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Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase
Journal of Biotechnology 63 (1998) 199 – 210
Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a b-mannanase Pia Ademark a, Arthur Varga a, Jo´zsef Medve a, Vesa Harjunpa¨a¨ b, Torbjo¨rn Drakenberg b, Folke Tjerneld a, Henrik Sta˚lbrand a,* a
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, PO Box 124, S-22100 Lund, Sweden b VTT Chemical Technology, FIN-02044 VTT (Espoo), Finland Received 19 December 1997; received in revised form 5 June 1998; accepted 12 June 1998
Abstract The enzymes needed for galactomannan hydrolysis, i.e. b-mannanase, a-galactosidase and b-mannosidase, were produced by the filamentous fungus Aspergillus niger. The b-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the b-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger b-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus b-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Aspergillus niger; b-Mannanase; a-Galactosidase; Hemicellulase
1. Introduction Hemicelluloses are complex polysaccharides which are abundant in higher plant cell walls. Galactoglucomannan, the major softwood hemi* Corresponding author. Fax: +46 46 2224534; e-mail: [email protected]
cellulose, contains b-1,4-linked D-mannopyranose and D-glucopyranose units. The residues in the main chain are partially substituted by a-1,6linked D-galactosyl side groups. The complete enzymatic degradation of hemicelluloses involves several specific activities. Endo-1,4-b-D-mannanase (EC 3.2.1.78) catalyzes the random cleavage of b-D-1,4-mannopyra-
0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00086-8
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nosyl linkages within the main chain of galactomannan, glucomannan, galactoglucomannan and mannan. Mannanases from a variety of different organisms have been studied, including bacteria, fungi, higher plants and animals (reviewed by Dekker and Richards, 1976 and Viikari et al., 1993). The interest in b-mannanase and other hemicellulose-degrading enzymes has recently increased, partly because of their potential applicability in the food and paper and pulp industries (Viikari et al., 1993, 1994). The degradation of galactomannan and galactoglucomannan by b-mannanase is greatly affected by the extent and pattern of substitution of the mannan backbone. The interference of Dgalactosyl side groups with hydrolysis has been carefully analyzed using b-mannanases from A. niger (McCleary and Matheson, 1983) and Trichoderma reesei (Tenkanen et al., 1997). The complete conversion of galactomannan into D-galactose and D-mannose requires the presence of two additional enzymes, a-galactosidase (EC 3.2.1.22) and b-mannosidase (EC 3.2.1.25). These enzymes catalyze the cleavage of terminal a-1,6linked D-galactosyl and b-1,4-linked D-mannopyranosyl residues, respectively. A considerable variation of the ability to hydrolyze galactosyl side groups from polymeric substrates exists among the a-galactosidases. A. niger, a filamentous fungus, is one of the most used organisms in the industrial production of fermented foods, organic acids, and enzymes (Barbesgaard, 1977, Bennett, 1985). The purification of b-mannanase from A. niger has been described earlier (Eriksson and Winell, 1968, Tsujisaka et al., 1972, Yamazaki et al., 1976, McCleary, 1979, 1988), but it has not been clear if more than one b-mannanase is secreted. A multiplicity of extracellular b-mannanases appears to be common among fungi and has been noticed, for example, in T. reesei (Sta˚lbrand et al., 1993, Sta˚lbrand, 1995) and Trichoderma harzianum (Torrie et al., 1990). In the present study the aim was to separate analytically the major galactoglucomannan-degrading enzymes from A. niger and to purify and characterize biochemically the bmannanase.
2. Materials and methods
2.1. Culture A. niger ATCC-46890 was obtained from the QM Culture Collection, Department of Botany, University of Massachusetts, Amherst. The culture was maintained on potato dextrose agar slants.
2.2. Screening of growth media The effect of different culture media on b-mannanase production was investigated. A. niger ATCC-46890 was grown at 28°C in shake-flasks containing one of the following carbon sources: 0.5–2% (w/v) locust bean gum galactomannan (Sigma), 0.5% Solka Floc cellulose, or 1% glucose. All media were supplemented with 4% Vogel’s medium N (Vogel, 1964), 0.1% peptone and 0.1% citric acid monohydrate. Samples were collected and assayed for b-mannanase activity at various times during the cultivation.
2.3. Enzyme production The fungus was cultivated in 1-liter shake-flasks on a medium containing 0.2% peptone, 2% locust bean gum, 4% Vogel’s medium N and 0.015% Tween 80; 100 ml of medium was inoculated with 5 ml of mycelia from a 3-day-old culture. After 7 days of growth at 28°C and 250 rpm, mycelia were removed and the culture fluid was filtered through a sterile 0.45-mm pore size membrane filter.
2.4. Acti6ity assays b-Mannanase activity was assayed as described by Sta˚lbrand et al. (1993) using locust bean gum galactomannan (Sigma G-0753) as substrate. aGalactosidase and b-mannosidase activities were assayed with p-nitrophenyl-a-D-galactopyranoside (Sigma N-0877) at pH 4.5 and p-nitrophenyl-b-D-mannopyranoside (Sigma N-1268) at pH 5.3, respectively, as described by Ra¨tto¨ and Poutanen (1988). All enzyme activities are expressed in SI units (katals).
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2.5. Protein assay Protein concentrations were measured by the Micro BCA Protein Assay (Pierce, Rockford, IL), using bovine serum albumin as standard. All chromatographic runs were monitored for protein by absorbance at 280 nm.
2.6. Gel electrophoresis and zymogram analysis Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were performed using the PhastSystem™ (Pharmacia Biotech, Uppsala, Sweden). Proteins were detected with silver staining as described in the Pharmacia PhastSystem™ instructions. Isoelectric focusing was carried out in the pH range 3–9 using gels of the type PhastGel® IEF 3-9 (Pharmacia). Marker proteins in the pI range 3.5–9.3 (Broad pI Kit, Pharmacia) were used as standards. SDS-PAGE was carried out on 12.5% polyacrylamide gels (PhastGel® Homogeneous 12.5, Pharmacia) using the Low Molecular Weight Calibration Kit (Pharmacia) as standard. b-Mannanase activity was detected by using an IEF zymogram technique with locust bean gum as substrate and staining with Congo red (Sta˚lbrand et al., 1993). a-Galactosidase activity was detected by using 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside (xagal, Boehringer Mannheim) as substrate. The zymogram was prepared as described by den Herder et al. (1992), with the exception that the incubation was made at pH 5.3 instead of 4.5. a-Galactosidase activity could be seen as light blue bands on the transparent gel.
2.7. Purification of the b-mannanase Culture filtrate (100 ml) was brought to 80% saturation by the addition of 56.1 g of solid ammonium sulfate. The precipitate was collected by centrifugation and then dissolved in 20 ml of citrate buffer, pH 5.3; 3 ml of this solution was desalted and equilibrated with 20 mM ammonium acetate buffer, pH 7.5, using a Fast Desalting Column (Pharmacia). The sample was then directly loaded on a Resource™ Q 1-ml anion-exchange column (Pharmacia) equilibrated with the
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same buffer. Proteins were eluted with a linear gradient (0–60%) of 1 M ammonium acetate, pH 7.5. Fractions of 0.25 ml were collected and assayed for b-mannanase, a-galactosidase and bmannosidase activities. The ten fractions most active in b-mannanase were pooled and further purified on a HiLoad™ 16/60 gel filtration column pre-packed with Superdex 200 prep grade (Pharmacia). Elution was achieved in 4 h using 200 mM NaCl in 100 mM phosphate buffer, pH 6.5. The flow rate was 0.5 ml min − 1. Fractions of 1 ml were collected and assayed for b-mannanase and a-galactosidase activities. Both ion-exchange chromatography and gel filtration were performed using the FPLC® System (Pharmacia).
2.8. pH optimum and stability The purified b-mannanase was incubated at 50°C at different pH values (pH 2.6–7: 50 mM citrate–phosphate buffer; pH 7.5–8: 50 mM phosphate buffer) at a concentration of 1.7 mg ml − 1. Bovine serum albumin (BSA, Sigma) was added at a concentration of 100 mg ml − 1. Samples were withdrawn at zero time and 24 h and assayed under standard conditions. The pH optimum was determined by using substrates with different pH values.
2.9. Temperature stability The purified b-mannanase was incubated (3.4 mg ml − 1) in citrate–phosphate buffer (50 mM, pH 5.3) including BSA (100 mg ml − 1) at different temperatures for 24 h. Following incubation, the remaining activities were measured as described above.
2.10. N-terminal sequence determination The N-terminal amino acid sequence of the purified b-mannanase was determined by automated Edman degradation, using an ABI 477A protein sequencer connected to an ABI 120A analyzer (Applied Biosystems, USA). The analysis was performed at the Biomolecular Resource Facility at Lund University.
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2.11. Adsorption studies The T. reesei mannanase (pI 5.4) was purified as described by Sta˚lbrand et al. (1993). Microcrystalline cellulose (Avicel, M 2331) was purchased from Merck (Darmstadt, Germany). The adsorption experiments were performed in 1.8-ml plastic tubes (Nunc, Denmark) at pH 5.3 in 50 mM sodium citrate buffer. The enzyme/substrate ratio was varied in the range 0.2 – 80 mmol g − 1 Avicel and the incubation was carried out at 4°C with continuous mixing by inversion of the tubes. After incubation for 60 min, the Avicel with bound enzyme was removed by filtration through a small syringe filter (Millex-GV4, pore size 0.22 mm; Millipore). The residual mannanase activity was measured and the adsorption was expressed as percent of the enzyme added. The time needed to achieve maximal adsorption at the given conditions was predetermined.
2.12. Hydrolysis of i6ory nut mannan Ivory nut (Phytelephas macrocarpa) mannan, an unbranched b-1,4-linked mannan polymer, was purchased from Megazyme, Australia. The polymer was suspended (2.5 g l − 1 in 20 mM sodium acetate buffer, pH 4.5) at 80°C with continuous stirring. After addition of the purified b-mannanase (2000 nkat g − 1 substrate), the solution was incubated at 40°C for 48 h with continuous mixing. Samples were removed at various times, heated at 100°C for 5 min and then stored at −20°C. The products formed during hydrolysis were analyzed on a Pharmacia HPLC system equipped with an Erma ERC-7515A refractive index detector (Erma, Tokyo, Japan); 20-ml samples were applied on an Aminex HPX-87H column (Bio-Rad, Richmond, CA). Elution was carried out at 65°C with 5 mM H2SO4 at a flow rate of 0.6 ml min − 1. Mannobiose and mannotriose (Megazyme, Australia) were used as standards.
concentration corresponding to a mannanase activity of 2000 nkat g − 1 substrate. The solution was then incubated at 40°C for 48 h with continuous mixing. Aliquots were removed at various times, heated at 100°C for 5 min, and then stored at − 20°C. The hydrolysis products were quantified by HPLC, using an Aminex HPX-87P column (Bio-Rad, Richmond, CA). Millipore water was used as eluant at 85°C at a flow rate of 0.6 ml min − 1. Mannose and galactose (Sigma) were used as standards.
2.14. Analysis of mannopentaose degradation by nuclear magnetic resonance (NMR) The hydrolysis reaction was carried out directly in the NMR tube. Mannopentaose was dissolved in 50 mM deuterated acetate buffer in 2H2O (pD 4.5) to a concentration of 3 mM. After accumulation of an initial spectrum, b-mannanase was added to the substrate solution in the NMR tube. The tube was quickly transferred to the spectrometer at 55°C and a new spectrum accumulation started as soon as possible. New spectra were thereafter started every 10 min. The 1H NMR spectra were recorded at 599.94 MHz using a Varian Unity 600 spectrometer. Each spectrum was the sum of 26 accumulations taken over 10 min. For details see Harjunpa¨a¨ et al. (1995).
2.15. Acid hydrolysis Acid hydrolysis of locust bean gum and ivory nut mannan was carried out. A volume of 5 ml of 0.25 M H2SO4 including 0.25% of the polymer was autoclaved for 2 h at 120°C. More H2SO4 was added to final concentration of 0.4 M and the solution was again heated for 2 h at 120°C. Samples of 100 ml were then analyzed by HPLC using Aminex HPX-87P and HPX-87H columns.
3. Results
2.13. Hydrolysis of locust bean gum 3.1. Screening of growth media Locust bean gum was prepared at a concentration of 0.25% (w/v) in 20 mM sodium acetate buffer, pH 4.5. Culture filtrate was added at a
b-Mannanase activity was assayed in the A. niger cultures containing either galactomannan,
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Fig. 1. Isoelectric focusing of the culture filtrate, silver stained (A) and activity stained for b-mannanase (B), and of the purified b-mannanase with silver staining (C). Isoelectric focusing with a-galactosidase activity staining of the a-galactosidase peak eluting adjacent to b-mannanase in ion-exchange chromatography (D), the culture filtrate (E) and the a-galactosidase peak eluting far ahead of b-mannanase in ion-exchange chromatography (F). Standards are marked at the sides. For experimental details see text.
cellulose or glucose, as described in Section 2. The growth was interrupted after 7 days when the b-mannanase production had reached a maximum in all flasks. A. niger produced bmannanase activity (32 – 56 nkat ml − 1) when grown on media containing locust bean gum galactomannan (results not shown). The highest activity (56 nkat ml − 1) was observed in the medium supplied with 2% locust bean gum. No b-mannanase activity could be detected when glucose and cellulose were used as sole carbon sources.
3.2. Characterization of the culture filtrate enzymes The following enzyme activities, necessary for complete hydrolysis of galactomannan, were detected in the culture filtrate of A. niger at the time for harvest: b-mannanase 90 nkat ml − 1, a-galactosidase 47 nkat ml − 1, b-mannosidase 8.1 nkat ml − 1. Isoelectric focusing and zymogram analysis of the culture filtrate showed several poorly separated a-galactosidase bands in the pI interval 4.2 – 6.6, with two major areas around pI 4.4 and 5.9 (Fig. 1E). A single clear single band of b-mannanase activity was detected at pI 3.7 (Fig. 1B).
3.3. Purification of the b-mannanase Ammonium sulfate precipitation was used mainly as a concentrating step before applying the sample to the Resource Q column; 97% of the b-mannanase activity was recovered in the precipitate. Analysis of the collected fractions from anion-exchange chromatography (Fig. 2) showed a single peak of b-mannanase activity. Two peaks of a-galactosidase activity, one eluting adjacent to the b-mannanase, and a single b-mannosidase peak were also resolved. Analysis of the b-mannanase fractions by SDS-PAGE (Fig. 3A) showed two major proteins, one of 40 kDa (b-mannanase) and one of 76 kDa. Further purification by gel filtration yielded a b-mannanase preparation that was pure as judged from IEF (Fig. 1C) and SDS-PAGE (Fig. 3B). The specific activity was determined to be 3860 nkat mg − 1 protein. No a-galactosidase activity could be detected in the mannanase peak after an assay time of 90 min. An overall recovery of 46% and a 46-fold purification of the b-mannanase were obtained. The purification is summarized in Table 1.
3.4. Enzyme properties The isoelectric point of the purified b-mannanase was 3.7 (Fig. 1C) and the apparent
90 436 350 103
15 3.0 2.5 6.0
Culture filtrate (NH4)2SO4 precipitation Ion-exchange chromatography Gel filtration
Activity (nkat ml−1)
Volume (ml)
Purification step
Table 1 Purification of b-mannanase from A. niger culture filtrate
618
1350 1308 875
Total activity (nkat)
0.16
16 6.7 0.70
3860
84 195 1250
Total protein (mg) Specific activity (nkat mg−1)
46
(100) 97 65
Yield (%)
46
(1) 2.3 15
Fold purification
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Fig. 2. Anion-exchange chromatography of the ammonium sulfate precipitated culture filtrate on a Resource™ Q 1-ml column. For experimental details see text. ——, absorbance at 280 nm; - - -, gradient; , b-mannanase ×0.1; , a-galactosidase; , b-mannosidase.
molecular weight 40 kDa (Fig. 3B). Zymogram analysis of the a-galactosidase peak eluting close to b-mannanase in ion-exchange chromatography showed a single band at pI 6.6 (Fig. 1D). The a-galactosidase peak eluting early in the chromatogram appeared as a broad, diffuse band at around pI 4.4 (Fig. 1F). Optimal pH for b-mannanase activity was 3.5 (Fig. 4). The enzyme proved to be stable in the pH range 3.5 – 7 (Fig. 4)
Fig. 3. SDS-PAGE with silver staining of the b-mannanase fractions after ion-exchange chromatography (A) and after gel filtration (B). Standards are marked at the sides. For experimental details see text.
and at temperatures of 50°C and below (data not shown). The N-terminal sequence of the b-mannanase is shown in Fig. 5 together with the gene deduced amino acid sequences of T. reesei (Sta˚lbrand et al., 1995) and A. aculeatus (Christgau et al., 1994).
Fig. 4. Effect of pH on b-mannanase activity () and stability (). The pH optimum, expressed in percent of maximum, was determined by measuring the activity under standard conditions using buffers of different pH. The pH stability was determined by incubating the purified enzyme at different pH values for 24 h at 50°C. The residual activity is shown as percent of the original activity.
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3.6. Hydrolysis experiments
Fig. 5. N-terminal amino acid sequence comparison of b-mannanases from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei. Identical amino acid residues are boxed. The A. niger b-mannanase sequence was determined by Edman degradation. The other two sequences are gene deduced. References: 1, Christgau et al. (1994); 2, Sta˚lbrand et al. (1995).
3.5. Adsorption on cellulose The adsorption of A. niger b-mannanase on cellulose was compared with that of a b-mannanase from T. reesei which has a cellulose binding domain (CBD) (Sta˚lbrand et al., 1995) (Fig. 6). The A. niger b-mannanase was unable to bind to cellulose at all the enzyme/substrate ratios tested. As expected, the T. reesei mannanase was readily adsorbed. About 80% of the enzyme was bound at a protein concentration of 80 mmol g − 1 Avicel, and it approached 100% bound when the concentration decreased below 10 mmol g − 1 Avicel. The amount of adsorbed enzyme reached a maximum after 30 min incubation; no further adsorption was detected even after 4 h (data not shown).
Fig. 6. Adsorption of Aspergillus niger and Trichoderma reesei b-mannanases on cellulose after 60 min of incubation at 4°C. The enzyme/substrate ratio was varied in the range 0.2–80 mmol g − 1 Avicel. , T. reesei b-mannanase; , A. niger b-mannanase.
Locust bean gum was incubated with H2SO4 as described in Section 2. An aliquot of 234 mg locust bean gum yielded 132 mg mannose and 32 mg galactose after acid hydrolysis. The mannose/ galactose ratio was determined as 4.1 on a molar basis, which is very close to previously reported values (Rol, 1973). Acid hydrolysis of 234 mg of ivory nut mannan yielded 194 mg of mannose. Incubation of locust bean gum with the culture filtrate yielded mainly mannose and galactose (Fig. 7A). The molar ratio of mannose/galactose produced by enzymatic hydrolysis for 48 h was 3.3 and the amount of released monomers 66% compared to the value obtained by total acid hydrolysis. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the purified b-mannanase for 48 h (Fig. 7B). The molar ratio of released mannobiose/mannotriose was 1.2. The hydrolysis of mannopentaose was analyzed by 1H NMR spectroscopy. The initial products were equal amounts of mannobiose and mannotriose (Fig. 8B), showing that the two central glycosidic bonds of the oligosaccharide are cleaved. Prolonged hydrolysis also results in the formation of mannose from the secondary hydrolysis of mannotriose. The progress curves in Fig. 8B show convincingly that the b-mannanase acts by the retaining mechanism (Sinnott, 1990) since there is an initial fast increase in the signal intensity from b-anomeric protons. The 1H NMR signal from the internal anomeric proton in mannotriose (shown in Fig. 8A) can be used to deduce the degradation pattern. If the hydrolysis occurs at the second glycosidic bond from the non-reducing end only, the mannotriose formed would have the equilibrium a/b ratio and the two resonances observed for the anomeric proton from the internal ring would have a time-independent ratio. This is clearly not the case. On the other hand, if only the third bond is hydrolyzed, the formed mannotriose would be completely in the b-
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Fig. 7. (a) HPLC analysis with the Aminex HPX-87P column of the products formed after hydrolysis of locust bean gum at 40°C for 48 h. Incubation of the substrate with culture filtrate (A) and without enzyme addition (B). Gal and Man indicate galactose and mannose, respectively. The large peak eluting at approximately 29 ml is acetate from the buffer. (B) HPLC analysis with the Aminex HPX-87H column of the products formed during hydrolysis of ivory nut mannan at 40°C for 48 h. Incubation of the substrate with purified b-mannanase (A) and without enzyme addition (B). M2 and M3 indicate mannobiose and mannotriose, respectively. The large peak eluting at approximately 18 ml is acetate from the buffer.
anomeric form with the a-form obtained through mutarotation. An equal probability for cleavage of the two central glycosidic bonds would result in the formation of 68% b and 32% a-mannotriose. It is clear that more of the b-form is formed than of the a-form; however, it is not straightforward to obtain this excess since the mutarotation can not be totally isolated from the hydrolysis. Model calculations, not shown, indicate a weak preference for cleavage of the third glycosidic bond over the second one.
4. Discussion The b-mannanase gene sequences of two fungal strains, A. aculeatus (Christgau et al., 1994) and T. reesei (Sta˚lbrand et al., 1995), have been published. The b-mannanase produced by A. aculeatus is 62 amino acids shorter and lacks the C-terminal CBD sequence present in the T. reesei enzyme. Alignment of the N-terminus of the purified A. niger b-mannanase with that of the A. aculeatus b-mannanase shows a high sequence
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identity (eight identical residues out of 12) (Fig. 5). The resemblance to the T. reesei mannanase is somewhat lower: five residues out of 12 are identical. The A. niger and A. aculeatus b-mannanases are both smaller than the one from T. reesei and
Fig. 8. (A) 1H NMR spectra of the region with the resonance from the anomeric proton from the internal ring in mannotriose. (B) Progress curves for the hydrolysis of mannopentaose by A. niger mannanase showing the intensities of various 1 H NMR resonances from the anomeric protons as a function of time. Conditions: 3 mM mannopentaose and 3.6 mM mannanase at 5°C. Symbols used in the figure are: , internal; , terminal; , reducing b; ", reducing a.
have very similar molecular weights (40000 and 45000, respectively). Consequently, judged from its size, it would not be surprising if the A. niger mannanase also lacks a CBD. This speculation is strongly supported by the adsorption data (Fig. 6). The T. reesei mannanase bound efficiently on cellulose whereas no binding could be observed for the A. niger mannanase. The strong adsorption of T. reesei mannanase on cellulose has previously been reported by Tenkanen et al. (1995). The addition of galactomannan (locust bean gum) to the culture media induced formation of b-mannanase, a-galactosidase and b-mannosidase, whereas no detectable b-mannanase activity was produced when the fungus was grown on cellulose or glucose as sole carbon source. It is worth mentioning that cellulose induces higher b-mannanase activity than galactomannan in T. reesei (Ra¨tto¨ and Poutanen, 1988, Arisan-Atac et al., 1993) and that the enzyme also contains a CBD (Sta˚lbrand et al., 1995). However, the A. niger b-mannanase in this study was not induced by cellulose, which is an interesting observation considering that it probably does not have a CBD. Purification of the A. niger mannanase was achieved using only three steps. The described method is very simple and yields a high recovery of mannanase activity (46%), suggesting that it might be suitable for upscaling trials. The specific activity using locust bean gum as substrate was 3860 nkat mg − 1 protein, which is six times the value obtained by McCleary (1988). The isoelectric point of the single b-mannanase detected was 3.7 and the molecular mass 40 kDa. In earlier investigations the pI has been determined to the following values: 3.2 (Tsujisaka et al., 1972), 4.0 (McCleary, 1979, 1988) and 4.1 (Yamazaki et al., 1976). The pI and the molecular mass for a bmannanase purified from a commercial enzyme preparation produced by a fungus from the Aspergillus niger-oryzae group have been determined to 3.95 (Ahlgren et al., 1967) and 42 kDa (Eriksson and Winell, 1968), respectively. The molecular weight has also been determined earlier to 45000 (McCleary, 1979, 1988). The A. niger b-mannanase has proved to be stable in the pH range 3–8 and below 70°C (McCleary, 1979,
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1988) and the pH optimum has been determined to 3.6 (Yamazaki et al., 1976) and 3.0 (McCleary, 1979). The pI and molecular weight data in the literature agree well with the results of the present investigation, and accordingly A. niger seems to secrete only one b-mannanase under the circumstances used. It can, however, not be ruled out that A. niger secretes additional b-mannanases which are present at very low levels or are induced if using other growth conditions. At least two enzymes with a-galactosidase activity, in the pI range 4.2–6.6, were detected in the A. niger culture filtrate. The production of multiple forms of a-galactosidase by A. niger is also confirmed by earlier investigations (Lee and Wacek, 1970, den Herder et al., 1992). The formation of mannobiose and mannotriose after hydrolysis of ivory nut mannan (Fig. 7B) indicates that the purified b-mannanase is an endoenzyme (Reese and Shibata, 1965). Similar results were obtained with b-mannanases purified from T. reesei (Sta˚lbrand et al., 1993), Aspergillus tamarii (Civas et al., 1984) and Aspergillus giganteus (Reese and Shibata, 1965). The ability of A. niger b-mannanase to attack manno-oligosaccharides with a degree of polymerization of three and higher was shown by Eriksson and Winell (1968). The purified mannanase partly hydrolyzed guar galactomannan to mannobiose and mannotriose, but showed no activity against mannobiose. Yamazaki et al. (1976) reported that the A. niger mannanase was able to degrade mannotriose and mannotetraose, but not mannobiose. Mannotriose is however hydrolyzed very slowly; a degree of polymerization of at least four is required for a significant hydrolysis rate (McCleary and Matheson, 1983). The results in Fig. 8 show that the A. niger b-mannanase has an activity towards mannopentaose that is very similar to that of the two major b-mannanases from T. reesei (Harjunpa¨a¨ et al., 1995). Like a T. reesei mannanase it acts according to the retaining mechanism and is therefore also able to perform transglycosylation. Furthermore, also like the T. reesei mannanase, there is no strong preference for hydrolysis of either the second or third glycosidic linkage. The transglycosylation capacity of A. niger b-mannanase was
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demonstrated by McCleary and Matheson (1983). A transient synthesis of higher oligosaccharides was detected during the early stages of reaction with mannopentaose and mannotetraose, and the final products after further hydrolysis were mainly mannobiose and mannotriose. Locust bean gum galactomannan was degraded to galactose and mannose when incubated with the culture filtrate enzymes (Fig. 7A). The complete enzymatic hydrolysis of this polymer requires the action of b-mannanase, a-galactosidase and b-mannosidase, which are all produced by A. niger. The detailed co-operative action of these enzymes on galactomannan and galactoglucomannan, however, remains to be investigated.
Acknowledgements This study was supported by the Swedish National Board for Industrial and Technical Development (NUTEK).
References Ahlgren, E., Eriksson, K.-E., Vesterberg, O., 1967. Characterization of cellulases and related enzymes by isoelectric focusing, gel filtration and zone electrophoresis. Acta Chem. Scand. 21, 937 – 944. Arisan-Atac, I., Hodits, R., Kristufek, D., Kubicek, C.P., 1993. Purification, and characterization of a b-mannanase of Trichoderma reesei C-30. Appl. Microbiol. Biotechnol. 39, 58 – 62. Barbesgaard, P., 1977. In: Smith, J.E., Pateman, J.A. (Eds.), Genetics and Physiology of Aspergillus (British Mycology Society Symposium Series No. 1). Academic Press, London, pp. 391 – 404. Bennett, J.W., 1985. In: Timberlake, W.E. (Ed.), Molecular Genetics of Filamentous Fungi. Alan R. Liss, New York, pp. 345 – 366. Christgau, S., Kauppinen, S., Vind, J., Kofod, L.V., Dalboge, H., 1994. Expression cloning, purification and characterization of a b-1,4-mannanase from Aspergillus aculeatus. Biochem. Mol. Biol. Int. 33, 917 – 925. Civas, A., Eberhard, R., Le Dizet, P., Petek, F., 1984. Glycosidases induced in Aspergillus tamarii. Secreted a-D-galactosidase and b-D-mannanase. Biochem. J. 857 – 863. Dekker, R.F.H., Richards, G.N., 1976. Hemicellulases: their occurrence, purification, properties, and mode of action. Adv. Carbohydr. Chem. Biochem. 32, 277 – 352.
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den Herder, I.F., Mateo Rosell, A.M., van Zuilen, C.M., Punt, P.J., van den Hondel, C.A.M.J.J., 1992. Cloning and expression of a member of the Aspergillus niger gene family encoding a-galactosidase. Mol. Gen. Genet. 233, 404–410. Eriksson, K.-E., Winell, M., 1968. Purification and characterization of a fungal b-mannanase. Acta Chem. Scand. 22, 1924 – 1934. Harjunpa¨a¨, V., Teleman, A., Drakenberg, T., 1995. Kinetic and stereochemical studies of manno-oligosaccharide hydrolysis catalysed by b-mannanases from Trichoderma reesei. Eur. J. Biochem. 234, 278–283. Lee, Y.C., Wacek, V., 1970. Galactosidases from Aspergillus niger. Arch. Biochem. Biophys. 138, 264–271. McCleary, B.V., 1979. Modes of action of b-mannanase enzymes of diverse origin on legume seed galactomannans. Phytochemistry 18, 757–763. McCleary, B.V., 1988. b-D-Mannanase. Methods Enzymol. 160, 596 – 610. McCleary, B.V., Matheson, N.K., 1983. Action patterns and substrate-binding requirements of b-D-mannanase with mannosaccharides and mannan-type polysaccharides. Carbohydr. Res. 119, 191–219. Ra¨tto¨, M., Poutanen, K., 1988. Production of mannan-degrading enzymes. Biotechnol. Lett. 10 (9), 661–664. Reese, E.T., Shibata, Y., 1965. b-Mannanases of fungi. Can. J. Microbiol. 11, 167 –183. Rol, F., 1973. Locust bean gum. In: Whistler, R.L. (Ed.), Industrial Gums, 2nd ed. Academic Press, New York, pp. 323 – 337. Sinnott, M.L., 1990. Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171–1202. Sta˚lbrand, H., 1995. Hemicellulose-degrading enzymes from fungi. Characterization of b-mannanase and the man1 gene of Trichoderma reesei. Doctoral Thesis, Lund University.
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Sta˚lbrand, H., Siika-aho, M., Tenkanen, M., Viikari, L., 1993. Purification and characterization of two b-mannanases from Trichoderma reesei. J. Biotechnol. 29, 229 – 242. Sta˚lbrand, H., Saloheimo, A., Vehmaanpera¨, J., Henrissat, B., Penttila¨, M., 1995. Cloning and expression in Saccharomyces cere6isiae of a Trichoderma reesei b-mannanase gene containing a cellulose binding domain. Appl. Env. Microbiol. 61, 1090 – 1097. Tenkanen, M., Buchert, J., Viikari, L., 1995. Binding of hemicellulases on isolated polysaccharide substrates. Enzyme Microb. Technol. 17, 499 – 505. Tenkanen, M., Makkonen, M., Perttula, M., Viikari, L., Teleman, A., 1997. Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp. J. Biotechnol. 57, 191 – 204. Torrie, J.P., Senior, D.J., Saddler, J.N., 1990. Production of b-mannanase by Trichoderma harzianum E58. Appl. Microbiol. Biotechnol. 34, 303 – 307. Tsujisaka, Y., Hiyama, K., Takenishi, S., Fukumoto, J., 1972. Studies on the hemicellulases part III. Purification and some properties of mannanase from Aspergillus niger van Tieghem sp. Nippon Nogei Kagaku Kaishi 46, 155 – 161. Viikari, L., Tenkanen, M., Buchert, J., Ra¨tto¨, M., Bailey, M., Siika-Aho, M., Linko, M., 1993. Hemicellulases for industrial applications. In: Saddler, J.N. (Ed.), Biotechnology in Agriculture, No. 9. Bioconversion of Forest and Agricultural Plant Residues. C.A.B. International, Wallingford, pp. 131 – 182. Viikari, L., Kantelinen, A., Sundquist, J., Linko, M., 1994. Xylanases in bleaching: from an idea to the industry. FEMS Microbiol. Rev. 13, 335 – 350. Vogel, H.J., 1964. Distribution of lysine pathways among fungi: evolutionary implications. Am. Nat. 98, 435 – 446. Yamazaki, N., Sinner, M., Dietrichs, H.H., 1976. Isolierung und eigenschaften einer b-1,4-Mannanase aus Aspergillus niger. Holzforschung 30, 101 – 109.
Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a b-mannanase Pia Ademark a, Arthur Varga a, Jo´zsef Medve a, Vesa Harjunpa¨a¨ b, Torbjo¨rn Drakenberg b, Folke Tjerneld a, Henrik Sta˚lbrand a,* a
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, PO Box 124, S-22100 Lund, Sweden b VTT Chemical Technology, FIN-02044 VTT (Espoo), Finland Received 19 December 1997; received in revised form 5 June 1998; accepted 12 June 1998
Abstract The enzymes needed for galactomannan hydrolysis, i.e. b-mannanase, a-galactosidase and b-mannosidase, were produced by the filamentous fungus Aspergillus niger. The b-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the b-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger b-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus b-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Aspergillus niger; b-Mannanase; a-Galactosidase; Hemicellulase
1. Introduction Hemicelluloses are complex polysaccharides which are abundant in higher plant cell walls. Galactoglucomannan, the major softwood hemi* Corresponding author. Fax: +46 46 2224534; e-mail: [email protected]
cellulose, contains b-1,4-linked D-mannopyranose and D-glucopyranose units. The residues in the main chain are partially substituted by a-1,6linked D-galactosyl side groups. The complete enzymatic degradation of hemicelluloses involves several specific activities. Endo-1,4-b-D-mannanase (EC 3.2.1.78) catalyzes the random cleavage of b-D-1,4-mannopyra-
0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00086-8
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nosyl linkages within the main chain of galactomannan, glucomannan, galactoglucomannan and mannan. Mannanases from a variety of different organisms have been studied, including bacteria, fungi, higher plants and animals (reviewed by Dekker and Richards, 1976 and Viikari et al., 1993). The interest in b-mannanase and other hemicellulose-degrading enzymes has recently increased, partly because of their potential applicability in the food and paper and pulp industries (Viikari et al., 1993, 1994). The degradation of galactomannan and galactoglucomannan by b-mannanase is greatly affected by the extent and pattern of substitution of the mannan backbone. The interference of Dgalactosyl side groups with hydrolysis has been carefully analyzed using b-mannanases from A. niger (McCleary and Matheson, 1983) and Trichoderma reesei (Tenkanen et al., 1997). The complete conversion of galactomannan into D-galactose and D-mannose requires the presence of two additional enzymes, a-galactosidase (EC 3.2.1.22) and b-mannosidase (EC 3.2.1.25). These enzymes catalyze the cleavage of terminal a-1,6linked D-galactosyl and b-1,4-linked D-mannopyranosyl residues, respectively. A considerable variation of the ability to hydrolyze galactosyl side groups from polymeric substrates exists among the a-galactosidases. A. niger, a filamentous fungus, is one of the most used organisms in the industrial production of fermented foods, organic acids, and enzymes (Barbesgaard, 1977, Bennett, 1985). The purification of b-mannanase from A. niger has been described earlier (Eriksson and Winell, 1968, Tsujisaka et al., 1972, Yamazaki et al., 1976, McCleary, 1979, 1988), but it has not been clear if more than one b-mannanase is secreted. A multiplicity of extracellular b-mannanases appears to be common among fungi and has been noticed, for example, in T. reesei (Sta˚lbrand et al., 1993, Sta˚lbrand, 1995) and Trichoderma harzianum (Torrie et al., 1990). In the present study the aim was to separate analytically the major galactoglucomannan-degrading enzymes from A. niger and to purify and characterize biochemically the bmannanase.
2. Materials and methods
2.1. Culture A. niger ATCC-46890 was obtained from the QM Culture Collection, Department of Botany, University of Massachusetts, Amherst. The culture was maintained on potato dextrose agar slants.
2.2. Screening of growth media The effect of different culture media on b-mannanase production was investigated. A. niger ATCC-46890 was grown at 28°C in shake-flasks containing one of the following carbon sources: 0.5–2% (w/v) locust bean gum galactomannan (Sigma), 0.5% Solka Floc cellulose, or 1% glucose. All media were supplemented with 4% Vogel’s medium N (Vogel, 1964), 0.1% peptone and 0.1% citric acid monohydrate. Samples were collected and assayed for b-mannanase activity at various times during the cultivation.
2.3. Enzyme production The fungus was cultivated in 1-liter shake-flasks on a medium containing 0.2% peptone, 2% locust bean gum, 4% Vogel’s medium N and 0.015% Tween 80; 100 ml of medium was inoculated with 5 ml of mycelia from a 3-day-old culture. After 7 days of growth at 28°C and 250 rpm, mycelia were removed and the culture fluid was filtered through a sterile 0.45-mm pore size membrane filter.
2.4. Acti6ity assays b-Mannanase activity was assayed as described by Sta˚lbrand et al. (1993) using locust bean gum galactomannan (Sigma G-0753) as substrate. aGalactosidase and b-mannosidase activities were assayed with p-nitrophenyl-a-D-galactopyranoside (Sigma N-0877) at pH 4.5 and p-nitrophenyl-b-D-mannopyranoside (Sigma N-1268) at pH 5.3, respectively, as described by Ra¨tto¨ and Poutanen (1988). All enzyme activities are expressed in SI units (katals).
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2.5. Protein assay Protein concentrations were measured by the Micro BCA Protein Assay (Pierce, Rockford, IL), using bovine serum albumin as standard. All chromatographic runs were monitored for protein by absorbance at 280 nm.
2.6. Gel electrophoresis and zymogram analysis Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were performed using the PhastSystem™ (Pharmacia Biotech, Uppsala, Sweden). Proteins were detected with silver staining as described in the Pharmacia PhastSystem™ instructions. Isoelectric focusing was carried out in the pH range 3–9 using gels of the type PhastGel® IEF 3-9 (Pharmacia). Marker proteins in the pI range 3.5–9.3 (Broad pI Kit, Pharmacia) were used as standards. SDS-PAGE was carried out on 12.5% polyacrylamide gels (PhastGel® Homogeneous 12.5, Pharmacia) using the Low Molecular Weight Calibration Kit (Pharmacia) as standard. b-Mannanase activity was detected by using an IEF zymogram technique with locust bean gum as substrate and staining with Congo red (Sta˚lbrand et al., 1993). a-Galactosidase activity was detected by using 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside (xagal, Boehringer Mannheim) as substrate. The zymogram was prepared as described by den Herder et al. (1992), with the exception that the incubation was made at pH 5.3 instead of 4.5. a-Galactosidase activity could be seen as light blue bands on the transparent gel.
2.7. Purification of the b-mannanase Culture filtrate (100 ml) was brought to 80% saturation by the addition of 56.1 g of solid ammonium sulfate. The precipitate was collected by centrifugation and then dissolved in 20 ml of citrate buffer, pH 5.3; 3 ml of this solution was desalted and equilibrated with 20 mM ammonium acetate buffer, pH 7.5, using a Fast Desalting Column (Pharmacia). The sample was then directly loaded on a Resource™ Q 1-ml anion-exchange column (Pharmacia) equilibrated with the
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same buffer. Proteins were eluted with a linear gradient (0–60%) of 1 M ammonium acetate, pH 7.5. Fractions of 0.25 ml were collected and assayed for b-mannanase, a-galactosidase and bmannosidase activities. The ten fractions most active in b-mannanase were pooled and further purified on a HiLoad™ 16/60 gel filtration column pre-packed with Superdex 200 prep grade (Pharmacia). Elution was achieved in 4 h using 200 mM NaCl in 100 mM phosphate buffer, pH 6.5. The flow rate was 0.5 ml min − 1. Fractions of 1 ml were collected and assayed for b-mannanase and a-galactosidase activities. Both ion-exchange chromatography and gel filtration were performed using the FPLC® System (Pharmacia).
2.8. pH optimum and stability The purified b-mannanase was incubated at 50°C at different pH values (pH 2.6–7: 50 mM citrate–phosphate buffer; pH 7.5–8: 50 mM phosphate buffer) at a concentration of 1.7 mg ml − 1. Bovine serum albumin (BSA, Sigma) was added at a concentration of 100 mg ml − 1. Samples were withdrawn at zero time and 24 h and assayed under standard conditions. The pH optimum was determined by using substrates with different pH values.
2.9. Temperature stability The purified b-mannanase was incubated (3.4 mg ml − 1) in citrate–phosphate buffer (50 mM, pH 5.3) including BSA (100 mg ml − 1) at different temperatures for 24 h. Following incubation, the remaining activities were measured as described above.
2.10. N-terminal sequence determination The N-terminal amino acid sequence of the purified b-mannanase was determined by automated Edman degradation, using an ABI 477A protein sequencer connected to an ABI 120A analyzer (Applied Biosystems, USA). The analysis was performed at the Biomolecular Resource Facility at Lund University.
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2.11. Adsorption studies The T. reesei mannanase (pI 5.4) was purified as described by Sta˚lbrand et al. (1993). Microcrystalline cellulose (Avicel, M 2331) was purchased from Merck (Darmstadt, Germany). The adsorption experiments were performed in 1.8-ml plastic tubes (Nunc, Denmark) at pH 5.3 in 50 mM sodium citrate buffer. The enzyme/substrate ratio was varied in the range 0.2 – 80 mmol g − 1 Avicel and the incubation was carried out at 4°C with continuous mixing by inversion of the tubes. After incubation for 60 min, the Avicel with bound enzyme was removed by filtration through a small syringe filter (Millex-GV4, pore size 0.22 mm; Millipore). The residual mannanase activity was measured and the adsorption was expressed as percent of the enzyme added. The time needed to achieve maximal adsorption at the given conditions was predetermined.
2.12. Hydrolysis of i6ory nut mannan Ivory nut (Phytelephas macrocarpa) mannan, an unbranched b-1,4-linked mannan polymer, was purchased from Megazyme, Australia. The polymer was suspended (2.5 g l − 1 in 20 mM sodium acetate buffer, pH 4.5) at 80°C with continuous stirring. After addition of the purified b-mannanase (2000 nkat g − 1 substrate), the solution was incubated at 40°C for 48 h with continuous mixing. Samples were removed at various times, heated at 100°C for 5 min and then stored at −20°C. The products formed during hydrolysis were analyzed on a Pharmacia HPLC system equipped with an Erma ERC-7515A refractive index detector (Erma, Tokyo, Japan); 20-ml samples were applied on an Aminex HPX-87H column (Bio-Rad, Richmond, CA). Elution was carried out at 65°C with 5 mM H2SO4 at a flow rate of 0.6 ml min − 1. Mannobiose and mannotriose (Megazyme, Australia) were used as standards.
concentration corresponding to a mannanase activity of 2000 nkat g − 1 substrate. The solution was then incubated at 40°C for 48 h with continuous mixing. Aliquots were removed at various times, heated at 100°C for 5 min, and then stored at − 20°C. The hydrolysis products were quantified by HPLC, using an Aminex HPX-87P column (Bio-Rad, Richmond, CA). Millipore water was used as eluant at 85°C at a flow rate of 0.6 ml min − 1. Mannose and galactose (Sigma) were used as standards.
2.14. Analysis of mannopentaose degradation by nuclear magnetic resonance (NMR) The hydrolysis reaction was carried out directly in the NMR tube. Mannopentaose was dissolved in 50 mM deuterated acetate buffer in 2H2O (pD 4.5) to a concentration of 3 mM. After accumulation of an initial spectrum, b-mannanase was added to the substrate solution in the NMR tube. The tube was quickly transferred to the spectrometer at 55°C and a new spectrum accumulation started as soon as possible. New spectra were thereafter started every 10 min. The 1H NMR spectra were recorded at 599.94 MHz using a Varian Unity 600 spectrometer. Each spectrum was the sum of 26 accumulations taken over 10 min. For details see Harjunpa¨a¨ et al. (1995).
2.15. Acid hydrolysis Acid hydrolysis of locust bean gum and ivory nut mannan was carried out. A volume of 5 ml of 0.25 M H2SO4 including 0.25% of the polymer was autoclaved for 2 h at 120°C. More H2SO4 was added to final concentration of 0.4 M and the solution was again heated for 2 h at 120°C. Samples of 100 ml were then analyzed by HPLC using Aminex HPX-87P and HPX-87H columns.
3. Results
2.13. Hydrolysis of locust bean gum 3.1. Screening of growth media Locust bean gum was prepared at a concentration of 0.25% (w/v) in 20 mM sodium acetate buffer, pH 4.5. Culture filtrate was added at a
b-Mannanase activity was assayed in the A. niger cultures containing either galactomannan,
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Fig. 1. Isoelectric focusing of the culture filtrate, silver stained (A) and activity stained for b-mannanase (B), and of the purified b-mannanase with silver staining (C). Isoelectric focusing with a-galactosidase activity staining of the a-galactosidase peak eluting adjacent to b-mannanase in ion-exchange chromatography (D), the culture filtrate (E) and the a-galactosidase peak eluting far ahead of b-mannanase in ion-exchange chromatography (F). Standards are marked at the sides. For experimental details see text.
cellulose or glucose, as described in Section 2. The growth was interrupted after 7 days when the b-mannanase production had reached a maximum in all flasks. A. niger produced bmannanase activity (32 – 56 nkat ml − 1) when grown on media containing locust bean gum galactomannan (results not shown). The highest activity (56 nkat ml − 1) was observed in the medium supplied with 2% locust bean gum. No b-mannanase activity could be detected when glucose and cellulose were used as sole carbon sources.
3.2. Characterization of the culture filtrate enzymes The following enzyme activities, necessary for complete hydrolysis of galactomannan, were detected in the culture filtrate of A. niger at the time for harvest: b-mannanase 90 nkat ml − 1, a-galactosidase 47 nkat ml − 1, b-mannosidase 8.1 nkat ml − 1. Isoelectric focusing and zymogram analysis of the culture filtrate showed several poorly separated a-galactosidase bands in the pI interval 4.2 – 6.6, with two major areas around pI 4.4 and 5.9 (Fig. 1E). A single clear single band of b-mannanase activity was detected at pI 3.7 (Fig. 1B).
3.3. Purification of the b-mannanase Ammonium sulfate precipitation was used mainly as a concentrating step before applying the sample to the Resource Q column; 97% of the b-mannanase activity was recovered in the precipitate. Analysis of the collected fractions from anion-exchange chromatography (Fig. 2) showed a single peak of b-mannanase activity. Two peaks of a-galactosidase activity, one eluting adjacent to the b-mannanase, and a single b-mannosidase peak were also resolved. Analysis of the b-mannanase fractions by SDS-PAGE (Fig. 3A) showed two major proteins, one of 40 kDa (b-mannanase) and one of 76 kDa. Further purification by gel filtration yielded a b-mannanase preparation that was pure as judged from IEF (Fig. 1C) and SDS-PAGE (Fig. 3B). The specific activity was determined to be 3860 nkat mg − 1 protein. No a-galactosidase activity could be detected in the mannanase peak after an assay time of 90 min. An overall recovery of 46% and a 46-fold purification of the b-mannanase were obtained. The purification is summarized in Table 1.
3.4. Enzyme properties The isoelectric point of the purified b-mannanase was 3.7 (Fig. 1C) and the apparent
90 436 350 103
15 3.0 2.5 6.0
Culture filtrate (NH4)2SO4 precipitation Ion-exchange chromatography Gel filtration
Activity (nkat ml−1)
Volume (ml)
Purification step
Table 1 Purification of b-mannanase from A. niger culture filtrate
618
1350 1308 875
Total activity (nkat)
0.16
16 6.7 0.70
3860
84 195 1250
Total protein (mg) Specific activity (nkat mg−1)
46
(100) 97 65
Yield (%)
46
(1) 2.3 15
Fold purification
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Fig. 2. Anion-exchange chromatography of the ammonium sulfate precipitated culture filtrate on a Resource™ Q 1-ml column. For experimental details see text. ——, absorbance at 280 nm; - - -, gradient; , b-mannanase ×0.1; , a-galactosidase; , b-mannosidase.
molecular weight 40 kDa (Fig. 3B). Zymogram analysis of the a-galactosidase peak eluting close to b-mannanase in ion-exchange chromatography showed a single band at pI 6.6 (Fig. 1D). The a-galactosidase peak eluting early in the chromatogram appeared as a broad, diffuse band at around pI 4.4 (Fig. 1F). Optimal pH for b-mannanase activity was 3.5 (Fig. 4). The enzyme proved to be stable in the pH range 3.5 – 7 (Fig. 4)
Fig. 3. SDS-PAGE with silver staining of the b-mannanase fractions after ion-exchange chromatography (A) and after gel filtration (B). Standards are marked at the sides. For experimental details see text.
and at temperatures of 50°C and below (data not shown). The N-terminal sequence of the b-mannanase is shown in Fig. 5 together with the gene deduced amino acid sequences of T. reesei (Sta˚lbrand et al., 1995) and A. aculeatus (Christgau et al., 1994).
Fig. 4. Effect of pH on b-mannanase activity () and stability (). The pH optimum, expressed in percent of maximum, was determined by measuring the activity under standard conditions using buffers of different pH. The pH stability was determined by incubating the purified enzyme at different pH values for 24 h at 50°C. The residual activity is shown as percent of the original activity.
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3.6. Hydrolysis experiments
Fig. 5. N-terminal amino acid sequence comparison of b-mannanases from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei. Identical amino acid residues are boxed. The A. niger b-mannanase sequence was determined by Edman degradation. The other two sequences are gene deduced. References: 1, Christgau et al. (1994); 2, Sta˚lbrand et al. (1995).
3.5. Adsorption on cellulose The adsorption of A. niger b-mannanase on cellulose was compared with that of a b-mannanase from T. reesei which has a cellulose binding domain (CBD) (Sta˚lbrand et al., 1995) (Fig. 6). The A. niger b-mannanase was unable to bind to cellulose at all the enzyme/substrate ratios tested. As expected, the T. reesei mannanase was readily adsorbed. About 80% of the enzyme was bound at a protein concentration of 80 mmol g − 1 Avicel, and it approached 100% bound when the concentration decreased below 10 mmol g − 1 Avicel. The amount of adsorbed enzyme reached a maximum after 30 min incubation; no further adsorption was detected even after 4 h (data not shown).
Fig. 6. Adsorption of Aspergillus niger and Trichoderma reesei b-mannanases on cellulose after 60 min of incubation at 4°C. The enzyme/substrate ratio was varied in the range 0.2–80 mmol g − 1 Avicel. , T. reesei b-mannanase; , A. niger b-mannanase.
Locust bean gum was incubated with H2SO4 as described in Section 2. An aliquot of 234 mg locust bean gum yielded 132 mg mannose and 32 mg galactose after acid hydrolysis. The mannose/ galactose ratio was determined as 4.1 on a molar basis, which is very close to previously reported values (Rol, 1973). Acid hydrolysis of 234 mg of ivory nut mannan yielded 194 mg of mannose. Incubation of locust bean gum with the culture filtrate yielded mainly mannose and galactose (Fig. 7A). The molar ratio of mannose/galactose produced by enzymatic hydrolysis for 48 h was 3.3 and the amount of released monomers 66% compared to the value obtained by total acid hydrolysis. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the purified b-mannanase for 48 h (Fig. 7B). The molar ratio of released mannobiose/mannotriose was 1.2. The hydrolysis of mannopentaose was analyzed by 1H NMR spectroscopy. The initial products were equal amounts of mannobiose and mannotriose (Fig. 8B), showing that the two central glycosidic bonds of the oligosaccharide are cleaved. Prolonged hydrolysis also results in the formation of mannose from the secondary hydrolysis of mannotriose. The progress curves in Fig. 8B show convincingly that the b-mannanase acts by the retaining mechanism (Sinnott, 1990) since there is an initial fast increase in the signal intensity from b-anomeric protons. The 1H NMR signal from the internal anomeric proton in mannotriose (shown in Fig. 8A) can be used to deduce the degradation pattern. If the hydrolysis occurs at the second glycosidic bond from the non-reducing end only, the mannotriose formed would have the equilibrium a/b ratio and the two resonances observed for the anomeric proton from the internal ring would have a time-independent ratio. This is clearly not the case. On the other hand, if only the third bond is hydrolyzed, the formed mannotriose would be completely in the b-
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Fig. 7. (a) HPLC analysis with the Aminex HPX-87P column of the products formed after hydrolysis of locust bean gum at 40°C for 48 h. Incubation of the substrate with culture filtrate (A) and without enzyme addition (B). Gal and Man indicate galactose and mannose, respectively. The large peak eluting at approximately 29 ml is acetate from the buffer. (B) HPLC analysis with the Aminex HPX-87H column of the products formed during hydrolysis of ivory nut mannan at 40°C for 48 h. Incubation of the substrate with purified b-mannanase (A) and without enzyme addition (B). M2 and M3 indicate mannobiose and mannotriose, respectively. The large peak eluting at approximately 18 ml is acetate from the buffer.
anomeric form with the a-form obtained through mutarotation. An equal probability for cleavage of the two central glycosidic bonds would result in the formation of 68% b and 32% a-mannotriose. It is clear that more of the b-form is formed than of the a-form; however, it is not straightforward to obtain this excess since the mutarotation can not be totally isolated from the hydrolysis. Model calculations, not shown, indicate a weak preference for cleavage of the third glycosidic bond over the second one.
4. Discussion The b-mannanase gene sequences of two fungal strains, A. aculeatus (Christgau et al., 1994) and T. reesei (Sta˚lbrand et al., 1995), have been published. The b-mannanase produced by A. aculeatus is 62 amino acids shorter and lacks the C-terminal CBD sequence present in the T. reesei enzyme. Alignment of the N-terminus of the purified A. niger b-mannanase with that of the A. aculeatus b-mannanase shows a high sequence
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identity (eight identical residues out of 12) (Fig. 5). The resemblance to the T. reesei mannanase is somewhat lower: five residues out of 12 are identical. The A. niger and A. aculeatus b-mannanases are both smaller than the one from T. reesei and
Fig. 8. (A) 1H NMR spectra of the region with the resonance from the anomeric proton from the internal ring in mannotriose. (B) Progress curves for the hydrolysis of mannopentaose by A. niger mannanase showing the intensities of various 1 H NMR resonances from the anomeric protons as a function of time. Conditions: 3 mM mannopentaose and 3.6 mM mannanase at 5°C. Symbols used in the figure are: , internal; , terminal; , reducing b; ", reducing a.
have very similar molecular weights (40000 and 45000, respectively). Consequently, judged from its size, it would not be surprising if the A. niger mannanase also lacks a CBD. This speculation is strongly supported by the adsorption data (Fig. 6). The T. reesei mannanase bound efficiently on cellulose whereas no binding could be observed for the A. niger mannanase. The strong adsorption of T. reesei mannanase on cellulose has previously been reported by Tenkanen et al. (1995). The addition of galactomannan (locust bean gum) to the culture media induced formation of b-mannanase, a-galactosidase and b-mannosidase, whereas no detectable b-mannanase activity was produced when the fungus was grown on cellulose or glucose as sole carbon source. It is worth mentioning that cellulose induces higher b-mannanase activity than galactomannan in T. reesei (Ra¨tto¨ and Poutanen, 1988, Arisan-Atac et al., 1993) and that the enzyme also contains a CBD (Sta˚lbrand et al., 1995). However, the A. niger b-mannanase in this study was not induced by cellulose, which is an interesting observation considering that it probably does not have a CBD. Purification of the A. niger mannanase was achieved using only three steps. The described method is very simple and yields a high recovery of mannanase activity (46%), suggesting that it might be suitable for upscaling trials. The specific activity using locust bean gum as substrate was 3860 nkat mg − 1 protein, which is six times the value obtained by McCleary (1988). The isoelectric point of the single b-mannanase detected was 3.7 and the molecular mass 40 kDa. In earlier investigations the pI has been determined to the following values: 3.2 (Tsujisaka et al., 1972), 4.0 (McCleary, 1979, 1988) and 4.1 (Yamazaki et al., 1976). The pI and the molecular mass for a bmannanase purified from a commercial enzyme preparation produced by a fungus from the Aspergillus niger-oryzae group have been determined to 3.95 (Ahlgren et al., 1967) and 42 kDa (Eriksson and Winell, 1968), respectively. The molecular weight has also been determined earlier to 45000 (McCleary, 1979, 1988). The A. niger b-mannanase has proved to be stable in the pH range 3–8 and below 70°C (McCleary, 1979,
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1988) and the pH optimum has been determined to 3.6 (Yamazaki et al., 1976) and 3.0 (McCleary, 1979). The pI and molecular weight data in the literature agree well with the results of the present investigation, and accordingly A. niger seems to secrete only one b-mannanase under the circumstances used. It can, however, not be ruled out that A. niger secretes additional b-mannanases which are present at very low levels or are induced if using other growth conditions. At least two enzymes with a-galactosidase activity, in the pI range 4.2–6.6, were detected in the A. niger culture filtrate. The production of multiple forms of a-galactosidase by A. niger is also confirmed by earlier investigations (Lee and Wacek, 1970, den Herder et al., 1992). The formation of mannobiose and mannotriose after hydrolysis of ivory nut mannan (Fig. 7B) indicates that the purified b-mannanase is an endoenzyme (Reese and Shibata, 1965). Similar results were obtained with b-mannanases purified from T. reesei (Sta˚lbrand et al., 1993), Aspergillus tamarii (Civas et al., 1984) and Aspergillus giganteus (Reese and Shibata, 1965). The ability of A. niger b-mannanase to attack manno-oligosaccharides with a degree of polymerization of three and higher was shown by Eriksson and Winell (1968). The purified mannanase partly hydrolyzed guar galactomannan to mannobiose and mannotriose, but showed no activity against mannobiose. Yamazaki et al. (1976) reported that the A. niger mannanase was able to degrade mannotriose and mannotetraose, but not mannobiose. Mannotriose is however hydrolyzed very slowly; a degree of polymerization of at least four is required for a significant hydrolysis rate (McCleary and Matheson, 1983). The results in Fig. 8 show that the A. niger b-mannanase has an activity towards mannopentaose that is very similar to that of the two major b-mannanases from T. reesei (Harjunpa¨a¨ et al., 1995). Like a T. reesei mannanase it acts according to the retaining mechanism and is therefore also able to perform transglycosylation. Furthermore, also like the T. reesei mannanase, there is no strong preference for hydrolysis of either the second or third glycosidic linkage. The transglycosylation capacity of A. niger b-mannanase was
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demonstrated by McCleary and Matheson (1983). A transient synthesis of higher oligosaccharides was detected during the early stages of reaction with mannopentaose and mannotetraose, and the final products after further hydrolysis were mainly mannobiose and mannotriose. Locust bean gum galactomannan was degraded to galactose and mannose when incubated with the culture filtrate enzymes (Fig. 7A). The complete enzymatic hydrolysis of this polymer requires the action of b-mannanase, a-galactosidase and b-mannosidase, which are all produced by A. niger. The detailed co-operative action of these enzymes on galactomannan and galactoglucomannan, however, remains to be investigated.
Acknowledgements This study was supported by the Swedish National Board for Industrial and Technical Development (NUTEK).
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