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Purification and characterization of a β-galactosidase from Sclerotinia sclerotiorum
FEMS MicrobiologyLetters 95 (19'92) 37-42 ;c: 1992 Federation of European MicrobiologicalSocieties1137S-1|1'97/92/$05.110 Published by Elsevier
FEMSLE 114971)
Purification and characterization of a/3-galactosidase from Sclerotinia sclerotiorum Christine R i o u ", G e o r g e s Frcyssinet b and Michel F~zvre " " Laboratoire d," Biologh' ('elluhtire Fongique. UMR ('NRS 106. Uniter~it~; I ,'on L I,illeurl~mne. France and t, Lal~oratoire &" Bi,dogie .$hdtf,'ulairt' et ('elhdaire I/~:g,:tales. Rh,;nc Poldenc .4grochimie. l.yon. France
Received I May 1992 Accepted 8 May 1`992 Key words: Sclerotinia sclerotiomm; fl-Galactosidasc: Enzyme production
2. I N T R O D U C T I O N
found in prokaryotic and eukaryotic microorganisms. Among the filamentous fungi, Neurospora crassa [1], Fusarium moniliforme [2], Aspergillus ot3"zae [3], Aspergillus fonsaceus [4], Aspergillus niger [5], Penicillium canescens [6], and Beauceria bassiana [7] arc known to produce /3-galactosidasc. Our studies on the production of polysaccharide depolymerases and glucoside hydrolases by Sclerotbua sclerotiorum have shown that this phytopathogenic fungus is able to secrete a large amount of /3-galactosidase when grown on various cell wall polysaccharides used as carbon sources [8]. As /3-galactosidase enzymes are of considerable importance [9], it was of interest to isolate, purify and characterize the extracellular enzyme from S. sclerotionem.
~-Galactosidase catalyzes the hydrolysis of lactose to glucose and galactose. This enzyme can be
3. M A T E R I A L S A N D M E T H O D S
i. S U M M A R Y Sclerotbua sclerotiomm secretes fl-galactosidase, giving very low activity when cultivated in a medium with glucose as the carbon source. This production is greatly enhanced during growth on different pectin related polysaccharidcs. An extracellular/3-galactosidase was purified 26-fold by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. This enzyme has a K m of 0.15 m g / m l , a M r of 120000 and a p l of 6.0. Enzyme activity was optimal at pH 4.0 and 50°C.
3.1. Organism attd culture conditions Sclerotinia sclerotiorum (Lb) de Bary was grown Corre,~pondetuv to: M. F~vre, Laboratoire de BiologieCellu-
laire Fongique, UMR CNRS 106, Universit~Lyon I, Brit. 405, bd du I 1 novembre 1918, 69622 Villeurbanne Cedex, France.
on liquid medium as previously described [8]. At daily intervals, samples of culture fluid were withdrawn from cultures grown in the presence of
38 citrus pectin, apple pectin, sodium polygalacturonatc, carboxymethylccllulose (CMC) or glucose. each used as carbon source 10.5c/~ w / v ) [10]. Large scale production of enzymes was carried out in 2-1 flasks containing 51X) ml of medium supplemented with citrus pectin (0.5% w/v). The culture fluids were separated from the mycclium by filtration, dialysed for 24 h at 4°C against distilled water and then freeze-dricd.
Rad) column ( 1.5 cm x 50 cm) equilibrated with (J.01 m sodium acetate buffer (pH 5.0)containing 0.05 M NaCI. The column was washed with the equilibrating buffer and the retained proteins were eluted with a linear NaCI gradient (0-0.4 M) in the same buffer at a flow rate of 15 m l / h . Active fractions (I.25 ml) were pooled and concentrated as described above.
3.4. Anah'tical teclmiques 3.2. Enzyme assay fl-Galactosidase activity was determined by measuring the 4-nitrophenol released from 4nitrophenyl fl-o-galactopyranoside (NPG). The reaction mixture (1 ml) contained 5-511 #1 of enzyme solution, depending on the enzyme concentration and 5 mM of substrate dissolved in tl.l M sodium acetate buffer, pH 5.11. After 15 min incubation at 50°C, reactions were stopped by the addition of 2 ml of 0.1 M Na_,CO 3 and the nitrophenol liberated was measured spectrophotometrically at 399 nm [5-8]. Enzyme activity is expressed as nmol of 4-nitrophenol m i n - t . Protein concentrations were determined by ti:,c method of Bradford [11].
3.3. Enzyme purification All procedures were done at 4°C. The powder from freeze-dried filtrates was solubilized in 0.1 M sodium acetate buffer (pH 5.11). The preparation was brought to 2 0 ~ saturation with ammonium sulfate and centrifuged at 8 t ~ ) × g for 30 rain. The supernatant fluid was brought to 8 0 ~ saturation with ammonium sulfate. The resulting precipitate was collected by centrifugation (801~1 × g , 30 min), dissolved in the smallest possible volume of sodium acetate buffer, then dialysed against distilled water then against sodium acetate buffer. The dialysed material (2 ml) was applied to an Ultrogel AcA 34 (1BF) column (2 cm × 95 cm) equilibrated with 0.01 M sodium acetate buffer (pH 5). Elution was performed with sodium acetate buffer at a flow rate of 20 m l / h and 2.5-ml fractions were collected. The fractions containing the enzyme activity were concentrated by centrifugation using Cf 25 Centriflo-ultrafiltration membrane cones (Am(con, USA), then applied to a CM Bio-Gel A (Bio
Molecular size and purity of the purified enzymes were examined by SDS-PAGE using 10('/t polyacrylamide resolving gels [12]. Molecular mass markers (Sigma or BioRad) were used to determine the M r of the enzyme. Proteins were made visible by silver staining using the Stratagene kit. Preparative isoelectric focusing (IEF) was performed in a Sephadex gel for 16 h. The pH gradient was formed with carrier ampholytes (pH range 3.11-10.0, Serva). Thirty fractions were collected and the proteins were remtwed by successive treatments with sodium acetate buffer and assayed for enzyme activity.
3.5. Enzyme chanwterization Estimates of optimal temperature and pH as well as thermal stability were made using a temperature range from 211° to 811°(` and a pH range from 2.11 to 8.11. The K m value was determined from the Lineweaver-Burk plot using NPG as a substrate at concentrations from 11.115 to 4 mg ml-- i. The ability of the purified enzyme to hydrolyze various substrates was assayed using NP-glycosides and polysaccharides at concentrations of 5 mM and 2 mg m l - ~, respectively.
3.6. hnmunological methods Antisera to the purified /3-galactosidase were prepared by immunizing rabbits with intradermal injections of 511 p,g of purified enzyme dissolved in 1 ml Freund's complete adjuvent followed by 3 injections at 20-day intervals. For immunoblotting, 2 /,tg of extracellular fungus proteins were separated on SDS-PAGE and then electroblotted onto nitrocellulose [13]. Remaining binding sites of nitrocellulose were blocked for 2 h with 5% non-fat dry milk in Tris-buffered saline (TBS). After three washes in TBS, protein blots were
incubated for 2 h in TBS with antiserum to /3galaetosidase (l:2(X~0) and specifically detected by staining with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (i : 1000), ! chloronaphthol (1.5 mg m l - ' and 0 . ( ) 1 % H.~O.,.
4. R E S U L T S A N D DISCUSSION
4. I. Prtnluction of/3-galactosidase The time course of secretion of /3-galactosidase activity in cultures grown on different carbon sources is represented in Fig. 1. A high /3-galactosidase activity was recorded in the presence of pectinolytic substrates and maximal enzymatic activities were reached after 8 days. /3Galactosidase activity was very low in the glucose medium and was only detected during the first week. T h e enzyme was also produced in medium containing CMC, the activity incre~ising slowly during the course of cultivation. In pectic or C M C media, growth yield was about seven times lower than in glucose medium. The differences observed in enzymatic activity were not associated with the growth yields and showed that pectinolytic substrates induced product'ion of extracellular /3-galactosidase. Similar observations have been made with Aspergillus or)'zae [14].
4.Z Galactosidase purification Purification of a /3-galactosidase was achieved in a series of three separation steps: (i) salt precipitaton, (it) gel filtration, and (iii) cation cx-
r
--
T
-
2
i
4
-
i
6
8
10
Fig. I. J:~-Galactosidaseproduction by S..~('lerotiorum during cultivation on citrus pectin ( 0 ) apple pectin ( • ) sodium pol.,,galacturonate ( • ) carboxymethylcellulose {o) carboxvincihylcellulosei o ) or glucose ( E3 ).
change chromatography. A main peak of /3galactosidase activity was retained on the cation~ exchange column and collected at 0.11 M NaCI during elution with a NaCI gradient. A lower peak was eluted at higher NaCI maturity but was not further studied. The purification data are summarized in Table I. The molecular mass of the enzyme was estimated to be 120 kDa by ultrogel AcA gel filtration column by comparing the elution volume to those of standard M, marker proteins (Fig. 2). After S D S - P A G E and silver staining of the purified enzyme, a single protein band was observed.
Table 1 Purification of/:Lgalactosidase from cultures of Sclen~tbda.~ch'rotiorum
Culture filtrate Ammonium sulfilte fractionation (20-8(IG.) Ultrogel AcA 34 CM Bio Gel A
Total activity x I 1)(•1U 4111
12
Days
Specific activity x l {100U/mg 5.7
Yield ("i) I[~}
Purification factor I.()
332
9,5
83
1.6
292 209
77.0 14~
73 52
13.4 26.2
10
0.5
f i 4.1 4.3
I 4.5
i 4.7
i 4.9
, 5.1
i 5.3
i 5.5
Log MW
O-gal I 4.3 4.5
I 4.7
, 4.9
,-,~, 5.1 5,3 Log MW
5.5
Fig. 2. Molecular mass determination of fl-galactosidase (A) gel filtration on Ultrogel AcA 34 Markers: (a) cytochrome c 125(X);(b) chicken ovalbumin 45(XX).(c) bovine serum albumin 660(X),(d) yeast hexokinase 100000, (e) eatalase 240(XI0.(B) SDS-polyacrylamide gel electrophoresis. Markers: (a) chicken ovalbumin 45000: (b) bovine serum albumin 66(h')0:(c) phosphorylase 97000- (d) /J-galactosidase 116(X~0;(e) myosin 2(10(XX).
The molecular mass estimated by S D S - P A G E was 120 kDa (Fig. 2). This indicates that the fl-galactosidase contains a single polypeptide. The molecular mass of the enzyme from $. sclerotiorum is close to those reported for A. niger [15], A. fonsecaeus [4], Curcu/aria inaequalis [16], and
A B ~ ~.~l
kDa 200 --.--116 ...... 9 7
,=--66 ~-45
P. canesca:s [6], which exhibit M r values ranging from l l 5 to 130 kDa. In analytical gel isoelectric focusing, fl-galactosidase was resolved as a single peak and had a p I value of pH 6.0. This p l is slightly higher than that of the enzymes from A. fonsecaeus (pH 4.2) [16] and N. crussa (pH 4.8) [1] but close to the p l of P. canescens enzyme (pH 6.7) [6]. Antibodies against the purified fl-galactosidase were raised in rabbits. 1 ng of purified proteins was detected with l : 1000 dilution in the most active antiserum. The abilities of the antibodies to cross-react with proteins from th¢ crude culture filtrate were examined by S D S - P A G E and immunoblotting of 2 p.g of extracellular proteins (Fig. 3). T h e antiserum detected one band of 120 kDa and revealed also a diffuse zone of higher Mr: this may be due to heterogenous glycosylation as shown for the fl-galactosidase from A. niger [15].
4.3. Characterization of the purified fl-galactosidase
Fig. 3. Eleetrophoretic analysis of the /J-galactosidase. A. SDS-PAGE of the purified extracellular fl-galactosidase from S. sclerotiomm. B. SDS-PAGE Western blot of extraeellular proteins from S. sclerotiontm. 2 pg of extracellular proteins were probed with the antiserum (1:2000) against the purified fl-galactosidase.
The enzyme showed a narrow pH activity curve: the optimal pH was 4.0. 50% of the maximal activity was found at about pH 3.0 and pH 5.0. In this respect the fl-galactosidase from S. sclerotiorum is similar to that from N. crussa [1], C. inaequalis [16] and A. fonsecaeus [4]. T h e optimum t e m p e r a t u r e for activity was 50°C but 25%
o f the maximal value was m e a s u r e d at 75°C. Thermostability was investigated by m e a s u r i n g the residual activity o f the enzyme after 4 h o f incubation at t e m p e r a t u r e s ranging from 20 to 80°C. T h e enzyme was stable below 45°C a n d exhibiting 50% activity after t r e a t m e n t at 65°C. A L i n e w e a v e r - B u r k plot gave a K m = 0.5 m M 4 NPG. N o significant alteration o f t h e enzyme activity was p r o d u c e d by metals such as Cu-" ÷ a n d Hg -'~ which inhibit the /3-galaetosidase from Aureobasidium pulhdans [17] or C o 2~ and Ca -'+ which activate the e n z y m e s from A. pulhdans [17] and B. bassiana [7]. H o w e v e r 4 - c h l o r o m e r c u r i b e n z o a t e a n d SDS strongly inhibited the /Jgalactosidase activity. T h e action o f the p u r i f i e d / 3 - g a l a c t o s i d a s e was tested on a large n u m b e r o f substrates. T h e enzyme h a d no d e t e c t a b l e glycosidase activity against the p N P - ~ - I i n k e d sugars pNP-/~-xyloside, pNP-/3-glucoside, pNP-/3-ceilobioside, pNP-/3lactoside a n d p N P - a - a r a b i n o s i d e . H o w e v e r pNP-/3 fucoside was weakly hydrolysed by the enzyme. This activity r e p r e s e n t e d about 15% o f the activity m e a s u r e d against p N P galactoside. T h e enzyme was not active against citrus pectin, apple pectin and Na polygalacturonate which are very efficient inducers o f its synthesis. T h e very high level o f induction o f /3-galactosidase in the p r e s e n c e o f pectic c o m p o u n d s suggests that this enzyme may be involved in the process of degradation of these heteropolysaccharides. Pectin s u b s t a n c e s contain a g a l a c t u r o n a n b a c k b o n e with side chains o f various sugar residues such as xylose, arabinose and galactose [18]. During growth o n pectic polymers, S. sclerotiomm secretes a wide range o f p e c t i n a s e s and polygalacturonases, t o g e t h e r with cellulolytic a n d hemicellulolytic enzymes, able to d e g r a d e plant cell wall polymers [8,10,19]. During hydrolysis, short side chains released from the pectic polym e r s may serve as inducers o f the fl-galactosidase
activity. As this enzyme attacks galactoside and, to a lesser extent, fucoside derivatives, /3-galactosidase may be involved in the final o f d e g r a d a tion o f the neutral side chains o f pectin.
REFERENCES [ I ] Johnson, tI.N and Gib Debusk. A. (19711)Arch. Biochem. Bioph~)s. 138, 4118-41I. [2] Macris, B.J. and Markakis, P. 119811 Appl. Environ. Microbiol. 4 I, 95h-t;58. [3] Mcga, T., Nishijima, T. and Ikenaka, T. (igq)) J. Biochem. 1117,641- h44. [4] Gonzales, R.R. and Monsan, P. {It~l)Enzyme Microb. Technol. 13, 349-352. [5] Lec, Y.C. and Wacek, V. (It,~70)Arch. Bkv,:hem. Biophys. 138, 264-271. [hi Nikolacv, I.V., Khodtwa, P.M., Timokhina. E.A., Aleksenko, A.Y. and Vinetskii, Y.P. {19891 Biochem. USSR 54, 1294-1299. [7] MacPherson, J.M. and Khatchatourian, G.G. 119911 Biotcchnol. Appl. Biochem. 13, 217-2311. [8] Riou, C., Frcyssinet, G, and F~vre, M. (19911 Appl. Environ. Microbiol. 57, !478-1484. [91 Gekas, V. and Lopez-Leiva, M. (It~85). Process Biochcm. 211.2-12. [lO] Riou, C., Fraissinct-Tachet, L., Freyssinet, G, lind F~:vrc, M. {It~'421FEMS Microbiol. Lett. tll, 231-238. [I II Bradfi)rd, M.M. (It~7h) Anal. Biochem. 72, 248-258. [12] Laemmli, U.K. 1197111 Nature 227. 6811-085. [131 Towbin, II.T,. Staehelin, T. lind Gordon, J. (It,~791 Proc. Natl. Acad. Sci. USA. 7h, 4350-4354. [14] Me Kay, A.M. 119911 : "It. Appl. Microbiol. 12, 75-77, [151 Kumar, V., Rama Krishnan, S.. Teeri, T,T., Knowles. J.K.C. and Hartley, B.S. 119921 Bio. Technol, I11.82-85. [161 Zagustina, N.A. and Tikhominwa, A.S. (197h) Biochem, USSR 41, 11161-1166. [17] Dcsphande, M.S., Dcopurkar, R.L. and Rale, VB. (19891 Lett. Appl. Microbiol. 9, 21-24. [18J Da~'ill, A.G., Albersheim. P., Mc Ncil. M., Lcu, J.M., York. W.S., Stevenson, T.T., Thomas. J., Doarc.~. S., Gollin, D.J., ('hell. P. and Davis, K. 119851 J. Cell. Sci. Suppl. 2. 2113-217. [10] Riou, ('., Freyssinet, G. and F~vre, M. (19921 Appl. Environ. Microbiol. 58, 578-583. [It~l Riou, C., Freyssinet, G. lind F6rc, M. {19921Appl. Environm Microbiol. 58. 578-583.
FEMSLE 114971)
Purification and characterization of a/3-galactosidase from Sclerotinia sclerotiorum Christine R i o u ", G e o r g e s Frcyssinet b and Michel F~zvre " " Laboratoire d," Biologh' ('elluhtire Fongique. UMR ('NRS 106. Uniter~it~; I ,'on L I,illeurl~mne. France and t, Lal~oratoire &" Bi,dogie .$hdtf,'ulairt' et ('elhdaire I/~:g,:tales. Rh,;nc Poldenc .4grochimie. l.yon. France
Received I May 1992 Accepted 8 May 1`992 Key words: Sclerotinia sclerotiomm; fl-Galactosidasc: Enzyme production
2. I N T R O D U C T I O N
found in prokaryotic and eukaryotic microorganisms. Among the filamentous fungi, Neurospora crassa [1], Fusarium moniliforme [2], Aspergillus ot3"zae [3], Aspergillus fonsaceus [4], Aspergillus niger [5], Penicillium canescens [6], and Beauceria bassiana [7] arc known to produce /3-galactosidasc. Our studies on the production of polysaccharide depolymerases and glucoside hydrolases by Sclerotbua sclerotiorum have shown that this phytopathogenic fungus is able to secrete a large amount of /3-galactosidase when grown on various cell wall polysaccharides used as carbon sources [8]. As /3-galactosidase enzymes are of considerable importance [9], it was of interest to isolate, purify and characterize the extracellular enzyme from S. sclerotionem.
~-Galactosidase catalyzes the hydrolysis of lactose to glucose and galactose. This enzyme can be
3. M A T E R I A L S A N D M E T H O D S
i. S U M M A R Y Sclerotbua sclerotiomm secretes fl-galactosidase, giving very low activity when cultivated in a medium with glucose as the carbon source. This production is greatly enhanced during growth on different pectin related polysaccharidcs. An extracellular/3-galactosidase was purified 26-fold by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. This enzyme has a K m of 0.15 m g / m l , a M r of 120000 and a p l of 6.0. Enzyme activity was optimal at pH 4.0 and 50°C.
3.1. Organism attd culture conditions Sclerotinia sclerotiorum (Lb) de Bary was grown Corre,~pondetuv to: M. F~vre, Laboratoire de BiologieCellu-
laire Fongique, UMR CNRS 106, Universit~Lyon I, Brit. 405, bd du I 1 novembre 1918, 69622 Villeurbanne Cedex, France.
on liquid medium as previously described [8]. At daily intervals, samples of culture fluid were withdrawn from cultures grown in the presence of
38 citrus pectin, apple pectin, sodium polygalacturonatc, carboxymethylccllulose (CMC) or glucose. each used as carbon source 10.5c/~ w / v ) [10]. Large scale production of enzymes was carried out in 2-1 flasks containing 51X) ml of medium supplemented with citrus pectin (0.5% w/v). The culture fluids were separated from the mycclium by filtration, dialysed for 24 h at 4°C against distilled water and then freeze-dricd.
Rad) column ( 1.5 cm x 50 cm) equilibrated with (J.01 m sodium acetate buffer (pH 5.0)containing 0.05 M NaCI. The column was washed with the equilibrating buffer and the retained proteins were eluted with a linear NaCI gradient (0-0.4 M) in the same buffer at a flow rate of 15 m l / h . Active fractions (I.25 ml) were pooled and concentrated as described above.
3.4. Anah'tical teclmiques 3.2. Enzyme assay fl-Galactosidase activity was determined by measuring the 4-nitrophenol released from 4nitrophenyl fl-o-galactopyranoside (NPG). The reaction mixture (1 ml) contained 5-511 #1 of enzyme solution, depending on the enzyme concentration and 5 mM of substrate dissolved in tl.l M sodium acetate buffer, pH 5.11. After 15 min incubation at 50°C, reactions were stopped by the addition of 2 ml of 0.1 M Na_,CO 3 and the nitrophenol liberated was measured spectrophotometrically at 399 nm [5-8]. Enzyme activity is expressed as nmol of 4-nitrophenol m i n - t . Protein concentrations were determined by ti:,c method of Bradford [11].
3.3. Enzyme purification All procedures were done at 4°C. The powder from freeze-dried filtrates was solubilized in 0.1 M sodium acetate buffer (pH 5.11). The preparation was brought to 2 0 ~ saturation with ammonium sulfate and centrifuged at 8 t ~ ) × g for 30 rain. The supernatant fluid was brought to 8 0 ~ saturation with ammonium sulfate. The resulting precipitate was collected by centrifugation (801~1 × g , 30 min), dissolved in the smallest possible volume of sodium acetate buffer, then dialysed against distilled water then against sodium acetate buffer. The dialysed material (2 ml) was applied to an Ultrogel AcA 34 (1BF) column (2 cm × 95 cm) equilibrated with 0.01 M sodium acetate buffer (pH 5). Elution was performed with sodium acetate buffer at a flow rate of 20 m l / h and 2.5-ml fractions were collected. The fractions containing the enzyme activity were concentrated by centrifugation using Cf 25 Centriflo-ultrafiltration membrane cones (Am(con, USA), then applied to a CM Bio-Gel A (Bio
Molecular size and purity of the purified enzymes were examined by SDS-PAGE using 10('/t polyacrylamide resolving gels [12]. Molecular mass markers (Sigma or BioRad) were used to determine the M r of the enzyme. Proteins were made visible by silver staining using the Stratagene kit. Preparative isoelectric focusing (IEF) was performed in a Sephadex gel for 16 h. The pH gradient was formed with carrier ampholytes (pH range 3.11-10.0, Serva). Thirty fractions were collected and the proteins were remtwed by successive treatments with sodium acetate buffer and assayed for enzyme activity.
3.5. Enzyme chanwterization Estimates of optimal temperature and pH as well as thermal stability were made using a temperature range from 211° to 811°(` and a pH range from 2.11 to 8.11. The K m value was determined from the Lineweaver-Burk plot using NPG as a substrate at concentrations from 11.115 to 4 mg ml-- i. The ability of the purified enzyme to hydrolyze various substrates was assayed using NP-glycosides and polysaccharides at concentrations of 5 mM and 2 mg m l - ~, respectively.
3.6. hnmunological methods Antisera to the purified /3-galactosidase were prepared by immunizing rabbits with intradermal injections of 511 p,g of purified enzyme dissolved in 1 ml Freund's complete adjuvent followed by 3 injections at 20-day intervals. For immunoblotting, 2 /,tg of extracellular fungus proteins were separated on SDS-PAGE and then electroblotted onto nitrocellulose [13]. Remaining binding sites of nitrocellulose were blocked for 2 h with 5% non-fat dry milk in Tris-buffered saline (TBS). After three washes in TBS, protein blots were
incubated for 2 h in TBS with antiserum to /3galaetosidase (l:2(X~0) and specifically detected by staining with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (i : 1000), ! chloronaphthol (1.5 mg m l - ' and 0 . ( ) 1 % H.~O.,.
4. R E S U L T S A N D DISCUSSION
4. I. Prtnluction of/3-galactosidase The time course of secretion of /3-galactosidase activity in cultures grown on different carbon sources is represented in Fig. 1. A high /3-galactosidase activity was recorded in the presence of pectinolytic substrates and maximal enzymatic activities were reached after 8 days. /3Galactosidase activity was very low in the glucose medium and was only detected during the first week. T h e enzyme was also produced in medium containing CMC, the activity incre~ising slowly during the course of cultivation. In pectic or C M C media, growth yield was about seven times lower than in glucose medium. The differences observed in enzymatic activity were not associated with the growth yields and showed that pectinolytic substrates induced product'ion of extracellular /3-galactosidase. Similar observations have been made with Aspergillus or)'zae [14].
4.Z Galactosidase purification Purification of a /3-galactosidase was achieved in a series of three separation steps: (i) salt precipitaton, (it) gel filtration, and (iii) cation cx-
r
--
T
-
2
i
4
-
i
6
8
10
Fig. I. J:~-Galactosidaseproduction by S..~('lerotiorum during cultivation on citrus pectin ( 0 ) apple pectin ( • ) sodium pol.,,galacturonate ( • ) carboxymethylcellulose {o) carboxvincihylcellulosei o ) or glucose ( E3 ).
change chromatography. A main peak of /3galactosidase activity was retained on the cation~ exchange column and collected at 0.11 M NaCI during elution with a NaCI gradient. A lower peak was eluted at higher NaCI maturity but was not further studied. The purification data are summarized in Table I. The molecular mass of the enzyme was estimated to be 120 kDa by ultrogel AcA gel filtration column by comparing the elution volume to those of standard M, marker proteins (Fig. 2). After S D S - P A G E and silver staining of the purified enzyme, a single protein band was observed.
Table 1 Purification of/:Lgalactosidase from cultures of Sclen~tbda.~ch'rotiorum
Culture filtrate Ammonium sulfilte fractionation (20-8(IG.) Ultrogel AcA 34 CM Bio Gel A
Total activity x I 1)(•1U 4111
12
Days
Specific activity x l {100U/mg 5.7
Yield ("i) I[~}
Purification factor I.()
332
9,5
83
1.6
292 209
77.0 14~
73 52
13.4 26.2
10
0.5
f i 4.1 4.3
I 4.5
i 4.7
i 4.9
, 5.1
i 5.3
i 5.5
Log MW
O-gal I 4.3 4.5
I 4.7
, 4.9
,-,~, 5.1 5,3 Log MW
5.5
Fig. 2. Molecular mass determination of fl-galactosidase (A) gel filtration on Ultrogel AcA 34 Markers: (a) cytochrome c 125(X);(b) chicken ovalbumin 45(XX).(c) bovine serum albumin 660(X),(d) yeast hexokinase 100000, (e) eatalase 240(XI0.(B) SDS-polyacrylamide gel electrophoresis. Markers: (a) chicken ovalbumin 45000: (b) bovine serum albumin 66(h')0:(c) phosphorylase 97000- (d) /J-galactosidase 116(X~0;(e) myosin 2(10(XX).
The molecular mass estimated by S D S - P A G E was 120 kDa (Fig. 2). This indicates that the fl-galactosidase contains a single polypeptide. The molecular mass of the enzyme from $. sclerotiorum is close to those reported for A. niger [15], A. fonsecaeus [4], Curcu/aria inaequalis [16], and
A B ~ ~.~l
kDa 200 --.--116 ...... 9 7
,=--66 ~-45
P. canesca:s [6], which exhibit M r values ranging from l l 5 to 130 kDa. In analytical gel isoelectric focusing, fl-galactosidase was resolved as a single peak and had a p I value of pH 6.0. This p l is slightly higher than that of the enzymes from A. fonsecaeus (pH 4.2) [16] and N. crussa (pH 4.8) [1] but close to the p l of P. canescens enzyme (pH 6.7) [6]. Antibodies against the purified fl-galactosidase were raised in rabbits. 1 ng of purified proteins was detected with l : 1000 dilution in the most active antiserum. The abilities of the antibodies to cross-react with proteins from th¢ crude culture filtrate were examined by S D S - P A G E and immunoblotting of 2 p.g of extracellular proteins (Fig. 3). T h e antiserum detected one band of 120 kDa and revealed also a diffuse zone of higher Mr: this may be due to heterogenous glycosylation as shown for the fl-galactosidase from A. niger [15].
4.3. Characterization of the purified fl-galactosidase
Fig. 3. Eleetrophoretic analysis of the /J-galactosidase. A. SDS-PAGE of the purified extracellular fl-galactosidase from S. sclerotiomm. B. SDS-PAGE Western blot of extraeellular proteins from S. sclerotiontm. 2 pg of extracellular proteins were probed with the antiserum (1:2000) against the purified fl-galactosidase.
The enzyme showed a narrow pH activity curve: the optimal pH was 4.0. 50% of the maximal activity was found at about pH 3.0 and pH 5.0. In this respect the fl-galactosidase from S. sclerotiorum is similar to that from N. crussa [1], C. inaequalis [16] and A. fonsecaeus [4]. T h e optimum t e m p e r a t u r e for activity was 50°C but 25%
o f the maximal value was m e a s u r e d at 75°C. Thermostability was investigated by m e a s u r i n g the residual activity o f the enzyme after 4 h o f incubation at t e m p e r a t u r e s ranging from 20 to 80°C. T h e enzyme was stable below 45°C a n d exhibiting 50% activity after t r e a t m e n t at 65°C. A L i n e w e a v e r - B u r k plot gave a K m = 0.5 m M 4 NPG. N o significant alteration o f t h e enzyme activity was p r o d u c e d by metals such as Cu-" ÷ a n d Hg -'~ which inhibit the /3-galaetosidase from Aureobasidium pulhdans [17] or C o 2~ and Ca -'+ which activate the e n z y m e s from A. pulhdans [17] and B. bassiana [7]. H o w e v e r 4 - c h l o r o m e r c u r i b e n z o a t e a n d SDS strongly inhibited the /Jgalactosidase activity. T h e action o f the p u r i f i e d / 3 - g a l a c t o s i d a s e was tested on a large n u m b e r o f substrates. T h e enzyme h a d no d e t e c t a b l e glycosidase activity against the p N P - ~ - I i n k e d sugars pNP-/~-xyloside, pNP-/3-glucoside, pNP-/3-ceilobioside, pNP-/3lactoside a n d p N P - a - a r a b i n o s i d e . H o w e v e r pNP-/3 fucoside was weakly hydrolysed by the enzyme. This activity r e p r e s e n t e d about 15% o f the activity m e a s u r e d against p N P galactoside. T h e enzyme was not active against citrus pectin, apple pectin and Na polygalacturonate which are very efficient inducers o f its synthesis. T h e very high level o f induction o f /3-galactosidase in the p r e s e n c e o f pectic c o m p o u n d s suggests that this enzyme may be involved in the process of degradation of these heteropolysaccharides. Pectin s u b s t a n c e s contain a g a l a c t u r o n a n b a c k b o n e with side chains o f various sugar residues such as xylose, arabinose and galactose [18]. During growth o n pectic polymers, S. sclerotiomm secretes a wide range o f p e c t i n a s e s and polygalacturonases, t o g e t h e r with cellulolytic a n d hemicellulolytic enzymes, able to d e g r a d e plant cell wall polymers [8,10,19]. During hydrolysis, short side chains released from the pectic polym e r s may serve as inducers o f the fl-galactosidase
activity. As this enzyme attacks galactoside and, to a lesser extent, fucoside derivatives, /3-galactosidase may be involved in the final o f d e g r a d a tion o f the neutral side chains o f pectin.
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