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Production and partial characterisation of feruloyl esterase by Sporotrichum thermophile in solid-state fermentation
Process Biochemistry 38 (2003) 1539 /1543 www.elsevier.com/locate/procbio
Production and partial characterisation of feruloyl esterase by Sporotrichum thermophile in solid-state fermentation Evangelos Topakas, Emanuel Kalogeris, Dimitris Kekos, Basil J. Macris, Paul Christakopoulos * Biotechnology Laboratory, Department of Chemical Engineering, National Technical University of Athens, Zografou Campus, Athens 157 80, Greece Received 15 July 2002; accepted 13 January 2003
Abstract A number of factors affecting production of feruloyl esterase an enzyme that hydrolyse ester linkages of ferulic acid (FA) in plant cell walls, by the thermophylic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimised. SSF in a laboratory horizontal bioreactor using the optimised medium allowed the production of 156 mU g 1 of carbon source, which compared favourably with those reported for the other micro-organisms. Optimal esterase activity was observed at pH 8 and 60 8C. The activity of the esterase was measured on an insoluble feruloylated hemicellulose substrate (de-starched wheat bran (DSWB)). De-esterification of wheat straw yielded loss of feruloyl esterase production even though the supplementation of free FA comparable to the alkali-extractable levels of FA found in wheat straw. Chromogenic (fluorogenic) 4-methylumbelliferyl ferulate was used to characterise the multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major esterase activity exhibiting pI and molecular mass values 5 and 27 kDa, respectively. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Feruloyl esterases; Solid state culture; Sporotrichum thermophile
1. Introduction Gramineous plant cell walls are known to contain phenolic constituents, such as ferulic acid (FA), covalently bound to the polysaccharides. FA is esterified to arabinose in the arabinoxylans of wheat bran, barley straw, maize and sugar-cane bagasse and to arabinose in the pectins of sugar beet and spinach. Feruloyl esterase (FAE) is an enzyme capable of hydrolysing sugarphenolic acid ester linkages. There has recently been considerable interest in FAE and its potential application in obtaining FA from agro-industrial waste materials such as those produced by the milling, brewing and sugar industries. Among processes used for enzyme production, solid state fermentation (SSF) is an attractive one because it presents many advantages, especially * Corresponding author. Tel.: /30-1-772-3231; fax: /30-1-7723163. E-mail address: [email protected] (P. Christakopoulos).
for fungal cultivations [1]. In SSF, the productivity per reactor volume is much higher than that in submerged culture [2]. The operational of costs are also lower, because simpler plant, machinery and energy are required [3]. The purpose of this study was to investigate the ability of Sporotrichum thermophile to produce feruloyl esterases in SSF using different agricultural wastes.
2. Materials and methods
2.1. Microorganism S. thermophile strain ATCC 34628 was used in the present study. The stock culture was maintained on potato-dextrose-agar (PDA) at 4 8C.
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00044-X
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E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543
2.2. Media and growth conditions A three-stage cultivation technique was employed. In the first, the fungus was grown on PDA slants for 4 days at 45 8C. In the second, 25 ml deionised sterile water, was added to a PDA slant and aliquots (5 ml) of the mixture were used to inoculate Erlenmeyer flasks (250 ml) containing 2.7 g corn cobs and 100 ml of salts solution containing 0.3% KH2PO4, 0.2% K2HPO4, 0.05% MgSO4 ×/ H2O, 0.01% CaCl2 ×/ 2H2O, 0.0005% FeSO4 ×/ 7H2O, 0.00016% MnSO4 ×/ 4H2O, 0.00014% ZnSO4 ×/ 7H2O, 0.00002% CoCl2 ×/ 6H2O [4] and 1% yeast extract as nitrogen source. The pH of the solution was adjusted to 5. The flasks were incubated at 50 8C for 3 days in an orbital shaker (150 rpm) for mycelium production. In the third stage, an inoculum of mycelial suspension (20% v/w) was added to the enzyme production medium (EPM) in 100 ml Erlenmeyer flasks and incubated at 50 8C for different periods of time under static conditions. The composition of EPM was 2.5 g of carbon source mixed with different volumes of the mineral solution adjusted to pH 5. The water during the cultivation was evaporated resulting the dryness of the initial moisture content at the seventh or ninth day depending the carbon source. The carbon sources, supplemented the mineral solution prior to heat sterilisation (121 8C, 20 min) were prepared as follows: a) De-starched wheat bran (DSWB). The wheat bran was destarched by incubation in 0.25% (w/v) potassium acetate for 10 min at 95 8C. The treated bran was then washed with water and air dried prior to use. b) De-esterified wheat straw was prepared by incubating wheat straw (0.75 g, containing 0.2% FA (w/w), alkali-extractable FA) overnight in 100 ml NaOH (0.1 M), in a cover flask, under gentle agitation. The whole mixture was dialysed extensively against water [5]. c) The other carbon sources that used were wheat bran and wheat straw, which were supplied by the Agricultural University of Athens. All materials were chopped in a laboratory hammer mill to a particle size smaller than 5 mm. 2.3. Cultivation in the bioreactor The bioreactor experiment was performed in a stainless steel horizontal bioreactor equipped with a perforated (pore size, 1 mm2) cylindrical drum (diameter, 0.15 m; length, 0.59 m; capacity, 10 l) where the temperature and the moisture content of the growth medium were effectively controlled at the set values [6]. The bioreactor drum was loaded with 0.5 kg of wheat straw particles, with the optimised mineral medium. The moisture
content of the growth medium was adjusted to 80% thus yielding 2.5 kg of wheat straw growth medium. The latter was sterilised in situ at 110 8C for 45 min. The inoculum was prepared as described above and added to the growth medium in a 1:10 ratio. The airflow rate in the bioreactor was adjusted to 5 l min 1 kg 1 dry matter. Rotating the drum at 10 rpm for 1 min every 3 h effected gentle agitation of growth medium. Sampling was carried out aseptically using a sterile sampler. 2.4. Enzyme extraction After suitable periods of growth time the esterases were extracted from the fermented carbon source with 10-fold (v/w) distilled water by shaking (150 rpm) at room temperature for 60 min. The suspended materials and fungal biomass were separated by centrifugation (10000 /g for 10 min) and the clarified supernatant was used as the source of crude enzyme. 2.5. Enzyme assay FAE activity was assayed by the analysis of free FA released from DSWB [7]. The assay was carried out in 100 mM MOPS buffer, pH 6, 50 8C. Ferulate release was analysed by HPLC using a Nucleosil C18, column. One unit (U) of activity was defined as the amount of enzyme that catalyses the release of 1 mmol FA per min. All assays results were expressed on a dry weight basis. Activity tests after analytical native and isoelectric focusing were performed by flooding the gels with solutions of methylumbelliferyl ferulate (MeUF) and UV-transillumination as described [8]. The preparation of the above chromogenic substrates will be described elsewhere (Nerinckx et al., in preparation). 2.6. Biomass estimation The biomass content was measured by the colorimetric method of Scotti et al. [9], based on glucosamine estimation of the fungal cell wall. 2.7. Determination of pH and temperature optima, pHstability and thermostability The effect of pH was measured using the following buffers: 0.1 M citrate /phosphate (pH 3.0 /6.0), 0.1 M MOPS/NaOH (pH 6.0 /8.0), 0.1 M Tris /HCL (pH 8.0 /9.0) and 0.1 M glycine /NaOH (pH 9.0 /10). The stability at different pH was determined using the above buffers at 4 8C for 1 h. The effect of temperature (40 / 70 8C) on esterase activity was determined using 0.1 M MOPS buffer, pH 6.0, while the thermostability was measured using the same buffer, between 40 and 70 8C for 1 h.
E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543
3. Results and discussion As the moisture content of the medium has a critical significance to SSF, the effect of the moisture content in the cultures after the addition of nutrients and inoculum on the production of enzymes was studied. The optimal initial moisture contents were 80.8, 64.3 and 77.3%, respectively, to wheat straw, wheat bran, and DSWB. The best results were obtained with wheat straw as carbon source regulated to the highest moisture content (80.8%; Fig. 1). As reported elsewhere [10 /13], high moisture enhanced fungal growth and enzyme production when wheat straw was used as carbon source in SSF. Kalogeris et al. [13] and Narag et al. [14] have identified the same substrate as being ideally suited for xylanase production in thermophilic fungi SSF. The same micro-organism has been identified as suitable for the production of xylanase using wheat straw as carbon source [15]. The production of the enzymes was studied in a solidstate horizontal bioreactor using the optimised culture medium and the optimal growth conditions. A typical growth associated enzyme production time course is shown in Fig. 2. At 48 h, very little enzyme activity (3 mU g 1) was detected. At 90 h, the secretion of esterase increased and continued to increase progressively up to 163 h, giving final esterase titres of 156 mU g 1 Quantitative comparison of feruloyl esterase activity produced in this work with that reported for the same enzyme produced by other micro-organisms is not always possible due to different substrates being used [16]. However, the feruloyl esterase activity observed in the present work from S. thermophile , grown on an
1541
inexpensive carbon source compared favourably with the activities reported for other microbial feruloyl esterases [5,16/18] In a separate experiment, the effect of the removal of FA, and other ester-linked groups, from wheat bran was examined. Alkali-treated wheat straw was used as the carbon source, as described in Section 2. Use of deesterified wheat straw as a growth substrate yielded loss of feruloyl esterase activity. The effect of adding free FA to a non-feruloylated growth substrate was examined. De-esterified wheat straw-containing medium was supplemented with 5 mg FA per 100 ml, which is comparable to the alkaliextractable levels of FA found in wheat straw. Since highest feruloyl esterase levels on wheat straw were reached over the first 7 days, cultures containing the supplementary FA were examined over the same period. Addition of free FA did not stimulate feruloyl esterase production by the cultures. These results show that FA has to be in a linked form to stimulate feruloyl esterase production by S. thermophile, free FA is not essential for the enzyme production. These results are in contrast with those reported in the literature where it was found that there is a direct response in feruloyl esterase production by Aspergillus niger to the presence of free FA in the growth medium [5,19]. The enzyme was optimally active at pH 8 and 60 8C. The enzyme was stable between pH 6.0 and 8.0 at 4 8C for 1 h, but under identical conditions, the stability over pH 9.0 /10.0 was only 75 /24%. At pH 6.0, the enzyme was stable to 40/50 8C for at least 1 h, and lost 60% of its original activity in 60 min at 60 8C.
Fig. 1. The effect of initial moisture content in the production of feruloyl esterase by S. thermophile . Wheat straw (m). Wheat bran (k). Destarched wheat bran (%).
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E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543
Fig. 2. Feruloyl esterase and biomass activities produced in the SSF bioreactor by S. thermophile under the optimum operating conditions; moisture content, 80%; temperature, 49 8C; airflow rate, 15 l min 1 kg 1 dry WS. Feruloyl esterase ('). Biomass (m).
The zymograms indicated one major esterase activity exhibiting pI and molecular mass values 5 and 27 kDa, respectively, (Fig. 3). These values were in accordance with these reported for fungal feruloyl esterases from Aspergillus awamori [20] and A. niger [21].
In conclusion, the fungus S. thermophile ATCC 34628 grown on a simple medium consisting of agricultural byproducts and a mineral source, proved to be a promising micro-organism for the production of feruloyl esterase. To the best of the author’s knowledge, no data for feruloyl esterase production in SSF bioreactors have been published so far. The fluorogenic substrates used in this paper allowed the characterisation of the major feruloyl esterase activity. Work is in progress to purify and characterise the enzymes participating in this system.
References
Fig. 3. Analysis by isoelectric focusing (A) and native PAGE electrophoresis (B) of the extracellular protein obtained after growth of S. thermophile for 7 days on wheat straw. Sample was desalted on a PD-10 column (Sephadex G-25) and concentrated by freeze drying. Identification of esterase activity in crude enzyme extract (lanes 2 and 4). Lane 1 and 3 have pI and LMW markers, respectively. Activities were detected with the fluorogenic substrate 4-methylumbelliferyl ferulate.
[1] Weiland P. Principles of solid state fermentation. In: Zadrazil F, Reiniger P, editors. Treatment of lignocellulosics with white rot fungi. London: Elsevier, 1988:64 /76. [2] Grajek W. Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid state fermentation. Appl Microbiol Biotechnol 1987;26:126 /9. [3] Roche N, Desgragnes C, Durand A. Study on the solid-state production of a thermostable a-L-arabinofuranosidase of Thermoascus aurantiacus on sugar beet pulp. J Biotechnol 1994;38:43 / 50. [4] Maheshwari R, Bhat KM. Sporotrichum thermophile growth, cellulose degradation, and cellulase activity. Appl Environ Microbiol 1987;53:2175 /82. [5] Faulds CB, de Vries RP, Kroon PA, Visser J, Williamson G. Influence of ferulic acid on the production of feruloyl esterases by Aspergillus niger . FEMS Microbiol Lett 1997;157:239 /44. [6] Kalogeris E, Fountoukides G, Kekos D, Macris BJ. Design of a solid-state bioreactor for thermophilic microorganisms. Bioresour Technol 1999;67:313 /5. [7] Cristov LP, O’Neill FH, Botes PJ, Prior BA. Rapid and simple assay for feruloyl and p -coumaroyl esterase. World J Microb Biotech 1996;12:239 /42.
E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543 [8] Tilbeurgh H, van Claeyssens M. Detection and differentiation of cellulose components using low molecular mass fluorogenic substrates. FEBS Lett 1985;187:282 /8. [9] Scotti CT, Vergoignan C, Feron G, Durand A. Glucosamine measurement as indirect method for biomass estimation of Cunninghamella elegans grown in solid state cultivation conditions. Biochem Eng J 2001;7:1 /5. [10] Abdulah AL, Tengerdy RP, Murphy VG. Optimization of solid substrate fermentation of wheat straw. Biotechnol Bioeng 1985;27:20 /7. [11] Dechamps F, Giuliano C, Asther M, Huet MC, Roussos S. Cellulase production by Trichoderma harzianum in static and mixed solid-state fermentation reactors under nonaseptic conditions. Biotechnol Bioeng 1985;27:1385 /8. [12] Roussos S, Raimbault M, Saucedo-Castaneda G, ViniegraGonzalez G, Lonsane BK. Kinetics and ratios of carboxymethyl cellulase and filter paper activities of the cellulolytic enzymes produced by Trichoderma harzianum on different substrates in solid state fermentation. Micol Neotrop Appl 1991;4:19 /40. [13] Kalogeris E, Christakopoulos P, Kekos D, Macris BJ. Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus : characterization of two isoenzymes. J Biotechnol 1998;60:155 /63. [14] Narang S, Sahai V, Bisaria VS. Optimization of xylanase production by Melanocarpus albomyces IIS68 in solid state fermentation using response surface methodology. J Biosci Bioeng 2001;91:425 /7.
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[15] Sudgen C, Bhat MK. Cereal straw and pure cellulose as carbon sources for growth and production of plant cell-wall degrading enzymes by Sporotrichum thermophile . J Microbiol Biotechnol 1994;10:444 /51. [16] Christov LP, Prior BA. Esterases of xylan-degrading microorganisms. Production, properties, and significance. Enzyme Microb Technol 1993;15:460 /75. [17] Garcia BL, Ball AS, Rodriguez J, Perez-Leblic MI, Arias ME, Copa-Patino JL. Induction of feruloyl esterase and xylanase activities in Streptomyces avermitilis UAH30. FEMS Microbiol Lett 1998;158:95 /9. [18] Garcia BL, Ball AS, Rodriguez J, Perez-Leblic MI, Arias ME, Copa-Patino JL. Production and characterization of feruloyl esterase activity in crude extracts by Streptomyces avermitilis CECT 3339. Appl Microbiol Biotechnol 1998;50:213 /8. [19] Faulds CB, Williamson G. Effect of hydroxycinnamates and benzoates on the production of feruloyl esterases by Aspergillus niger . J Sci Food Agric 1999;79:450 /2. [20] Koseki T, Furuse S, Iwano K, Matsuzawa H. Purification and characterization of a feruloyl esterase from Aspergillus awamori . Biosci Biotechnol Biochem 1998;62(10):2032 /4. [21] Faulds CB, Williamson G. Feruloyl esterase from Aspergillus niger : purification and partial characterization of two forms from a commercial source of pectinase. Biotechnol Appl Biochem 1993;17:349 /59.
Production and partial characterisation of feruloyl esterase by Sporotrichum thermophile in solid-state fermentation Evangelos Topakas, Emanuel Kalogeris, Dimitris Kekos, Basil J. Macris, Paul Christakopoulos * Biotechnology Laboratory, Department of Chemical Engineering, National Technical University of Athens, Zografou Campus, Athens 157 80, Greece Received 15 July 2002; accepted 13 January 2003
Abstract A number of factors affecting production of feruloyl esterase an enzyme that hydrolyse ester linkages of ferulic acid (FA) in plant cell walls, by the thermophylic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimised. SSF in a laboratory horizontal bioreactor using the optimised medium allowed the production of 156 mU g 1 of carbon source, which compared favourably with those reported for the other micro-organisms. Optimal esterase activity was observed at pH 8 and 60 8C. The activity of the esterase was measured on an insoluble feruloylated hemicellulose substrate (de-starched wheat bran (DSWB)). De-esterification of wheat straw yielded loss of feruloyl esterase production even though the supplementation of free FA comparable to the alkali-extractable levels of FA found in wheat straw. Chromogenic (fluorogenic) 4-methylumbelliferyl ferulate was used to characterise the multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major esterase activity exhibiting pI and molecular mass values 5 and 27 kDa, respectively. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Feruloyl esterases; Solid state culture; Sporotrichum thermophile
1. Introduction Gramineous plant cell walls are known to contain phenolic constituents, such as ferulic acid (FA), covalently bound to the polysaccharides. FA is esterified to arabinose in the arabinoxylans of wheat bran, barley straw, maize and sugar-cane bagasse and to arabinose in the pectins of sugar beet and spinach. Feruloyl esterase (FAE) is an enzyme capable of hydrolysing sugarphenolic acid ester linkages. There has recently been considerable interest in FAE and its potential application in obtaining FA from agro-industrial waste materials such as those produced by the milling, brewing and sugar industries. Among processes used for enzyme production, solid state fermentation (SSF) is an attractive one because it presents many advantages, especially * Corresponding author. Tel.: /30-1-772-3231; fax: /30-1-7723163. E-mail address: [email protected] (P. Christakopoulos).
for fungal cultivations [1]. In SSF, the productivity per reactor volume is much higher than that in submerged culture [2]. The operational of costs are also lower, because simpler plant, machinery and energy are required [3]. The purpose of this study was to investigate the ability of Sporotrichum thermophile to produce feruloyl esterases in SSF using different agricultural wastes.
2. Materials and methods
2.1. Microorganism S. thermophile strain ATCC 34628 was used in the present study. The stock culture was maintained on potato-dextrose-agar (PDA) at 4 8C.
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00044-X
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E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543
2.2. Media and growth conditions A three-stage cultivation technique was employed. In the first, the fungus was grown on PDA slants for 4 days at 45 8C. In the second, 25 ml deionised sterile water, was added to a PDA slant and aliquots (5 ml) of the mixture were used to inoculate Erlenmeyer flasks (250 ml) containing 2.7 g corn cobs and 100 ml of salts solution containing 0.3% KH2PO4, 0.2% K2HPO4, 0.05% MgSO4 ×/ H2O, 0.01% CaCl2 ×/ 2H2O, 0.0005% FeSO4 ×/ 7H2O, 0.00016% MnSO4 ×/ 4H2O, 0.00014% ZnSO4 ×/ 7H2O, 0.00002% CoCl2 ×/ 6H2O [4] and 1% yeast extract as nitrogen source. The pH of the solution was adjusted to 5. The flasks were incubated at 50 8C for 3 days in an orbital shaker (150 rpm) for mycelium production. In the third stage, an inoculum of mycelial suspension (20% v/w) was added to the enzyme production medium (EPM) in 100 ml Erlenmeyer flasks and incubated at 50 8C for different periods of time under static conditions. The composition of EPM was 2.5 g of carbon source mixed with different volumes of the mineral solution adjusted to pH 5. The water during the cultivation was evaporated resulting the dryness of the initial moisture content at the seventh or ninth day depending the carbon source. The carbon sources, supplemented the mineral solution prior to heat sterilisation (121 8C, 20 min) were prepared as follows: a) De-starched wheat bran (DSWB). The wheat bran was destarched by incubation in 0.25% (w/v) potassium acetate for 10 min at 95 8C. The treated bran was then washed with water and air dried prior to use. b) De-esterified wheat straw was prepared by incubating wheat straw (0.75 g, containing 0.2% FA (w/w), alkali-extractable FA) overnight in 100 ml NaOH (0.1 M), in a cover flask, under gentle agitation. The whole mixture was dialysed extensively against water [5]. c) The other carbon sources that used were wheat bran and wheat straw, which were supplied by the Agricultural University of Athens. All materials were chopped in a laboratory hammer mill to a particle size smaller than 5 mm. 2.3. Cultivation in the bioreactor The bioreactor experiment was performed in a stainless steel horizontal bioreactor equipped with a perforated (pore size, 1 mm2) cylindrical drum (diameter, 0.15 m; length, 0.59 m; capacity, 10 l) where the temperature and the moisture content of the growth medium were effectively controlled at the set values [6]. The bioreactor drum was loaded with 0.5 kg of wheat straw particles, with the optimised mineral medium. The moisture
content of the growth medium was adjusted to 80% thus yielding 2.5 kg of wheat straw growth medium. The latter was sterilised in situ at 110 8C for 45 min. The inoculum was prepared as described above and added to the growth medium in a 1:10 ratio. The airflow rate in the bioreactor was adjusted to 5 l min 1 kg 1 dry matter. Rotating the drum at 10 rpm for 1 min every 3 h effected gentle agitation of growth medium. Sampling was carried out aseptically using a sterile sampler. 2.4. Enzyme extraction After suitable periods of growth time the esterases were extracted from the fermented carbon source with 10-fold (v/w) distilled water by shaking (150 rpm) at room temperature for 60 min. The suspended materials and fungal biomass were separated by centrifugation (10000 /g for 10 min) and the clarified supernatant was used as the source of crude enzyme. 2.5. Enzyme assay FAE activity was assayed by the analysis of free FA released from DSWB [7]. The assay was carried out in 100 mM MOPS buffer, pH 6, 50 8C. Ferulate release was analysed by HPLC using a Nucleosil C18, column. One unit (U) of activity was defined as the amount of enzyme that catalyses the release of 1 mmol FA per min. All assays results were expressed on a dry weight basis. Activity tests after analytical native and isoelectric focusing were performed by flooding the gels with solutions of methylumbelliferyl ferulate (MeUF) and UV-transillumination as described [8]. The preparation of the above chromogenic substrates will be described elsewhere (Nerinckx et al., in preparation). 2.6. Biomass estimation The biomass content was measured by the colorimetric method of Scotti et al. [9], based on glucosamine estimation of the fungal cell wall. 2.7. Determination of pH and temperature optima, pHstability and thermostability The effect of pH was measured using the following buffers: 0.1 M citrate /phosphate (pH 3.0 /6.0), 0.1 M MOPS/NaOH (pH 6.0 /8.0), 0.1 M Tris /HCL (pH 8.0 /9.0) and 0.1 M glycine /NaOH (pH 9.0 /10). The stability at different pH was determined using the above buffers at 4 8C for 1 h. The effect of temperature (40 / 70 8C) on esterase activity was determined using 0.1 M MOPS buffer, pH 6.0, while the thermostability was measured using the same buffer, between 40 and 70 8C for 1 h.
E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543
3. Results and discussion As the moisture content of the medium has a critical significance to SSF, the effect of the moisture content in the cultures after the addition of nutrients and inoculum on the production of enzymes was studied. The optimal initial moisture contents were 80.8, 64.3 and 77.3%, respectively, to wheat straw, wheat bran, and DSWB. The best results were obtained with wheat straw as carbon source regulated to the highest moisture content (80.8%; Fig. 1). As reported elsewhere [10 /13], high moisture enhanced fungal growth and enzyme production when wheat straw was used as carbon source in SSF. Kalogeris et al. [13] and Narag et al. [14] have identified the same substrate as being ideally suited for xylanase production in thermophilic fungi SSF. The same micro-organism has been identified as suitable for the production of xylanase using wheat straw as carbon source [15]. The production of the enzymes was studied in a solidstate horizontal bioreactor using the optimised culture medium and the optimal growth conditions. A typical growth associated enzyme production time course is shown in Fig. 2. At 48 h, very little enzyme activity (3 mU g 1) was detected. At 90 h, the secretion of esterase increased and continued to increase progressively up to 163 h, giving final esterase titres of 156 mU g 1 Quantitative comparison of feruloyl esterase activity produced in this work with that reported for the same enzyme produced by other micro-organisms is not always possible due to different substrates being used [16]. However, the feruloyl esterase activity observed in the present work from S. thermophile , grown on an
1541
inexpensive carbon source compared favourably with the activities reported for other microbial feruloyl esterases [5,16/18] In a separate experiment, the effect of the removal of FA, and other ester-linked groups, from wheat bran was examined. Alkali-treated wheat straw was used as the carbon source, as described in Section 2. Use of deesterified wheat straw as a growth substrate yielded loss of feruloyl esterase activity. The effect of adding free FA to a non-feruloylated growth substrate was examined. De-esterified wheat straw-containing medium was supplemented with 5 mg FA per 100 ml, which is comparable to the alkaliextractable levels of FA found in wheat straw. Since highest feruloyl esterase levels on wheat straw were reached over the first 7 days, cultures containing the supplementary FA were examined over the same period. Addition of free FA did not stimulate feruloyl esterase production by the cultures. These results show that FA has to be in a linked form to stimulate feruloyl esterase production by S. thermophile, free FA is not essential for the enzyme production. These results are in contrast with those reported in the literature where it was found that there is a direct response in feruloyl esterase production by Aspergillus niger to the presence of free FA in the growth medium [5,19]. The enzyme was optimally active at pH 8 and 60 8C. The enzyme was stable between pH 6.0 and 8.0 at 4 8C for 1 h, but under identical conditions, the stability over pH 9.0 /10.0 was only 75 /24%. At pH 6.0, the enzyme was stable to 40/50 8C for at least 1 h, and lost 60% of its original activity in 60 min at 60 8C.
Fig. 1. The effect of initial moisture content in the production of feruloyl esterase by S. thermophile . Wheat straw (m). Wheat bran (k). Destarched wheat bran (%).
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E. Topakas et al. / Process Biochemistry 38 (2003) 1539 /1543
Fig. 2. Feruloyl esterase and biomass activities produced in the SSF bioreactor by S. thermophile under the optimum operating conditions; moisture content, 80%; temperature, 49 8C; airflow rate, 15 l min 1 kg 1 dry WS. Feruloyl esterase ('). Biomass (m).
The zymograms indicated one major esterase activity exhibiting pI and molecular mass values 5 and 27 kDa, respectively, (Fig. 3). These values were in accordance with these reported for fungal feruloyl esterases from Aspergillus awamori [20] and A. niger [21].
In conclusion, the fungus S. thermophile ATCC 34628 grown on a simple medium consisting of agricultural byproducts and a mineral source, proved to be a promising micro-organism for the production of feruloyl esterase. To the best of the author’s knowledge, no data for feruloyl esterase production in SSF bioreactors have been published so far. The fluorogenic substrates used in this paper allowed the characterisation of the major feruloyl esterase activity. Work is in progress to purify and characterise the enzymes participating in this system.
References
Fig. 3. Analysis by isoelectric focusing (A) and native PAGE electrophoresis (B) of the extracellular protein obtained after growth of S. thermophile for 7 days on wheat straw. Sample was desalted on a PD-10 column (Sephadex G-25) and concentrated by freeze drying. Identification of esterase activity in crude enzyme extract (lanes 2 and 4). Lane 1 and 3 have pI and LMW markers, respectively. Activities were detected with the fluorogenic substrate 4-methylumbelliferyl ferulate.
[1] Weiland P. Principles of solid state fermentation. In: Zadrazil F, Reiniger P, editors. Treatment of lignocellulosics with white rot fungi. London: Elsevier, 1988:64 /76. [2] Grajek W. Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid state fermentation. Appl Microbiol Biotechnol 1987;26:126 /9. [3] Roche N, Desgragnes C, Durand A. Study on the solid-state production of a thermostable a-L-arabinofuranosidase of Thermoascus aurantiacus on sugar beet pulp. J Biotechnol 1994;38:43 / 50. [4] Maheshwari R, Bhat KM. Sporotrichum thermophile growth, cellulose degradation, and cellulase activity. Appl Environ Microbiol 1987;53:2175 /82. [5] Faulds CB, de Vries RP, Kroon PA, Visser J, Williamson G. Influence of ferulic acid on the production of feruloyl esterases by Aspergillus niger . FEMS Microbiol Lett 1997;157:239 /44. [6] Kalogeris E, Fountoukides G, Kekos D, Macris BJ. Design of a solid-state bioreactor for thermophilic microorganisms. Bioresour Technol 1999;67:313 /5. [7] Cristov LP, O’Neill FH, Botes PJ, Prior BA. Rapid and simple assay for feruloyl and p -coumaroyl esterase. World J Microb Biotech 1996;12:239 /42.
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