Chi • Martin Hofrichter • Christiane Liers

Received: 13 August 2014 / Accepted: 11 March 2015 / Published online: 18 March 2015 Ó The Korean Society for Applied Biological Chemistry 2015

Abstract Three ascomycetous soft-rot fungi belonging to Keywords Esterase Á Ascomycetous Á Xylaria the family were found to produce high levels polymorpha Á Lignocelluloses Á Water-soluble lignin of a p-nitrophenyl acetate-hydrolyzing activity during fragments growth on lignocellulosic materials, i.e., wheat straw and beech wood chips. Accordingly, Kretzschmaria deusta, elata, Xylaria polymorpha were seemingly most Introduction noteworthy acetyl esterase producers and, of which, X. polymorpha (strain A35) was chosen for further studies. Lignocellulose degrading-microorganisms play a pivotal Induction study indicated that raw carbohydrate sources, role in maintaining the global carbon cycle owing to effi- such as beech wood, rape straw, birch wood, and wheat cient solubilization plant material by various hydrolases. straw, were extremely important for acetyl esterase pro- One of the major reasons that lignocellulosic materials are duction. Acetyl esterase of X. polymorpha was produced in resistant to hydrolysis is the great amount of acetylation solid-state culture on wheat straw and purified by different present in the hemicellulose fraction (Chungool et al. steps of anion-exchange and size-exclusion chromatogra- 2008). Acetyl groups are present in wood hemicellulose as phy. This purified enzyme (MW = 44 kDa and pI values of side chain moieties, such as xylopyranosyl residues of 3.5–3.6) exhibited the capability to solubilise in vitro beech angiosperms (hardwood) and glucuronoxylan, mannopy- wood to release water-soluble lignin fragments with ranosyl residues of gymnosperms (softwood) (Timell 1967; molecular masses of 1–3 kDa as analyzed by high perfor- Joseleau et al. 1993). Similarly acetylation occurs at annual mance size exclusion chromatography. plant xylans; the main chains of which are substituted mainly with L-arabinofuranosyl residues. In hardwood xy- lan, some xylosyl residues are 2,3-di-O-acetylated and 3-O- acetylation is to a high degree observed on xylopyranosyl Electronic supplementary material The online version of this residues carrying a-1,2-linked 4-O-methyl-D-glucuronic article (doi:10.1007/s13765-015-0061-7) contains supplementary acid (MeGlcA). In addition, the 3-O-acetylation of material, which is available to authorized users. MeGlcA-substituted xylosyl residues has also been re- D. H. Nghi (&) Á L. M. Huong Á V. D. Giap Á D. H. Chi ported in corn stover xylan (reviewed by Biely 2012). Institute of Natural Products Chemistry, Vietnam Academy Therefore, enzymes that are capable of deacetylation may of Science and Technology, Hanoi, Vietnam have a promising potential for industrial applications, e.g., e-mail: [email protected] in the pulp and paper industry and fuel ethanol production, R. Ullrich Á M. Hofrichter Á C. Liers as environment-friendly catalysts. Unit of Environmental Biotechnology, International Graduate Acetyl esterases (EC 3.1.1.6) are hydrolases that catalyze School of Zittau (IHI Zittau), 03583 Zittau, Germany liberation of acetyl groups from acetylated polysaccharides, such as pectins and xylan. This enzyme from several as- F. Moritz Institut fu¨rO¨ kologische Chemie, Helmholtz Zentrum Mu¨nchen comycetes has been described, such as Trichoderma reesei GmbH, 85764 Neuherberg, Germany (Poutanen and Sundberg 1988), Aspergillus awamori 123 416 J Korean Soc Appl Biol Chem (2015) 58(3):415–421

-1 -1 (Koseki et al. 1997), A. niger (Kormelink et al. 1993), p-nitrophenyl acetate at 405 nm (e405 = 2.5 mM cm ) Penicillium purpurogenum (Egana et al. 1996), Fusarium with a SpectraMax Plate Reader (NanoQuant, Tecan, oxysporum (Christakopoulos et al. 1999). However, inves- Austria). Final substrate concentration was 1 mM in pH 6.8 tigation on biochemical properties of Xylaria acetyl esterase phosphate buffer and the reaction was occurred for 10 min as well as contribution of this enzyme to solubilisation of at 37 °C. One unit of acetyl esterase activity was defined as wood material is still not published so far. We describe the amount of enzyme releasing 1 lmol of p-nitrophenol herein the production and characterization of acetyl esterase min-1 at 37 °C and pH 6.8. from Xylaria polymorpha, a wood-rotting ascomycete that demonstrated secreting considerable amounts of feruloyl Induction study improved esterase production esterase, laccase, and mineralizing synthetic 14C-labeled lignin (Liers et al. 2006, 2007; Nghi et al. 2012). 100-ml Erlenmeyer flasks each containing 50 ml of mini- mal medium (pH 6.0) supplemented with 2 % (w/v) of different carbohydrate sources: birch wood xylan, birch Materials and methods wood, beech wood xylan, beech wood, wheat arabinoxylan, wheat bran, wheat straw, rape straw, oat spelt, olive oil, Microorganism and growth conditions and triacetin. The samples were taken following every 3 days for 21 days from fungal cultures and retained for Thirteen laboratory strains of basidiomycetes and as- acetyl esterase activity measurement. comycetes belonging to seven families were obtained from the culture collection of the American Type Culture Col- Production and purification of acetyl esterase from X. lection (Manassas, USA), the German Collection of Mi- polymorpha croorganisms and Cell Cultures (Braunschweig, Germany), the International Graduate School of Zittau (Zittau, Ger- Approximately 2.0-kg wheat straw was pre-soaked with many), and the University of Helsinki (Finland). The fun- distilled water over night, then filled in large autoclavable gal pre-culture was incubated at 23 °C for 3 weeks on malt plastic bags and sterilized at 121 °C for 30 min. For extract agar plates (MA) containing 20-g malt extract per inoculation, the content of two overgrown agar plates of X. litter to serve the subsequent experiments. polymorpha (strain A35) was homogenized in a sterile NaCl solution (0.9 %) and then added to each straw bag. Screening on agar plates After 4 weeks of incubation at 23 °C, the colonized wheat- straw bags were harvested and extracted with distilled An agar plug ([ 1 cm) from fungal pre-culture was chopped water. The enzyme-containing straw extract was concen- and transferred on double-layer agar plates with modified Kirk trated and dialyzed in a ultrafiltration system (10 kDa cut- medium (Kirk et al. 1978). The upper layer contained glucose off; Pall-Filtron, Dreieich, Germany) at 11 °C. b-D-pentaacetate (2 %, w/v) as indicator substrates for acetyl Crude enzyme preparation that exhibited acetyl esterase esterase. A halo zone formed around the fungal colony by activity was purified by two steps of fast protein liquid deacetylation indicates the action of acetyl esterase. chromatography (FPLC) using an A¨ KTATM system (GE Healthcare, Freiburg, Germany). The first separation step Screening on solid-state cultures on DEAE Sepharose was carried out using sodium acetate buffer (10 mM, pH 4.5) as mobile phase and an increasing To estimate the acetyl esterase activity in lignocellulosic sodium chloride gradient from 0 to 2.5 M. The second substrates, the cultivation of fungi proceeded in 100-ml Er- purification step was performed by size exclusion chro- lenmeyer flasks containing 3 g chopped wheat straw or milled matography (SuperdexTM 75) using sodium acetate buffer beech wood that was moistened with 10-ml distilled water. (50 mM, pH 6.5) containing sodium chloride (100 mM) as The cultures were incubated at 23 °C for 4 weeks. Following eluent. Acetyl esterase containing fractions were collected, every 5 days, enzyme activity of crude aqueous extracts from pooled, concentrated, and dialyzed against 10 mM sodium fungal cultures was determined in triplicate. The maximal acetate buffer (pH 6.0) and stored at -80 °C. activity of each fungal strain during the growth on solid media compared to those of other strains for fungal selection. Effects of temperature and pH on enzyme stability

Enzyme assay The pH stability of purified Xylaria acetyl esterase was tested in citrate/phosphate buffer (50 mM) at pH 3.0; 7.0; Acetyl esterase activity was assayed spectrophotometri- 9.0 and at 37 °C. The effect of temperature on the enzyme cally by measuring the release of p-nitrophenol from stability was studied at 45 °C and 60 °C in MOPS buffer 123 J Korean Soc Appl Biol Chem (2015) 58(3):415–421 417

(100 mM, pH 6.0). After incubation intervals of 1, 2, 3, 4, Accordingly, all ascomycetes belonging to the family 6, and 8 h, aliquots of the sample were taken and the Xylariaceae such as Kretzschmaria deusta, Morchella residual enzyme activity was measured as described above. elata, X. polymorpha (strain A35) were seemingly most All experiments were performed in triplicate. noteworthy acetyl esterase producers. Nevertheless, the focus of the present study has been on the X. polymorpha HPSEC analysis of lignin fragments (strain A35) that showed the highest levels of another esterase (feruloyl esterase) activity in the previous ex- High performance size exclusion chromatography periment (Nghi et al. 2012). (HPSEC) was used to determine the molecular mass dis- As shown in Fig. 1, acetyl esterase production by X. tribution of lignin fragments formed after enzymatic action polymorpha (strain A35) was detectable after 5 days of of X. polymorpha acetyl esterase on milled beech wood. cultivation. During the solid-state cultivation with wheat Beech-wood meal (0.5 %; w/v) was incubated with en- straw as growth substrate, enzyme activity increased just till zyme acetyl esterase from X. polymorpha at 37 °C and the 10th culture day (from 88 to 1039 mU g-1) and then continuously shaken at 200 rpm for 48 h. The reaction was decreased but remained relatively high ([670 mU g-1) till carried out in 100 mM MOPS buffer (pH 6.0), and for the end of cultivation. The pH of the culture medium comparison purposes, a control with heat-inactivated en- slightly decreased from the initial pH 7.0 to a weak acidic zyme (95 °C for 30 min) was used. The analysis was pH of 6.2. performed on a HPLC system (HP 1200 liquid chro- matography, Agilent) fitted with a HEMA-Bio linear col- Induction of carbohydrate sources on the acetyl esterase umn (8 mm 9 300 mm, Polymer Standard Service, Mainz, production Germany). The elution at pH 10.0 by the mobile phase consisting of 20 % acetonitrile and 80 % of an aqueous Carbohydrate sources had very different effects on the solution (0.34 % NaCl and 0.2 % K2HPO4) was monitored acetyl esterase production by X. polymorpha in the agitated at 280 nm by a diode array detector (DAD). Sodium liquid culture. Thus, acetyl esterase activity was hardly polystyrene sulfonates (1.3–168 kDa, Polymer Standard detected in the minimal medium supplemented with beech Service) and biphenyl dicarboxylic acid (0.246 kDa) wood xylan, triacetin, wheat arabinoxylan, and wheat bran served as molecular mass standards (Hofrichter et al. 2001; as growth substrates. Specially, X. polymorpha was abso- Liers et al. 2006). lutely unable to grow in the 2 % triacetin medium and therefore acetyl esterase was not released in this culture. On the contrary, raw carbohydrate sources such as beech Results wood, rape straw, birch wood, and wheat straw strongly stimulated the production of enzyme acetyl esterase. Fungal screening Whereby, the max acetyl esterase activity reached 93.5 U l-1 in the medium containing 2 % chopped rape A representative selection of 13 asco- and basidiomycetous straw on day fifteenth of incubation. Additionally, high fungi was screened for the production of extracellular acetyl esterase activities were also obtained during growth acetyl esterase. A main finding has been that 10 out of 13 on beech wood, birch wood, and wheat straw at nearly fungi formed halo zones around their colonies on agar equal of 80.0 U l-1 (Fig. 2). This result suggested that the plates containing insoluble glucose b-D-pentaacetate (2 %, raw carbohydrate sources were extremely important to in- w/v) as indicator substrate. This is an indication for the duce acetyl esterase production by X. polymorpha. secretion of acetyl esterase by the respective fungi causing the deacetylation of glucose acetate (Table 1). Purification and characterization of X. polymorpha During solid-state cultivation of natural lignocelluloses acetyl esterase (beech wood and wheat straw), all test fungi produced acetyl esterase to a certain extent (40–1568 mU g-1). Aqueous extract of 4-week fungal culture with wheat straw However, wheat straw seems to be more appropriate for as growth substrate was concentrated by ultrafiltration with acetyl esterase production in comparison to enzyme ac- 10 kDa cut-off filter. Thereafter, it was purified for acetyl tivities detected in beech-wood culture, at least in these esterase by fast protein liquid chromatography (FPLC) experimental conditions. Overall, the ascomycetes secreted using an A¨ KTA system (GE HealthCare, Freiburg, Ger- markedly higher amounts of acetyl esterase than white-rot many). First, the elution occurred on weak anion exchange fungi did. Interestingly, the results obtained in agar-plate DEAE Sepharose column and three fractions of enzyme screenings correlated well with the enzymatic activities AE were detected that numbered I–III. Total elution vol- measured in aqueous extracts of wheat-straw cultures. ume of highest activity fraction (III) was pooled and 123 418 J Korean Soc Appl Biol Chem (2015) 58(3):415–421

Table 1 Screening of acetyl esterase activity using agar plate assays and solid state cultures (SSCs) with wheat straw and beech wood as growth substrates Fungal strains Agar-plate test Acetyl estrase activities on SSCs (mU g-1) Wheat straw Beech wood

Basidiomycota Agrocybe aegerita DSMZ22459 ? 408 47 Coprinellus radians DSMZ888 - 288 43 Mycetinis alliacens Zi06 ? 449 87 Pleurotus ostreatus K5 - 288 47 Pycnoporus cinnabarinus ATCC200478 ? 690 87 Daldinia concentrica A20 - 616 53 Daldinia vernicosa A31 ? 80 53 Kretzschmaria deusta A29 ? 1568 63 Morchella elata A30 ? 1561 50 A38 ? 1394 67 Xylaria polymorpha A34 ? 998 57 Xylaria polymorpha A35 ? 1039 40 Xylaria polymorpha A32 ? 858 47

(?) means a halo zone was formed around the colony by deacetylation of glucose b-D-pentaacetate, (-) no halo zone was found (growth period: 3–7 days) The solid-state cultures (SSCs) contained 3 g of chopped wheat straw or beech-wood meals. Every fifth day, three flasks of each series were harvested and used for measurement of acetyl esterase activity

Fig. 2 Time-course of acetyl esterase production by X. polymorpha during 21-day liquid cultivation. Medium contained of different carbohydrates: wheat arabinoxylan (WAXyl), wheat straw (WS), oat spelt xylan (OSXyl), wheat bran (WBr), beech wood xylan (BeeXyl), birch wood xylan (BirXyl), beech wood (BeeW), rape straw (RS), Fig. 1 Time courses of acetyl esterase production by X. polymorpha birch wood (BirW), triacetin (Tria), and olive oil (Oliv). Fermentation (strain A35) in solid-state cultures with wheat straw (solid line)as broths were harvested every 3 days for enzyme measurement and the growth substrate. All experiments were performed in triplicate and maximal esterase activities of each fungal strain during the growth for standard deviation is displayed by error bars; medium pH (dashed 21 days were compared to those of other strains. All experiments line) were performed in triplicate and standard deviation is displayed by error bars reloaded on the SuperdexTM 75 column. After this gel fil- tration chromatographic step, one enzyme activity fraction distinct band of purified acetyl esterase with apparent was purified 18-fold and represented a 26.8 % yield (Fig. 3 molecular mass was 44 kDa as visualized by SDS poly- and the information in the supplemental material). This acrylamide gel electrophoresis. Isoelectric focusing re- purified acetyl esterase would be used for subsequent vealed two contiguous bands of acetyl esterase at pI values characterization and in vitro conversion studies. One of 3.5 and 3.6 (Fig. 4).

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Fig. 4 Purified protein (1, 3) was visualized by SDS-PAGE (a) and IEF gel (b); Protein marker (2, 4)

1 A 30 2 24 18 12 Fig. 3 Purification of an acetyl esterase of X. polymorpha by FPLC. 6 Ion exchange chromatography on DEAE Sepharose (a) and size 0 exclusion chromatography. (b). Solid lines absorbance at 280 nm, 4 5 6 7 8 9 10 11 12 black circles esterase activity measured with p-nitrophenyl acetate, dashed line NaCl Absorbance at 280 nm (mAU) Retention time (min) B 2 Xylaria acetyl esterase was relatively stable at 45 °C 1 after 3 h of incubation and thereafter lost more than 50 % of the initial activity on prolonged exposure to this tem- perature. A rapid decrease in activity was observed at 60 °C where the enzyme lost almost all its activity within 1 h of incubation (see Fig. S1A in the supplemental ma- terial). The purified enzyme was found to be stable at neutral and alkaline pH but lost more than 90 % of its activity within 1 h at pH 3 (see Fig. S1B in the supple- Fig. 5 a HPSEC-elution profile of water-soluble lignin fragments mental material). released from milled beech wood material after in vivo conversion with X. polymorpha (after 7 culture days, thick black line) and in vitro conversion with Xylaria acetyl-esterase (after 48 h incubation time, Conversion of milled beech wood by acetyl esterase thin black line). Control: heat-denaturated Xylaria acetyl esterase of X. polymorpha with milled beech wood (after 48 h incubation time, dashed line). b UV–Vis spectra of water-soluble lignin fragments (black solid line) in comparison to reference spectra (black dashed line) of alkaline HPSEC-elution profile (Fig. 5) indicates the liberation of lignin (1) and lignin sulfonic acid (2) water-soluble aromatic fragments from beech wood after the fungal- and enzymatic incubation. The major changes were observed for the small-sized fractions in comparison fragments were detected, whereas amount of smaller-sized with control that consisting of denatured enzyme and ones (*1 kDa) was relatively low. milled beech wood. After 48 h of incubation with Xylaria The UV–Vis spectra of the released aromatic fragments acetyl esterase, high amount of 3-kDa molecular-mass indicated a lignin-like character with typical absorption maxima 123 420 J Korean Soc Appl Biol Chem (2015) 58(3):415–421 between 250 and 280 nm in comparison to water soluble model et al. 1996) and Absidia corymbifera (Zhoua et al. 2014). lignins, i.e., alkaline lignin (peak 1) and a lignin sulfonic acid Acetyl esterase purified from solid-state culture of X. poly- (peak 2). Interestingly, the elution profile of the in vitro con- morpha showed a discrete protein band on SDS-PAGE gel version with the purified enzyme of X. polymorpha was similar with the MW of 44 kDa, and it was in the range of that of other to that of the 7-day aged in vivo culture of the whole fungus. acetyl esterases (34–67 kDa; Poutanen and Sundberg 1988; Especially, the main fragments with a molecular size of *1and Kormelink et al. 1993; Basaran and Hang 2000; Neumu¨ller 3 kDa appeared in both chromatograms, probably indicating the et al. 2014; Zhoua et al. 2014). Its isoelectric point (pI of enzyme influence during fungal attack of the woody material. 3.5–3.6) was nearly identical to that of acetyl(xylan) esterase purified from A. niger (Kormelink et al. 1993) and lower than that reported for T. Reesei, Candida guilliermondii (pI of Discussion 6.8–7.6; Margolles-Clarke et al. 1996; Basaran and Hang 2000) and thermophilic soil bacterium Thermobifida fusca Soft rot (type II) fungi belonging to the family of Xy- (pI of 6.6; Yang and Liu 2008). The Xylaria acetyl esterase lariaceae are known to substantially degrade hardwood by was stable at temperatures lower than 45 °C and pH 7.0–9.0. means of their poorly understood lignocellulolytic enzyme Its thermal and pH stabilities were similar to those of acetyl system comprising different hydrolases, e.g., esterases, esterase purified from A. corymbifera (Zhoua et al. 2014), xylanases or cellulases, and laccases. In the present study, while those from C. guilliermondii exhibited thermal sta- the wood-colonizing ascomycete X. polymorpha was found bility up to 60 °C (Basaran and Hang 2000). Since their to produce an extracellular ester-cleaving hydrolase during cosmopolitan distribution, particularly in the tropics and its growth on agar plates contained glucose b-D-pentaac- sub-tropics, the question of whether xylariaceaous fungi are etate as well as on solid-state cultures with wheat-straw and capable of extensive lignin degradation is important in an beech-wood substrates. The working group of Tsujiyama ecological context. The Xylaria species are generally known and Nakano (1996) reported a selection of 30 test strains of to have a strong capacity to solubilize lignin of beech wood basidiomycetes (e.g., Pleurotus ostreatus, Pycnoporus causing similar wood decay like white-rot basidiomycetes coccineus) showed positive results in a similar screening, (Pointing et al. 2003). In this study, Xylaria acetyl esterase suggesting the ubiquitous occurrence of acetyl esterase can act on beech-wood meal to release water-soluble lignin among wood-rotting fungi. Enzyme assay showed that fragments. The elution profiles of the released aromatic extracellular acetyl(xylan) esterase activity was present in fragments on one hand caused by the enzymatic attack the filtrates of wood-meal culture of all these fungi. in vitro and of those on the other hand extracted from a 7-day Regarding the induction study, the raw lignocellulosic aged in vivo culture of X. polymorpha were found to be quite materials (e.g., wheat straw, beech wood, rape straw, and similar and the development of characteristic, distinct low birch wood) seem to be efficient inducers for acetyl esterase molecular mass fragments (*1–3 kDa; Hofrichter et al. secretion by X. polymorpha. The complex-material wheat 2001) indicated for the enzymatic influence. This result is straw was chosen for large-scale production of acetyl ester- good agree with the previous studies that reported the wood- ase by X. polymorpha, which was also used as growth sub- colonizing ascomycete X. polymorpha seemingly lacked strate for feruloyl esterase production by the same fungus peroxidases but produced laccase as sole ligninolytic (Nghi et al. 2012). The inducing effect of xylans (e.g., wheat oxidoreductase and caused the formation of large lignocel- arabinoxylan, oat spelt xylan, beech wood xylan, birch wood lulose fragments along with the aggressive production of xylan) was relatively low. In contrast, xylans were assumed esterases (Liers et al. 2007; Nghi et al. 2012). being the best carbon sources for fungal growth and acetyl esterase production by T. reesei, Schizophyllum commune Acknowledgments This study was partly supported by Vietnam (Biely et al. 1988), Termitomyces clypeatus (Mukhopadhyay Academy of Science and Technology (under Grant No. VAST.ÐTL.01/14-15) and National Foundation for Science and et al. 1997), and A. awamori (Koseki et al. 1997). In general, Technology Development (NAFOSTED; No. 106-NN.02-2013.44). acetyl esterase activity of X. polymorpha culture filtrate is We thank the colleagues at the INPC (VAST) and IHI Zittau for equal to or lower than that reported for these fungi, but en- useful comments and technical assistance. zyme production in the present study could be significantly stimulated by using inexpensive and ubiquitous raw carbon sources like rape- and wheat straw. Nevertheless, Xylaria acetyl esterase exhibited specific activity on p-nitrophenyl References acetate considerably higher than various enzyme prepara- tions of T. reesei, T. viride, A. niger, and was comparable Basaran P, Hang YD (2000) Purification and characterization of with that of A. pullulans (reviewed by Biely et al. 1985)but acetyl esterase from Candida guilliermondii. Lett Appl Micro- much lower than those of Schizophyllum commune (Biely biol 30:167–171 123 J Korean Soc Appl Biol Chem (2015) 58(3):415–421 421

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