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Purification and regulation of the synthesis of a β-xylosidase from Aspergillus nidulans
FEMS Microbiology
Letters 13.5 (1996) 287-293
Purification and regulation of the synthesis of a P_xylosidase from Aspergillus nidulans Sudeep Kumar, Daniel Ran-h Departmemo
*
de Biotecnologpiude Alimentos, Institute de Agroquimica y Tecnologia de Alimentos, Consejo Superior de Irwestigaciones Cientifcas, Apartado de Correos 73, 46100 Burjassot, Valencia, Spain Received 3 November
1995; revised 17 November
1995; accepted
17 November
1995
Abstract PXylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50°C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-P-D-xylopyranoside as substrate, the K, and V,,,,, values have been found to be 1.l mM and 25.6 wrnol min- ’ (mg protein)- ‘, respectivdy. Enzyme activity was inhibited by Hg’+, Ag2+, and CL?+ at a concentration of 1 X lop3 M. The synthesis of /3-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the creA gene product. Keywrds:
Aspergillus nidulans; P-Xylosidase;
Purification:
Carbon catabolite
1. Introduction Xylan is the major constituent of wood and agricultural residues. In nature, the complete degradation of xylan requires the synergistic action of several enzymes, mainly endo /3- l+xylanases (EC 3.2.1.8) which cleave the p-1,4 glycosidic bond between two different xylose residues to produce xylooligosaccharides, and Pxylosidase (EC 3.2.1.37) which cleaves xylooligosaccharides to yield xylose [l]. The hydrolysis of xylan is of great interest for various biotechnological applications [2]. Filamentous fungi are well known producers of xylan degrading enzymes. Several species of the
* Corresponding author. Tel: + 34 (6) 390 0022; Fax: + 34 (6) 363 6301: E-mail: dramon_iata.csic.es. 0378-1097/96/$12.00 0 1996 Federation SSDI 0378-1097(95)00468-E
of European
Microbiological
repression:
CREA
genus Aspergillus are efficient producers of such enzymes and of these A. nidulans has become a model system for studying the mechanisms of control of gene expression in filamentous fungi [3]. Recently the xylanolytic complex of A. nidtlilans has been identified [4]. In the presence of xylan as sole carbon source A. nidulans secretes at least three endo-l&3-xylanases with molecular masas of 22, 24, and 34 kDa, named respectively X,, , X 24 and X,. The three proteins have been purified and characterised [5-71. X,, is a nc4&al xylanrtse while the other two xylanases = acidic pnMcins. Thd synthesis of the xylanolytic complex of A. n&lans is induced by xylan and xylooIigosaccharitW such as xylobiose, xylotriose and xylotetraose and tiepressed by glucose. This carbon catabolite repressi~ is apparently mediated by the CREA protein [8] and only affects the X,, and X,, xylanases [9]. Societies.
All rights reserved
288
S. Kumar, D. Ram& / FEh4S Microbiology
Previously, an extracellular /I-xylosidase was purified from the culture supematants of a natural isolate of Emericellu niduluns [lo]. In this paper we report the purification and biochemical characterization of a mycelial bound P-xylosidase from A. niduluns exhibiting different properties to the enzyme isolated from E. niduluns as well as the induction and carbon catabolite repression of the synthesis of the A. niduluns enzyme.
2. Materials and methods
2.1. Strains AspergiZZus niduluns CECT2544 was obtained from the Spanish Type Culture Collection and maintained on complete agar medium [ 1 I]. The strain creAd30 [12] was a generous gift of Prof. H.N. Arst. 2.2. Culture conditions Culture conditions for enzyme purification were followed as described earlier [7]. The regulation of P-xylosidase production was investigated in replacement cultures as described previously [9]. At different intervals, 5 ml culure aliquots were withdrawn, filtered and the mycelium used for analysing pxylosidase activity. 2.3. Enzyme assays Reaction mixtures containing 250 ~1 of 2 mM p-nitrophenyl-P-r+xylopyranoside (pNpX) in water, 250 ~1 of suitably diluted enzyme or mycelia in sodium acetate buffer (50 mM pH 5.0) were incubated at 50°C for 30 min. The reaction was stopped by adding 1 ml of 2 M sodium carbonate solution and the release of p-nitrophenol was measured at 400 nm. One unit of P-xylosidase activity was defined as the amount of enzyme which liberates 1 pmol of p-nitrophenol/min/ml or g of mycelia under the conditions described above.
Letters 135 (19961 287-293
fractions were estimated 280 nm.
by measuring
absorbance
at
2.5. Purification Mycelium from a xylan grown culture was extracted twice with saline phosphate buffer containing 0.05% (w/v) Triton X100. The mycelial extract was concentrated in a Minitan Ultrafiltration system (Millipore Corp., Beldford, MA) using a 10000 molecular mass cut-off polysulfone filter. The concentrate was loaded onto a 2.5 X 30 cm Q-Sepharose column equilibrated with 50 mM sodium acetate buffer, pH 3.5. Unbound proteins were eluted with equilibrating buffer and bound proteins were eluted with a linear gradient up to 0.5 M NaCl in sodium acetate buffer. The flow rate was 30 ml/h and 5 ml fractions were collected. Fractions showing @xylosidase activity were pooled, dialysed, concentrated and then applied to a Mono-Q column (Pharmacia, Uppsala, Sweden) equilibrated with 10 mM piperazine buffer, pH 4.5. Fractions showing P-xylosidase activity were eluted with a linear gradient up to 0.5 M NaCl. The flow rate was 30 ml/h and 2 ml fractions were collected. Finally the Pxylosidase activity recovered was applied to a Superdex 200 HR lo/30 molecular gel filtration prepacked column (Pharmacia, Uppsala, Sweden) equilibrated with 10 mM piperazine buffer, pH 5.5. The flow rate was 30 ml/h and 2 ml fractions were collected. The molecular mass of the protein was estimated by the gel filtration chromatography described above using molecular mass standards. 2.6. Electrophoresis
and isoelectric focussing
SDS polyacrylamide gel electrophoresis was performed according to Smith [14] using an acrylamide concentration of 10% (w/v). The gel was stained with silver as described by Merril et al. [ 151. The low molecular mass calibration mixture from Pharmacia was used as standard. Analytical isoelectric focussing was performed as described previously [7].
2.4. Protein estimation
2.7. Determination
Protein content was estimated by the method of Bradford [ 131 using bovine serum albumin (fraction V) as standard. Protein content in chromatography
The Michaelis-Menten constants were determined by non-linear regression, using pNpX at concentrations ranging from 0.2 to 3.0 mM.
of kinetic constants
S. Kumar,
2.8. Temperature
D. Ram&
/ FEMS Microbiology
and pH relationships
The optimum temperature for P-xylosidase activity was determined by incubating the enzyme preparation with pNpX at different temperatures ranging from 30 to 60°C. Thermal stability was assessed by incubating the enzyme preparation at 50, 55 and 60°C and assaying activity at various time points using the standard assay. The pH optimum for P-xylosidase activity was determined by incubating the enzyme preparation with pNpX in Teorell and Stenhagen universal buffer [ 161 at various pH values between 3.5 and 9.0 at 50°C. pH stability was assessed by incubating the enzyme at different pH at 25°C and measuring activity at various times using the standard protocol. 2.9. l$hect.s
of carious reagents
An appropriate amount of enzyme was mixed with 50 mM sodium acetate buffer (pH 5.5) containing 1 mM of various reagents. The enzyme activity was measured as described earlier. 2.10. Hydrolysis
experiments
End products of the hydrolysis of xylobiose, xylotriose and xylotetraose (Megazyme, Sidney, Australia) were determined by HPLC on a Sugar Pack column (Waters Associates, Milford, MA) equilibrated and eluted with Milli-Q water. Peaks were detected by differential refractometry and identified by comparing elution times to those of appropriate standards. 2. Il. Antibodies
lam
and Western blotting
Antibodies against the /3-xylosidase of A. niduwere raised in rabbit. Cross-reactivity between
Table I Purification Step Crude extract Q-Sepharose Mono-Q Superdex-200
of P-xylosidase
Letters
135
f IYY61
289
287-293
the antisera and the enzyme was tested by western blotting as described previously [7]. @Xylosidase was extracted from the mycelia of the xylan grown cultures of A. niger, A. terreus and A. nidulans with saline phosphate buffer containing 0.05% (w/v> Triton X- 100.
3. Results and discussion 3. I. Pur$cation Unlike the situation reported in Emericella nidulans [lo], no extracellular /3-xylosidase activity was detected when A. nidulans was grown on xylan as sole carbon source. All P-xylosidase activity was found to be mycelium bound. The details of the purification of /3-xylosidase, following the protocol described in Materials and methods, are presented in Table 1. Most of the protein content in the crude extract was eluted from Q-sepharose with the equilibrating buffer. P-Xylosidase activity was eluted at 180-200 mM NaCl (Fig. 1). Fractions showing pxylosidase activity were concentrated, dialysed and loaded onto a Mono-Q ion exchange column. pXylosidase activity was eluted by 200 mM NaCl. P-Xylosidase was further purified using a Superdex200 gel filtration column. The biochemical characterization and substrate hydrolysis pattern studies reported below were performed with this purified /3xylosidase preparation, stored at - 70°C. 3.2. Biochemical
characterization
Purified P-xylosidase showed a single sllverstained protein band on SDS-PAGE. corresponding to a molecular mass of 85000 (result not shown). Gel filtration column chromatography indicated that the molecular mass of native P-xylosidase was
from A. nidulans
Total activity
Total protein
Specific activity
Recovery
(U/ml)
(mg)
KJ/mg)
(%)
44.2 22.3 20.0 12.0
36.5 2.9 0.6 0.1
1.2 7.6 34.1 107.1
loo 50 45.2 27.2
Fold purification
I 6.3 28.4 89.2
S. Kumur, D. Ramo’n/ FEMS Microbiology Letters 13.5 (19%) 287-293
290
optimum temperature (50°C) inactivated within 4 to 6 h. examine thermostability it was period of 30 min the enzyme 60°C but at 55°C it retained activity.
the enzyme became In an experiment to observed that over a lost all its activity at 50% of its original
3.3. Kinetic parameters
0
20
40
60
80
0.00
100
Fraction Number
Fig. 1. Elution pattern of S-xylosidase by Q-Sepharose column chromatography. Experimental details are described in the text. Solid line, absorbance at 280 nm; (A ) Pxylosidase activity.
180 000 suggesting that the native enzyme may be dimeric in nature. The estimated molecular mass of the A. niduluns &xylosidase is similar to those of P-xylosidase reported from other fungi [ 10,17,18]. The dimeric nature of Pxylosidase has also been reported in E. niduluns [lo], Aspergillus niger [19], Penicillium wortmanni [20] and Chaetomium trilatetute [21]. Analytical IEF data showed the P-xylosidase to be an acidic protein with an isoelectric point of approximately 3.4. Similar isoelectric points have been observed for the P-xylosidase of E. niduluns [ 101 and P-xylosidase II of Aspergillus puluerulentus [lS], although the p1 of A. niger P-xylosidase has been reported to be 4.9 [19]. The pH optimum of the purified P-xylosidase was found to be 5.0. This is the same as that observed for E. niduluns [lo] and P-xylosidase II of A. puluerulentus [ 181 but different to that of A. niger which has been reported to be 4.0 [19,22]. The enzyme is stable over the pH range 4.0 to 6.0 for up to 8 h at room temperature. Stability of @xylosidase was also reported in A. niger [19,22]. The optimum temperature for the purified P_ xylosidase activity at pH 5.0 was found to be 50°C. This was rather lower than the optimum temperature for the A. niger /3-xylosidase [ 191 but very similar to that of E. niduluns [lo]. The enzyme was quite unstable above 45°C. At lower temperatures (4045°C) the enzyme was stable up to 8 h, while at the
The Michaelis constants determined on pNpX were different to those found earlier in E. nidulans [lo]. The calculated K, and V,,, values of pxylosidase were found to be 1.1 mM and 25.6 ~mol/min/mg of protein, respectively. The lower K, value of Pxylosidase from A. nidulans shows that it has an higher substrate affinity than that of E. niduluns [ 1O] but less than that of A. niger [ 191. 3.4. Effects actiuity
of various
reagents
on
P-qlosidase
The effects of various metallic ions and reagents on the activity of purified &xylosidase were investigated. As shown in Table 2, activity was dramatically inhibited by Cu*+, Ag*+ and Hg*+ and also some inhibition was observed in the presence of Zn*+ and Fe*+. Some inhibition was also observed in the presence of SDS. The enzymatic activity was
Table 2 Effects of various reagents (at 1 mM) on the enzyme activity of purified Pxylosidase of A. nidulans Reagents
Relative activity (o/o)
Control CoCl
100 108& I+
2
AgW
CUCI, ZnCl 2 MgCl2 I&Cl, CaClz CdCI? FeCl, EDTA Cysteine DTT SDS
9* 50+ 114+
4
1 1 I 7
7* 1 111+ 8 96k 5 47* 3 126k 7 130* 9 125+ 9 71* 10
The results presented are the average three different experiments.
(+ standard
deviation)
of
4. Kumar, D. Ram& / FEMS Microbiology Leiters 135 (1996) 287-293
0 hours
16 hours
x1 II r
291
slightly stimulated in the presence of Co*+, Mg’+ and Ca*+. In the presence of DTT, EDTA and cysteine /3-xylosidase activity was incresed by approximately 30%. In comparison to the enzyme from E. nidulans [lo], the A. nidulans P-xylosidase shows some resistance towards SDS but high sensitivity to cu’+. The effects of D and L forms of xylose were also studied at two different concentrations (25 and 100 mM1. /?-Xylosidase activity was not affected by L-xylose even at 100 mM concentration but inhibition was very clear in the presence of D-xylose at 25 mM (44% inhibition). 3.5. Hydrolysis patterns The hydrolysis patterns of xylobiose, xylotriose and xylotetraose with the purified P-xylosidase are shown in Fig. 2. With xylobiose as substrate, the enzyme effected 90% degradation to xylose within 6 to 9 h while with xylotriose hydrolysis took longer and the enzyme only effected 70 to 80% degradation after 16 h. When xylotetraose was used as a substrate the hydrolysis was much slower and only 20 to 30% of the substrate was converted to xylose after 16 h. This result indicates that the best substrates for pxylosidase are short oligomers of xylose and that as chain length increases the hydrolysis becomes progressively less efficient. No hydrolysis products were identified with oat, spelt or birch wood xylans (results not shown). Similar results have been obtained with the /3-xylosidases from Trichoderma r:iride [ 171 and E. nidulans [lo]. In all these cases, the rate of hydrolysis of xylooligosaccharides decreased with increasing chain length. 3.6. Western blotting Antibody raised against the purified P-xylosidase from A. nidulans was used to try to detect similar
Fig. 2. Time course of enzymatic hydrolysis of (A) xylobiose, (B) xylotriose and (C) xylotetraose. Different substrates (1.5 mg/ml final concentration) were incubated with 100 ~1 of purified P-xylosidase in 50 mM sodium acetate buffer, pH 5.0 at 50°C. At 0 h and 16 h 100 ~1 aliquots were withdrawn and used for analysing the end products. Peaks denote: Xl, xylose; X2, xylobiose; X3, xylotriose; and X4, xylotetraose.
292
S. Kumar, D. Ram& / FEMS Microbiology
Pxylosidases in other Aspergillus species. Enzymatic assays had shown the presence of pxylosidase activities in A. niger and A. terreus, however no @-xylosidase bands were detected in A. niger and A. terreus by western blotting using the A. niduluns antibody (results not shown). This indicates that the P-xylosidases present in the other two species of Aspergihs are immunologically different to the P-xylosidase purified from A. niduhns. 3.7. Induction P-xylosidase
and carbon
catabolite
repression
of
Using culture replacement, the influences of different carbon compounds on P-xylosidase produc-
‘#Ad-type
I
0
6
12
16
1
I
24
Time ( h)
Letters 135 (1996) 287-293
tion were followed in the A. nidulans wild-type strain. The enzymatic activity was found to be associated with the mycelium and no activity was detected in culture supematants. The greatest levels of P-xylosidase production were observed in the presence of arabinose (0.35 U/mg of mycelium) and xylose (0.31 U/mg of mycelium). The inducing abilities of xylan (0.22 U/mg of mycelium) and arabitol (0.17 U/mg of mycelium) were less than those of arabinose and xylose. Xylitol acts as non-inducing carbon source. In order to study carbon catabolite repression, transfer experiments were carried out with both wild-type and the creAd30 mutant strain of A. nidulam. Cultures were transferred to xylan or xylan plus glucose. As seen in Fig. 3, the presence of glucose represses the synthesis of P-xylosidase in the wildtype strain but in the case of the creAd30 mutant this repression was partially overcome and the synthesis of &xylosidase started at around 6 h after transfer. This result indicates that the synthesis of p-xylosidase in A. niduluns is carbon catabolite repressible, mediated, at least in part, by CREA. The carbon catabolite repression effect is similar to that observed on xylanase synthesis in A. nidulans 191. The P-xylosidase purified from A. niduluns in the present investigation appears to be different to that previously isolated from E. niduhs [ 101 not only in its cellular location but also in its kinetic characteristics.
Acknowledgements
0
6
12
18
24
This work has been supported by grants from D.G. XII of the European Commission (BIOTECH BI02-CT93-0174) and the Comisi6n Interministerial de Ciencia y Tecnologia of the Spanish Government (ALI93-0809). S.K. is the recipient of a Fellowship of the Ministerio de Education y Ciencia. The A. niduluns creAd30 strain was provided by Dr. H.N. Arst. Thanks are due to Dr A.P. MacCabe for critical reading of the manuscript.
Time (h) Fig. 3. Time course of Pxylosidase production during incubation of washed, fructose grown mycelia of the A. nidufans wild-type strain and creAd30 mutant incubated in minimal medium containing 1% (w/v) xylan (A) or 1% (w/v) xylan plus 1% (w/v) glucose ( W1.
References [l] Biely, P. (1985) Acetyl xylan esterases Trends Biotechnol. 3, 286-290.
in cellulolytic
fungi.
S. Kumar, D. Ram&
[2] Visser,
/ FEMS Microbiology
J., Beldman, G., Kusters-Van Someren, M.A. and Voragen, A.G.J. (1992) Xylan and Xylanases. Elsevier, Amsterdam. [3] Amt. H.N., Jr. and Scazzocchio, C. (1985) Formal genetics and molecular biology of the control of gene expression in Aspergillus niduluns. In: Gene Manipulations in Fungi. (Bennett, J.W. and Lasure, L.L., Eds.), pp. 309-343. Academic Press, New York. [4] Femandez-Espinar, M.T., Ramon, D., Piiiaga, F. and Vallts, S. (1992) Xylanase production by Aspergillus nidulans. FEMS Microbial. Lett. 91, 91-96. [S] Fernandez-Espinar. M.T., Piiiaga, F., Sam P., Ram&, D. and VallCs, S. (1993) Purification and characterization of a neutral endoxylanase from Aspergillus niduluns. FEMS Microbiol. Lett. 113, 223-228. [6] Fernandez-Espinar, M.T., VallCs, S., Piiiaga, F., PtrezGonzalez, J.A. and Ram&, D. Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use to purify the minor X,, xylanase. Appl. Microbiol. Biotechnol. In press, [7] Fernandez-Espinar, M.T., Pihaga, F., de Graaff, L., Visser. J., Ram&r, D. and VallCs, S. (1994) Purification, characterization and regulation of the synthesis of an Aspergillus niduluns acidic xylanase. Appl. Microbial. Biotechnol. 42, 555-562. [8] Kulmburg, P.. Mathieu, M., Dowzer, C., Kelly, J. and Felenbok, B. (1993) Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus niduluns. Mol. Microbial. 7, 847-857. [9] Piilaga, F., Femandez-Espinar, M.T., Vallts, S. and Ramon, D. (1994) Xylanase production in Aspergillus nidulans: Induction and carbon catabolite repression. FEMS Microbial. Lett. 115, 3 19-324. [IO] Matsuo, M. and Yasui, T. (19841 Purification and some properties of P-xylosidase from Emericella nidulans. Agric. Biol. Chem. 48, 1853-1860. [I 11 Pontecorvo, Cl., Roper, J.A. Hemmons, L.M., Macdonald, K.D. and Bufton, A.W.J. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141-238.
Letters
135 (1996) 287-293
293
[12] Arst, H.N., Jr., Tollervey, D., Dowzer, C.E.A. and Kelly, J.M. (19901 An inversion truncating the creA gene of Aspergillus nidulans results in carbon catabolite repression. Mol. Microbial. 4, 851-854. [13] Bradford, M.M. (19761 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein dye-binding. Anal. Biochem. 72, 248254. [14] Smith, B.J. (1984) SDS polyacrylamide gel electoplhoresis of proteins. In: Methods in Molecular Biology. 1 Protein (Walker, J.M., Ed.), pp. 41-56. Human Press, New Jersey. [15] Merril, CR., Goldman, D., Sedman, S.A. and Ebert, M.H. (1981) Ultrasensitive stain for proteins in polyacrylamide gel shows regional variation in cerebrospinal fluid proteins, Science 21 I, 304-307. [16] Stauffer, C.E. (19891 Enzyme assays for food scientists. pp. 77. Van Nostrand Reinhold, New York. [17] Matsuo M, and Yasui, T. (19841 Purification and some properties of /3-xylosidase from Trichoderma rliride. Agric. Biol. Chem. 48, 1845-1852. [ 181 Sulistyo, J., Kamiyama, Y. and Yasui, T. (1995) Purification and some properties of Aspergillus pulcerulentus /3-xylosidase with transxylosylation capacity. J. Ferment. Bioengeen. 79, 17-22. 1191 Rodionova, N.A.. Tavobilov, J.M. and Bezborodov, A.M. (1983) P-xylosidase from A. niger 15: Purification and properties. J. Appl. B&hem. 5, 300-312. [20] Matsuo, M., Fujie, A., Win, M. and Yasui, T. (1987) Four types of /3-xylosidase from Penicillium wortmanni IFO. 7237. Agric. Biol. Chem. 5 1, 2367-2379. [21] Uziie, M., Matsuo, M. and Yasui, T. (1985) Purification and some properties of fi-xylosidase from Chaetonium trilaterale. Agric. Biol. Chem. 49, 1159-l 166. [22] Takenishi, S., Tsujisaka, Y. and Fukumoto, J. (1973) Studies on Hemicellulose IV. Purification and properties of the pxylosidase produced by A. niger Van Tieghem. J. Biochem. 73. 335-343.
Letters 13.5 (1996) 287-293
Purification and regulation of the synthesis of a P_xylosidase from Aspergillus nidulans Sudeep Kumar, Daniel Ran-h Departmemo
*
de Biotecnologpiude Alimentos, Institute de Agroquimica y Tecnologia de Alimentos, Consejo Superior de Irwestigaciones Cientifcas, Apartado de Correos 73, 46100 Burjassot, Valencia, Spain Received 3 November
1995; revised 17 November
1995; accepted
17 November
1995
Abstract PXylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50°C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-P-D-xylopyranoside as substrate, the K, and V,,,,, values have been found to be 1.l mM and 25.6 wrnol min- ’ (mg protein)- ‘, respectivdy. Enzyme activity was inhibited by Hg’+, Ag2+, and CL?+ at a concentration of 1 X lop3 M. The synthesis of /3-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the creA gene product. Keywrds:
Aspergillus nidulans; P-Xylosidase;
Purification:
Carbon catabolite
1. Introduction Xylan is the major constituent of wood and agricultural residues. In nature, the complete degradation of xylan requires the synergistic action of several enzymes, mainly endo /3- l+xylanases (EC 3.2.1.8) which cleave the p-1,4 glycosidic bond between two different xylose residues to produce xylooligosaccharides, and Pxylosidase (EC 3.2.1.37) which cleaves xylooligosaccharides to yield xylose [l]. The hydrolysis of xylan is of great interest for various biotechnological applications [2]. Filamentous fungi are well known producers of xylan degrading enzymes. Several species of the
* Corresponding author. Tel: + 34 (6) 390 0022; Fax: + 34 (6) 363 6301: E-mail: dramon_iata.csic.es. 0378-1097/96/$12.00 0 1996 Federation SSDI 0378-1097(95)00468-E
of European
Microbiological
repression:
CREA
genus Aspergillus are efficient producers of such enzymes and of these A. nidulans has become a model system for studying the mechanisms of control of gene expression in filamentous fungi [3]. Recently the xylanolytic complex of A. nidtlilans has been identified [4]. In the presence of xylan as sole carbon source A. nidulans secretes at least three endo-l&3-xylanases with molecular masas of 22, 24, and 34 kDa, named respectively X,, , X 24 and X,. The three proteins have been purified and characterised [5-71. X,, is a nc4&al xylanrtse while the other two xylanases = acidic pnMcins. Thd synthesis of the xylanolytic complex of A. n&lans is induced by xylan and xylooIigosaccharitW such as xylobiose, xylotriose and xylotetraose and tiepressed by glucose. This carbon catabolite repressi~ is apparently mediated by the CREA protein [8] and only affects the X,, and X,, xylanases [9]. Societies.
All rights reserved
288
S. Kumar, D. Ram& / FEh4S Microbiology
Previously, an extracellular /I-xylosidase was purified from the culture supematants of a natural isolate of Emericellu niduluns [lo]. In this paper we report the purification and biochemical characterization of a mycelial bound P-xylosidase from A. niduluns exhibiting different properties to the enzyme isolated from E. niduluns as well as the induction and carbon catabolite repression of the synthesis of the A. niduluns enzyme.
2. Materials and methods
2.1. Strains AspergiZZus niduluns CECT2544 was obtained from the Spanish Type Culture Collection and maintained on complete agar medium [ 1 I]. The strain creAd30 [12] was a generous gift of Prof. H.N. Arst. 2.2. Culture conditions Culture conditions for enzyme purification were followed as described earlier [7]. The regulation of P-xylosidase production was investigated in replacement cultures as described previously [9]. At different intervals, 5 ml culure aliquots were withdrawn, filtered and the mycelium used for analysing pxylosidase activity. 2.3. Enzyme assays Reaction mixtures containing 250 ~1 of 2 mM p-nitrophenyl-P-r+xylopyranoside (pNpX) in water, 250 ~1 of suitably diluted enzyme or mycelia in sodium acetate buffer (50 mM pH 5.0) were incubated at 50°C for 30 min. The reaction was stopped by adding 1 ml of 2 M sodium carbonate solution and the release of p-nitrophenol was measured at 400 nm. One unit of P-xylosidase activity was defined as the amount of enzyme which liberates 1 pmol of p-nitrophenol/min/ml or g of mycelia under the conditions described above.
Letters 135 (19961 287-293
fractions were estimated 280 nm.
by measuring
absorbance
at
2.5. Purification Mycelium from a xylan grown culture was extracted twice with saline phosphate buffer containing 0.05% (w/v) Triton X100. The mycelial extract was concentrated in a Minitan Ultrafiltration system (Millipore Corp., Beldford, MA) using a 10000 molecular mass cut-off polysulfone filter. The concentrate was loaded onto a 2.5 X 30 cm Q-Sepharose column equilibrated with 50 mM sodium acetate buffer, pH 3.5. Unbound proteins were eluted with equilibrating buffer and bound proteins were eluted with a linear gradient up to 0.5 M NaCl in sodium acetate buffer. The flow rate was 30 ml/h and 5 ml fractions were collected. Fractions showing @xylosidase activity were pooled, dialysed, concentrated and then applied to a Mono-Q column (Pharmacia, Uppsala, Sweden) equilibrated with 10 mM piperazine buffer, pH 4.5. Fractions showing P-xylosidase activity were eluted with a linear gradient up to 0.5 M NaCl. The flow rate was 30 ml/h and 2 ml fractions were collected. Finally the Pxylosidase activity recovered was applied to a Superdex 200 HR lo/30 molecular gel filtration prepacked column (Pharmacia, Uppsala, Sweden) equilibrated with 10 mM piperazine buffer, pH 5.5. The flow rate was 30 ml/h and 2 ml fractions were collected. The molecular mass of the protein was estimated by the gel filtration chromatography described above using molecular mass standards. 2.6. Electrophoresis
and isoelectric focussing
SDS polyacrylamide gel electrophoresis was performed according to Smith [14] using an acrylamide concentration of 10% (w/v). The gel was stained with silver as described by Merril et al. [ 151. The low molecular mass calibration mixture from Pharmacia was used as standard. Analytical isoelectric focussing was performed as described previously [7].
2.4. Protein estimation
2.7. Determination
Protein content was estimated by the method of Bradford [ 131 using bovine serum albumin (fraction V) as standard. Protein content in chromatography
The Michaelis-Menten constants were determined by non-linear regression, using pNpX at concentrations ranging from 0.2 to 3.0 mM.
of kinetic constants
S. Kumar,
2.8. Temperature
D. Ram&
/ FEMS Microbiology
and pH relationships
The optimum temperature for P-xylosidase activity was determined by incubating the enzyme preparation with pNpX at different temperatures ranging from 30 to 60°C. Thermal stability was assessed by incubating the enzyme preparation at 50, 55 and 60°C and assaying activity at various time points using the standard assay. The pH optimum for P-xylosidase activity was determined by incubating the enzyme preparation with pNpX in Teorell and Stenhagen universal buffer [ 161 at various pH values between 3.5 and 9.0 at 50°C. pH stability was assessed by incubating the enzyme at different pH at 25°C and measuring activity at various times using the standard protocol. 2.9. l$hect.s
of carious reagents
An appropriate amount of enzyme was mixed with 50 mM sodium acetate buffer (pH 5.5) containing 1 mM of various reagents. The enzyme activity was measured as described earlier. 2.10. Hydrolysis
experiments
End products of the hydrolysis of xylobiose, xylotriose and xylotetraose (Megazyme, Sidney, Australia) were determined by HPLC on a Sugar Pack column (Waters Associates, Milford, MA) equilibrated and eluted with Milli-Q water. Peaks were detected by differential refractometry and identified by comparing elution times to those of appropriate standards. 2. Il. Antibodies
lam
and Western blotting
Antibodies against the /3-xylosidase of A. niduwere raised in rabbit. Cross-reactivity between
Table I Purification Step Crude extract Q-Sepharose Mono-Q Superdex-200
of P-xylosidase
Letters
135
f IYY61
289
287-293
the antisera and the enzyme was tested by western blotting as described previously [7]. @Xylosidase was extracted from the mycelia of the xylan grown cultures of A. niger, A. terreus and A. nidulans with saline phosphate buffer containing 0.05% (w/v> Triton X- 100.
3. Results and discussion 3. I. Pur$cation Unlike the situation reported in Emericella nidulans [lo], no extracellular /3-xylosidase activity was detected when A. nidulans was grown on xylan as sole carbon source. All P-xylosidase activity was found to be mycelium bound. The details of the purification of /3-xylosidase, following the protocol described in Materials and methods, are presented in Table 1. Most of the protein content in the crude extract was eluted from Q-sepharose with the equilibrating buffer. P-Xylosidase activity was eluted at 180-200 mM NaCl (Fig. 1). Fractions showing pxylosidase activity were concentrated, dialysed and loaded onto a Mono-Q ion exchange column. pXylosidase activity was eluted by 200 mM NaCl. P-Xylosidase was further purified using a Superdex200 gel filtration column. The biochemical characterization and substrate hydrolysis pattern studies reported below were performed with this purified /3xylosidase preparation, stored at - 70°C. 3.2. Biochemical
characterization
Purified P-xylosidase showed a single sllverstained protein band on SDS-PAGE. corresponding to a molecular mass of 85000 (result not shown). Gel filtration column chromatography indicated that the molecular mass of native P-xylosidase was
from A. nidulans
Total activity
Total protein
Specific activity
Recovery
(U/ml)
(mg)
KJ/mg)
(%)
44.2 22.3 20.0 12.0
36.5 2.9 0.6 0.1
1.2 7.6 34.1 107.1
loo 50 45.2 27.2
Fold purification
I 6.3 28.4 89.2
S. Kumur, D. Ramo’n/ FEMS Microbiology Letters 13.5 (19%) 287-293
290
optimum temperature (50°C) inactivated within 4 to 6 h. examine thermostability it was period of 30 min the enzyme 60°C but at 55°C it retained activity.
the enzyme became In an experiment to observed that over a lost all its activity at 50% of its original
3.3. Kinetic parameters
0
20
40
60
80
0.00
100
Fraction Number
Fig. 1. Elution pattern of S-xylosidase by Q-Sepharose column chromatography. Experimental details are described in the text. Solid line, absorbance at 280 nm; (A ) Pxylosidase activity.
180 000 suggesting that the native enzyme may be dimeric in nature. The estimated molecular mass of the A. niduluns &xylosidase is similar to those of P-xylosidase reported from other fungi [ 10,17,18]. The dimeric nature of Pxylosidase has also been reported in E. niduluns [lo], Aspergillus niger [19], Penicillium wortmanni [20] and Chaetomium trilatetute [21]. Analytical IEF data showed the P-xylosidase to be an acidic protein with an isoelectric point of approximately 3.4. Similar isoelectric points have been observed for the P-xylosidase of E. niduluns [ 101 and P-xylosidase II of Aspergillus puluerulentus [lS], although the p1 of A. niger P-xylosidase has been reported to be 4.9 [19]. The pH optimum of the purified P-xylosidase was found to be 5.0. This is the same as that observed for E. niduluns [lo] and P-xylosidase II of A. puluerulentus [ 181 but different to that of A. niger which has been reported to be 4.0 [19,22]. The enzyme is stable over the pH range 4.0 to 6.0 for up to 8 h at room temperature. Stability of @xylosidase was also reported in A. niger [19,22]. The optimum temperature for the purified P_ xylosidase activity at pH 5.0 was found to be 50°C. This was rather lower than the optimum temperature for the A. niger /3-xylosidase [ 191 but very similar to that of E. niduluns [lo]. The enzyme was quite unstable above 45°C. At lower temperatures (4045°C) the enzyme was stable up to 8 h, while at the
The Michaelis constants determined on pNpX were different to those found earlier in E. nidulans [lo]. The calculated K, and V,,, values of pxylosidase were found to be 1.1 mM and 25.6 ~mol/min/mg of protein, respectively. The lower K, value of Pxylosidase from A. nidulans shows that it has an higher substrate affinity than that of E. niduluns [ 1O] but less than that of A. niger [ 191. 3.4. Effects actiuity
of various
reagents
on
P-qlosidase
The effects of various metallic ions and reagents on the activity of purified &xylosidase were investigated. As shown in Table 2, activity was dramatically inhibited by Cu*+, Ag*+ and Hg*+ and also some inhibition was observed in the presence of Zn*+ and Fe*+. Some inhibition was also observed in the presence of SDS. The enzymatic activity was
Table 2 Effects of various reagents (at 1 mM) on the enzyme activity of purified Pxylosidase of A. nidulans Reagents
Relative activity (o/o)
Control CoCl
100 108& I+
2
AgW
CUCI, ZnCl 2 MgCl2 I&Cl, CaClz CdCI? FeCl, EDTA Cysteine DTT SDS
9* 50+ 114+
4
1 1 I 7
7* 1 111+ 8 96k 5 47* 3 126k 7 130* 9 125+ 9 71* 10
The results presented are the average three different experiments.
(+ standard
deviation)
of
4. Kumar, D. Ram& / FEMS Microbiology Leiters 135 (1996) 287-293
0 hours
16 hours
x1 II r
291
slightly stimulated in the presence of Co*+, Mg’+ and Ca*+. In the presence of DTT, EDTA and cysteine /3-xylosidase activity was incresed by approximately 30%. In comparison to the enzyme from E. nidulans [lo], the A. nidulans P-xylosidase shows some resistance towards SDS but high sensitivity to cu’+. The effects of D and L forms of xylose were also studied at two different concentrations (25 and 100 mM1. /?-Xylosidase activity was not affected by L-xylose even at 100 mM concentration but inhibition was very clear in the presence of D-xylose at 25 mM (44% inhibition). 3.5. Hydrolysis patterns The hydrolysis patterns of xylobiose, xylotriose and xylotetraose with the purified P-xylosidase are shown in Fig. 2. With xylobiose as substrate, the enzyme effected 90% degradation to xylose within 6 to 9 h while with xylotriose hydrolysis took longer and the enzyme only effected 70 to 80% degradation after 16 h. When xylotetraose was used as a substrate the hydrolysis was much slower and only 20 to 30% of the substrate was converted to xylose after 16 h. This result indicates that the best substrates for pxylosidase are short oligomers of xylose and that as chain length increases the hydrolysis becomes progressively less efficient. No hydrolysis products were identified with oat, spelt or birch wood xylans (results not shown). Similar results have been obtained with the /3-xylosidases from Trichoderma r:iride [ 171 and E. nidulans [lo]. In all these cases, the rate of hydrolysis of xylooligosaccharides decreased with increasing chain length. 3.6. Western blotting Antibody raised against the purified P-xylosidase from A. nidulans was used to try to detect similar
Fig. 2. Time course of enzymatic hydrolysis of (A) xylobiose, (B) xylotriose and (C) xylotetraose. Different substrates (1.5 mg/ml final concentration) were incubated with 100 ~1 of purified P-xylosidase in 50 mM sodium acetate buffer, pH 5.0 at 50°C. At 0 h and 16 h 100 ~1 aliquots were withdrawn and used for analysing the end products. Peaks denote: Xl, xylose; X2, xylobiose; X3, xylotriose; and X4, xylotetraose.
292
S. Kumar, D. Ram& / FEMS Microbiology
Pxylosidases in other Aspergillus species. Enzymatic assays had shown the presence of pxylosidase activities in A. niger and A. terreus, however no @-xylosidase bands were detected in A. niger and A. terreus by western blotting using the A. niduluns antibody (results not shown). This indicates that the P-xylosidases present in the other two species of Aspergihs are immunologically different to the P-xylosidase purified from A. niduhns. 3.7. Induction P-xylosidase
and carbon
catabolite
repression
of
Using culture replacement, the influences of different carbon compounds on P-xylosidase produc-
‘#Ad-type
I
0
6
12
16
1
I
24
Time ( h)
Letters 135 (1996) 287-293
tion were followed in the A. nidulans wild-type strain. The enzymatic activity was found to be associated with the mycelium and no activity was detected in culture supematants. The greatest levels of P-xylosidase production were observed in the presence of arabinose (0.35 U/mg of mycelium) and xylose (0.31 U/mg of mycelium). The inducing abilities of xylan (0.22 U/mg of mycelium) and arabitol (0.17 U/mg of mycelium) were less than those of arabinose and xylose. Xylitol acts as non-inducing carbon source. In order to study carbon catabolite repression, transfer experiments were carried out with both wild-type and the creAd30 mutant strain of A. nidulam. Cultures were transferred to xylan or xylan plus glucose. As seen in Fig. 3, the presence of glucose represses the synthesis of P-xylosidase in the wildtype strain but in the case of the creAd30 mutant this repression was partially overcome and the synthesis of &xylosidase started at around 6 h after transfer. This result indicates that the synthesis of p-xylosidase in A. niduluns is carbon catabolite repressible, mediated, at least in part, by CREA. The carbon catabolite repression effect is similar to that observed on xylanase synthesis in A. nidulans 191. The P-xylosidase purified from A. niduluns in the present investigation appears to be different to that previously isolated from E. niduhs [ 101 not only in its cellular location but also in its kinetic characteristics.
Acknowledgements
0
6
12
18
24
This work has been supported by grants from D.G. XII of the European Commission (BIOTECH BI02-CT93-0174) and the Comisi6n Interministerial de Ciencia y Tecnologia of the Spanish Government (ALI93-0809). S.K. is the recipient of a Fellowship of the Ministerio de Education y Ciencia. The A. niduluns creAd30 strain was provided by Dr. H.N. Arst. Thanks are due to Dr A.P. MacCabe for critical reading of the manuscript.
Time (h) Fig. 3. Time course of Pxylosidase production during incubation of washed, fructose grown mycelia of the A. nidufans wild-type strain and creAd30 mutant incubated in minimal medium containing 1% (w/v) xylan (A) or 1% (w/v) xylan plus 1% (w/v) glucose ( W1.
References [l] Biely, P. (1985) Acetyl xylan esterases Trends Biotechnol. 3, 286-290.
in cellulolytic
fungi.
S. Kumar, D. Ram&
[2] Visser,
/ FEMS Microbiology
J., Beldman, G., Kusters-Van Someren, M.A. and Voragen, A.G.J. (1992) Xylan and Xylanases. Elsevier, Amsterdam. [3] Amt. H.N., Jr. and Scazzocchio, C. (1985) Formal genetics and molecular biology of the control of gene expression in Aspergillus niduluns. In: Gene Manipulations in Fungi. (Bennett, J.W. and Lasure, L.L., Eds.), pp. 309-343. Academic Press, New York. [4] Femandez-Espinar, M.T., Ramon, D., Piiiaga, F. and Vallts, S. (1992) Xylanase production by Aspergillus nidulans. FEMS Microbial. Lett. 91, 91-96. [S] Fernandez-Espinar. M.T., Piiiaga, F., Sam P., Ram&, D. and VallCs, S. (1993) Purification and characterization of a neutral endoxylanase from Aspergillus niduluns. FEMS Microbiol. Lett. 113, 223-228. [6] Fernandez-Espinar, M.T., VallCs, S., Piiiaga, F., PtrezGonzalez, J.A. and Ram&, D. Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use to purify the minor X,, xylanase. Appl. Microbiol. Biotechnol. In press, [7] Fernandez-Espinar, M.T., Pihaga, F., de Graaff, L., Visser. J., Ram&r, D. and VallCs, S. (1994) Purification, characterization and regulation of the synthesis of an Aspergillus niduluns acidic xylanase. Appl. Microbial. Biotechnol. 42, 555-562. [8] Kulmburg, P.. Mathieu, M., Dowzer, C., Kelly, J. and Felenbok, B. (1993) Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus niduluns. Mol. Microbial. 7, 847-857. [9] Piilaga, F., Femandez-Espinar, M.T., Vallts, S. and Ramon, D. (1994) Xylanase production in Aspergillus nidulans: Induction and carbon catabolite repression. FEMS Microbial. Lett. 115, 3 19-324. [IO] Matsuo, M. and Yasui, T. (19841 Purification and some properties of P-xylosidase from Emericella nidulans. Agric. Biol. Chem. 48, 1853-1860. [I 11 Pontecorvo, Cl., Roper, J.A. Hemmons, L.M., Macdonald, K.D. and Bufton, A.W.J. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141-238.
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[12] Arst, H.N., Jr., Tollervey, D., Dowzer, C.E.A. and Kelly, J.M. (19901 An inversion truncating the creA gene of Aspergillus nidulans results in carbon catabolite repression. Mol. Microbial. 4, 851-854. [13] Bradford, M.M. (19761 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein dye-binding. Anal. Biochem. 72, 248254. [14] Smith, B.J. (1984) SDS polyacrylamide gel electoplhoresis of proteins. In: Methods in Molecular Biology. 1 Protein (Walker, J.M., Ed.), pp. 41-56. Human Press, New Jersey. [15] Merril, CR., Goldman, D., Sedman, S.A. and Ebert, M.H. (1981) Ultrasensitive stain for proteins in polyacrylamide gel shows regional variation in cerebrospinal fluid proteins, Science 21 I, 304-307. [16] Stauffer, C.E. (19891 Enzyme assays for food scientists. pp. 77. Van Nostrand Reinhold, New York. [17] Matsuo M, and Yasui, T. (19841 Purification and some properties of /3-xylosidase from Trichoderma rliride. Agric. Biol. Chem. 48, 1845-1852. [ 181 Sulistyo, J., Kamiyama, Y. and Yasui, T. (1995) Purification and some properties of Aspergillus pulcerulentus /3-xylosidase with transxylosylation capacity. J. Ferment. Bioengeen. 79, 17-22. 1191 Rodionova, N.A.. Tavobilov, J.M. and Bezborodov, A.M. (1983) P-xylosidase from A. niger 15: Purification and properties. J. Appl. B&hem. 5, 300-312. [20] Matsuo, M., Fujie, A., Win, M. and Yasui, T. (1987) Four types of /3-xylosidase from Penicillium wortmanni IFO. 7237. Agric. Biol. Chem. 5 1, 2367-2379. [21] Uziie, M., Matsuo, M. and Yasui, T. (1985) Purification and some properties of fi-xylosidase from Chaetonium trilaterale. Agric. Biol. Chem. 49, 1159-l 166. [22] Takenishi, S., Tsujisaka, Y. and Fukumoto, J. (1973) Studies on Hemicellulose IV. Purification and properties of the pxylosidase produced by A. niger Van Tieghem. J. Biochem. 73. 335-343.