Appl Microbiol Biotechnol (2001) 56:762–766 DOI 10.1007/s002530100716
O R I G I N A L PA P E R
B. C. Saha
Xylanase from a newly isolated Fusarium verticillioides capable of utilizing corn fiber xylan
Received: 7 November 2000 / Received revision: 2 April 2001 / Accepted: 20 April 2001 / Published online: 30 June 2001 © Springer-Verlag 2001
Abstract A fungus, Fusarium verticillioides (NRRL 26518), was isolated by screening soil samples using corn fiber xylan as carbon source. The extracellular xylanase from this fungal strain was purified to apparent homogeneity from the culture supernatant by ultrafiltration using a 30,000 cut-off membrane, octyl-Sepharose chromatography and Bio gel A-0.5 m gel filtration. The purified xylanase (specific activity 492 U/mg protein; MW 24,000; pI 8.6) displayed an optimum temperature at 50 °C and optimum pH at 5.5, a pH stability range from 4.0 to 9.5 and thermal stability up to 50 °C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability.
Introduction Corn fiber xylan is one of the complex heteroxylans containing β-(1,4)-linked xylose residues (Saha 2000). This backbone is highly substituted with monomeric side-chains of arabinose or glucuronic acid linked to O-2 and/or O-3 of xylose residues, and also by oligomeric side chains containing arabinose, xylose and sometimes galactose residues (Saulnier et al. 1995). It is highly branched with high levels of ferulic acid (5%) esterified to it as well as high level of cross-linking through diferulic acid bridges between the heteroxylan chains Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. B.C. Saha (✉) Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, Agricultural Research service, United States Department of Agriculture, Peoria, Illinois 61604, USA e-mail:
[email protected] Tel.: +1-309-6816276, Fax: + 1-309-6816427
(Saulnier and Thibault 1999). Recently, we have demonstrated that xylan in corn fiber is highly resistant to enzymatic degradation and that commercial hemicellulase preparations do not effectively saccharify corn fiber xylan (Saha and Bothast 1999a). A number of soil samples from various corn fields were screened for the purpose of isolating an organism that could produce enzymes useful for saccharification of corn fiber xylan. Three cultures capable of utilizing corn fiber xylan as growth substrate were isolated. Xylanase (β-1,4-D-xylan xylanohydrolase, EC 3.2.1.8) is the key enzyme for xylan depolymerization. A number of xylanases have been purified from a wide variety of microorganisms such as Bacillus sp., Clostridium sp., Streptomyces sp., Aspergillus sp., Fusarium sp. and Trichoderma sp. (Saha and Bothast 1999b). In this report, the production, purification and characterization of a xylanase from one newly isolated fungal strain is described.
Materials and methods Materials Oat spelt xylan, birch wood xylan, larch wood xylan, Sigma cell 50, all aryl-glycosides and molecular weight markers for gel filtration were purchased from Sigma (St. Louis, Mo.). Wheat arabinoxylan, rye arabinoxylan and xylooligosaccharides [xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5) and xylohexaose (X6)] were purchased from MegaZyme (North Rocks, Australia). Molecular weight markers and precast gels for SDS-PAGE, Bio Gel A-0.5m, and the Aminex HPX-87C column for HPLC were obtained from Bio-Rad (Hercules, Calif.). Octyl-Sepharose 4, Mono P, PBE-94 and Polybuffer 96 were from Amersham Pharmacia (Piscataway, N.J.). Corn fiber xylan was prepared from corn fiber obtained from a corn wet-milling facility (supplied by Williams Energy Corp., Pekin, Ill.) according to the method described by Hespell (1998). Corn cob xylans A and B, prepared by the procedure of Whistler et al. (1948), were provided by Patricia O’Bryan. Econase HC was supplied by Enzyme Development Corp. (New York, N.Y.). Screening, isolation and identification of the fungal strain The fungal strain used in this study was isolated by screening 132 soil samples surrounding decaying corn and wood collected from
763 the Peoria, Ill., area. The screening medium contained (per l): 2 g NaNO3, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4· 7H2O, 1.0 g KH2PO4, 0.4 g yeast extract and 10 g corn fiber xylan. Corn fiber xylan was sterilized (121 °C, 15 min) separately. The pH of the medium was adjusted to 5.0 with 1 M HCl before inoculation. The collected soil samples (~0.5 g) were placed in test tubes (1.5×15 cm, 10 ml medium with 1% (w/v) corn fiber xylan as energy source) and incubated at 30 °C and 200 rpm for 4 days. After five transfers of only well-grown cultures (0.5 ml) in liquid culture (10 ml), samples of culture broth were serially diluted and grown on agar plates (2%, w/v agar) containing the screening medium for 2–3 days. Subsequently, after ten transfers in agar plates, single isolated colonies were transferred to test tubes containing 10 ml of the screening medium. These procedures were repeated several times to ensure purity of the culture. The isolated culture was maintained throughout this study at 4 °C on 2% (w/v) agar slants with YMP medium, which contained (per 100 ml): 0.3 g yeast extract, 0.3 g malt extract, 0.3 g peptone and 1 g oat spelt xylan. Three cultures were eventually isolated that had the capability to utilize corn fiber xylan. These were identified as a strain of Fusarium proliferatum (Matsushima) Nirenberg, Fusarium verticillioides (Saccardo) Nirenberg and Mucor circinelloides v. Tieghem by Centralbureau voor schimmelcultures, Institute of the Royal Netherlands Academy of Arts and Sciences, and were deposited in the ARS Culture Collection, NCAUR, Peoria, Ill. (designated as NRRL 26517, NRRL 26518 and NRRL 26519, respectively). In this study, Fusarium verticillioides NRRL 26518 was used because of its higher level of enzyme production compared with the other two isolated cultures. Cultivation and enzyme production The YMP medium was used for seed culture and enzyme production. Oat spelt xylan was sterilized separately. The pH of the medium was adjusted to 5.0 with 1 M HCl before inoculation. A 125-ml Erlenmeyer flask containing 50 ml of YMP medium was inoculated with a loopful of cells taken from a stock slant and incubated at 30 °C on a rotary shaker (200 rpm) for 2 days. Shake flasks (1-l Erlenmeyer flask containing 400 ml medium) were inoculated with 8 ml of this culture and cultivated on a rotary shaker (200 rpm) at 30 °C. After 4 days, the cells were removed from the culture broth by centrifugation (18,000 g, 20 min). The resulting supernatant solution was used as the crude enzyme preparation. Enzyme assays Xylanase activity was assayed in a reaction mixture (0.5 ml) containing 1% (w/v) boiled oat spelt xylan, 50 mM acetate buffer, pH 5.0 and appropriately diluted enzyme solution. After a 30-min incubation at 50 °C, the reducing sugar liberated in the reaction mixture was measured by the dinitrosalicylic acid method (Miller 1959). One unit (U) of xylanase activity is defined as the amount of enzyme which produces 1 µmol reducing sugar as xylose in the reaction mixture per min under the specified conditions. β-Xylosidase (1,4-β-D-xyloside xylanohydrolase, EC 3.2.1.37) and α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) activities were assayed in the reaction mixture (1 ml) containing 2 mM p-nitrophenyl β-D-xyloside and 1 mM p-nitrophenyl-α-L-arabinofuranoside, respectively, 50 mM acetate buffer, pH 5.0 and appropriately diluted enzyme solutions. After incubation at 50 °C for 30 min, the reaction was stopped by adding 1 ml of ice-cold 0.5 M Na2CO3 and the color that developed as a result of p-nitrophenol (pNP) liberation was measured at 405 nm. One unit (U) of each enzyme activity is defined as the amount of enzyme which releases 1 µmol pNP per min in the reaction mixture under these assay conditions. Purification of xylanase All purification steps were performed at 4 °C unless otherwise stated.
Ultrafiltration. The culture supernatant was subjected to ultrafiltration with a stirred cell (model 202; Amicon, Beverly, Mass.) equipped with a PM 30 membrane under nitrogen pressure of 20 lb/in2 and the filtrate was used as a source of xylanase. This was then concentrated about two-fold using a YM 5 membrane. Octyl-Sepharose chromatography. The concentrated culture filtrate was equilibrated with 50 mM citrate-phosphate buffer, pH 5.0, plus 20% (NH4)2SO4 (buffer A). Octyl-Sepharose pre-equilibrated with buffer A was added to the culture filtrate, stirred gently for 2–3 h and then left overnight. Octyl-Sepharose was then packed in a column (2.5×26 cm). The column was washed extensively with buffer A and eluted with the same buffer without (NH4)2SO4. The highly active xylanase fractions were pooled and concentrated by ultrafiltration using a YM 5 membrane. Gel filtration on Bio-Gel A-0.5m. The xylanase was further purified by gel filtration on a Bio-Gel A-0.5m column (1.5×120 cm) preequilibrated with 50 mM acetate buffer, pH 5.0. The enzyme solution in 50 mM acetate buffer, pH 5.0, was applied to the column and eluted with the same buffer. Xylanase activity was eluted as a single protein and enzyme peak. The highly active xylanase fractions were pooled, concentrated by ultrafiltration (YM 5 membrane) and used as purified xylanase for subsequent studies. Other methods Protein was estimated by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Protein in the column effluents was monitored by measuring absorbance at 280 nm. The isoelectric point (pI) of xylanase was determined by chromatofocusing, which was carried out in a PBE-Mono P anion exchange resin packed in a 1.0×30.0 cm column. An enzyme sample in 25 mM ethanolamine with 10% glycerol, pH 9.4, was applied to the column, which was pre-equilibrated with the same buffer. The proteins were eluted with diluted (1:10) Polybuffer 96, pH 6.0. SDS-PAGE was done on a 12% gel according to Laemmli (1970). The molecular weight of the native enzyme was determined by gel filtration on Bio-Gel A-0.5m as described by Andrews (1965). The Km value was determined by the double-reciprocal plot method of Lineweaver-Burk using the KINET software program. Xylan hydrolysis product analysis was done by HPLC (Spectra-Physics, San Jose, Calif.) using an ion-moderated partition chromatography column (Aminex HPX-87C). The column was maintained at 85 °C, and the sugars were eluted with Milli-Q (Millipore, Bedford, Mass.) water at a flow rate of 0.6 ml/min. Peaks were detected by refractive index and identified and quantified by comparison to retention times of authentic standards (xylose, X2–X6).
Results Production of xylanase The time courses of corn fiber xylan utilization and production of extracellular xylanolytic enzymes by the newly isolated F. verticillioides NRRL-26518 were studied (Fig. 1). There was a lag in corn fiber xylan utilization (monitored by the disappearance of xylan peak using HPLC) as well as in enzyme production and both increased sharply during the 24–48 h growth period, after which the xylanase level remained almost the same. β-Xylosidase and α-L-arabinofuranosidase production continued to increase during the time period studied. The fungus utilized about 60% corn fiber xylan and produced extracellularly 8 U of xylanase, 95 mU of β-xylosidase and 111 mU of α-L-arabinofuranosidase per ml in 4 days.
764 Table 1 Purification of xylanase from Fusarium verticillioides NRRL 26518
Step
Total Total Specific activity Recovery Purification protein (mg) activity (U) (U/mg protein) (%) fold
Culture supernatant 5463 Ultrafiltration (PM 30) 1093 Octyl-Sepharose 20.5 Bio Gel A-0.5m gel filtration 0.5
7268 3282 834 246
1.3 3.0 40.7 492
100 45.2 11.5 3.4
1 2 31 378
Fig. 1 Time course of corn fiber xylan (1%, w/v) utilization and extracellular enzyme production by Fusarium verticillioides NRRL 26518 at 30 °C. ● Corn fiber xylan, ● xylanase, ▲ β-xylosidase, ■ α-L-arabinofuranosidase. Values are averages from duplicate experiments
It produced 1.1 U of xylanase, 27 mU of β-xylosidase and 10 mU of α-L-arabinofuranosidase per ml in 4 days when grown on oat spelt xylan. When birchwood xylan was used as substrate, F. verticillioides produced 1.2 U of xylanase, 15 mU of β-xylosidase and 3 mU of α-Larabinofuranosidase per ml in 4 days. It produced very little or no enzyme when grown on xylose. It is clear that the fungal strain produced more xylanolytic enzymes when grown on corn fiber xylan as substrate. Purification of xylanase An extracellular xylanase was purified to apparent homogeneity from the culture filtrates of F. verticillioides grown on oat spelt xylan. A summary of the purification procedures is presented in Table 1. The xylanase activity showed a single peak of activity during the purification. Upon SDS-PAGE of the purified xylanase, a single band was visualized when stained with Coomassie brilliant blue (Fig. 2). The final purification resulted in a yield of 3.4% of the activity, 0.01% retention of total protein and a 378-fold increase in specific activity (Table 1).
Fig. 2 SDS-PAGE of purified xylanase from Fusarium verticillioides NRRL 26518. The enzyme (~20 µg protein) was electrophoresed at pH 8.3 on a 12% acrylamide gel and stained with Coomassie brilliant blue R-250. 1 Molecular weight standards, 2 purified xylanase. The standards were: phosphorylase B (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), trypsin inhibitor (21,500) and lysozyme (14,400)
of the enzyme as determined by chromatofocusing was 8.6. pH and temperature dependence. The thermostability and thermoactivity of the purified xylanase are shown in Fig. 3. The purified enzyme in 50 mM acetate buffer, pH 5.0 (0.7 U/ml; 1.4 µg protein/ml) was fairly stable up to 50 °C for 30 min. It exhibited maximum activity at 50 °C with 81% relative activity at 60 °C and 18% activity at 70 °C under the assay conditions used. The enzyme (0.9 U/ml) was also fairly stable at pH 4.0–9.5 (1 h at 40 °C). It displayed an optimum activity at pH 5.5 with no activity at pH 3.0 and 50% relative activity at pH 8.0. This means that the xylanase from F. verticillioides NRRL 26518 is fairly stable at alkaline pH conditions.
Characterization of xylanase Molecular weight The apparent molecular weight of the native xylanase estimated by gel filtration on Bio-Gel A-0.5m was 35,000. By SDS-PAGE analysis, the molecular weight of the enzyme was about 24,000 (Fig. 2). The pI
Substrate specificity and kinetic analysis. Relative initial rates of hydrolysis of various xylan substrates by the purified enzyme (1.25 U/ml) are shown in Table 2. The purified xylanase did not degrade corn fiber xylan although it degraded corn cob xylan well. The crude
765 Table 2 Comparative rates of xylan hydrolysis and product formation by purified xylanase from Fusarium verticillioides NRRL 26518. A 100% activity corresponds to 1.25 U of xylanase (per ml) under standard assay conditions (1%, w/v oat spelt xylan as substrate, 50 °C, pH 5.0). X2 Xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentaose, – not determined Substrate
Relative rate of hydrolysis (%)
Product (72 h)
Corn fiber xylan Corn cob xylan A Corn cob xylan B Birch wood xylan Larch wood xylan Oat spelt xylan Rye arabinoxylan
0.0 87±6 99±0 65±2 58±2 100±2 –
None X2 X2, X 3 X 2, X 3, X 5 X 2, X 3, X 4 X2, X3, X4 X2
was followed by analyzing the reaction products by HPLC (Table 2). It released mainly X2 and in addition X3–X5 depending on the substrate. No xylose was detected. The purified enzyme did not bind to microcrystalline cellulose (Sigma cell 50) tested at pH 5.0. Effect of metal ions and reagents on activity. The influence of certain inhibitors or activators on xylanase activity (50 °C, pH 5.0, 30 min reaction) was studied. The enzyme did not require Ca2+, Mg2+, Mn2+ (each at 5 mM) or Co2+ (0.5 mM) for activity. Enzyme activity was not affected by EDTA (10 mM), dithiothreitol (10 mM) or p-chloromercuribenzoic acid (0.2 mM). Fig. 3a, b Effect of temperature (a) and pH (b) on stability (● ●) and activity (●) of purified xylanase from Fusarium verticillioides NRRL 26518. For thermal stability, the enzyme solution in 50 mM acetate buffer, pH 5.0, was incubated for 30 min at various temperatures, and the residual enzyme activities were then assayed under standard conditions. For thermoactivity, the enzyme activity was assayed at various temperatures by the standard assay method. Enzyme used, 0.7 U/ml. For pH stability, the enzyme solutions in buffers at various pH values were incubated for 30 min at 50 °C. After adjustment of pH, the residual activity was assayed. The enzyme activity was assayed by changing the buffer to obtain the desired pH. Buffer used (100 mM): citrate-phosphate, pH 3.0–7.0, phosphate buffer (7.5–8.0) and glycine buffer (8.5–10.0). Enzyme used, 0.9 U/ml. All values are averages from duplicate experiments
enzyme preparation (xylanase 2.0 U/ml, 1% substrate) released 59 mg arabinose, 64 mg xylose and 18 mg xylobiose per g corn fiber xylan in 24 h. Econase HC (xylanase 2 U/ml), a commercial hemicellulase preparation, released only 57 mg arabinose, 21 mg xylose and 11 mg xylobiose in a 96-h reaction at 50 °C and pH 5.0. However, the addition of this crude enzyme preparation (2 U/ml) from F. verticillioides to Econase HC (xylanase 2 U/ml) significantly enhanced the saccharification reaction (232 mg arabinose, 132 mg xylose, 32 mg xylobiose and 28 mg xylotriose in a 96-h reaction). The rate dependence of the enzymatic reaction on the oat spelt xylan concentration at pH 5.5 and 50 °C followed MichaelisMenten kinetics. The reciprocal plot showed an apparent Km value of 9.5 mg/ml for hydrolysis of oat spelt xylan and 2.2 mg/ml for the hydrolysis of birch wood xylan. The degradation of various xylans by purified xylanase
Discussion This is the first report on the purification and characterization of xylanase from F. verticillioides, a newly isolated fungus capable of utilizing corn fiber xylan as growth substrate. The fungus makes significantly more xylanolytic enzymes when grown on xylan from corn fiber than other sources. Reports are available on the purification and characterization of multiple xylanases from Fusarium oxysporum (Alconada and Martinez 1994; Christakopoulos et al 1996a, b). However, the xylanase from F. verticillioides differs significantly from these xylanases with respect to certain physical and biochemical properties. The molecular weight and pI of a major xylanase from F. oxysporum F3 were 60,200 and 6.6, respectively (Christakopoulos et al. 1996a). The enzyme showed an optimum pH at 7.4 and released xylose in addition to xylobiose and other saccharides from xylan substrates. Christakopoulos et al. (1996b) also purified and characterized two, low molecular weight (20,800 and 23,500) alkaline xylanases (pI 9.5, 8.45–8.7) from the same strain. During purification of the xylanase from F. verticillioides, only one peak of xylanase activity was obtained. This indicates that the xylanase from F. verticillioides may exist in only one form and is a low molecular weight alkaline enzyme. The production of multiple xylanases is characteristic of many microorganisms (Saha and Bothast 1999b). The purified xylanase from F. oxysporum f. sp. melonis
766
had a molecular weight of 80,000, an optimum pH at 5.0 and an optimum temperature at 50 °C (Alconada and Martinez 1994). A xylanase from F. oxysporum F3 binds on crystalline cellulose (Christakopoulus et al. 1996c), whereas the xylanase from F. verticillioides did not bind to cellulose. The native molecular weight (35,000) of the purified xylanase from F. verticillioides was higher than that (24,000) calculated from SDS-PAGE. Several investigators have reported the abnormal behavior of xylanase on gel filtration columns (Lin et al. 1999). The addition of EDTA did not affect the activity, suggesting that no metals are needed for the enzymatic reaction. The products of xylan hydrolysis are a mixture of xylooligosaccharides (X2–X5). This indicates that this xylanase may have practical utility in making xylooligosaccharides from xylan substrates (Chen et al. 1997). Further, xylose was not detected in any case of xylan hydrolysis, which indicates that the purified enzyme was not contaminated with β-xylosidase activity. The crude enzyme preparation hydrolyzed corn fiber xylan significantly but the purified xylanase did not attack the substrate. It did degrade corn cob xylan (Table 2). The structural complexity between the two xylans from corn cob and corn fiber is quite different (Ebringerova et al. 1992; Saulnier and Thibault 1999). These results indicate that for effective hydrolysis of xylan substrates, a proper mix of xylanase with accessory enzymes such as β-xylosidase and α-L-arabinofuranosidase is essential. Thus, in order to make enzymatic hydrolysis of hemicellulosic substrates a reality, a hemicellulase preparation having all the accessory enzymes in proper quantities (enzyme cocktail) has to be developed. Acknowledgement The author thanks Sarah E. Campagna for excellent technical assistance.
References Alconada TM, Martinez MJ (1994) Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis. FEMS Microbiol Lett 118:305–310 Andrews P (1965) Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem J 91:595–606
Chen C, Chen J-L, Lin T-Y (1997) Purification and characterization of a xylanase from Trichoderma longibrachiatum for xylooligosaccharide production. Enzyme Microb Technol 21: 91–96 Christakopoulos P, Kekos D, Macris BJ, Claeyssens M, Bhat MK (1996a) Purification and characterization of a major xylanase with cellulase and transferase activities from Fusarium oxysporum F3. Carbohydrate Res 289:91–104 Christakopoulos P, Nerinckx W, Kekos D, Macris B, Claeyssens M (1996b) Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3. J Biotechnol 51:181–189 Christakopoulos P, Nerinckx W, Samyn B, Kekos D, Macris B, Breeumen JV, Claeyssens M (1996c) Functional characterization of a cellulose binding xylanase from Fusarium oxysporum. Biotechnol Lett 18:349–354 Ebringerova A, Hromadkova Z, Alfoldi J, Berth G (1992) Structural and solutions properties of corn cob heteroxylans. Carbohydr Polym 19:99–105 Hespell RB (1998) Extraction and characterization of hemicellulose from the corn fiber produced by corn wet-milling processes. J Agric Food Chem 46:2615–2619 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 Lin J, Ndlovu LM, Singh S, Pillay B (1999) Purification and biochemical characterization of β-D-xylanase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. Biotechnol Appl Biochem 30:73–79 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275 Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428 Saha BC (2000) α-L-Arabinofuranosidase, biochemistry, molecular biology, and application in biotechnology. Biotechnol Adv 18: 403–423 Saha BC, Bothast RJ (1999a) Pretreatment and enzymatic saccharification of corn fiber. Appl Biochem Biotechnol 76:65–77 Saha BC, Bothast, RJ (1999b) Enzymology of xylan degradation. In: Imam SH, Greene RV, Zaidi BR (eds) Biopolymers: utilizing nature’s advanced materials. American Chemical Society, Washington, DC, pp 167–194 Saulnier L, Thibault JF (1999) Ferulic acid and diferulic acids as components of sugar-beet pectins and maize bran heteroxylans. J Sci Food Agric 79:396–402 Saulnier L, Marot C, Chanliaud E, Thibault JF (1995) Cell wall polysaccharide interactions in maize bran. Carbohyd Polymers 26:279–287 Whistler RL, Bachrach J, Bowman DR (1948) Preparation and properties of corn cob holocellulose. Arch Biochem 19:25–33
