534 C PARTIAL PURIFICATION AND CHARACTERISATION OF · (1) CB-Departamento de Biologia, Universidade...
1
29,8 800 Chitin 5 28 1150 S-2 4 9 450 S-1 4 1,6 84 P-2 4 12,8 640 P-1 4 % Wt (mg) Fraction Table 2: Fractionation of P. guillermondii cell walls 4) Glucans were extracted and partially purified according to Manners et al. 5) Chitin were extracted and partially purified as described by Fleet. PARTIAL PURIFICATION AND CHARACTERISATION OF PARTIAL PURIFICATION AND CHARACTERISATION OF CANDIDA NODAENSIS CANDIDA NODAENSIS KILLER TOXIN KILLER TOXIN da Silva S .(1), Aguiar, C. (1), Veríssimo, P. (2), Pires, E. (2) and Lucas, C. (1) CB-Departamento de Biologia, Universidade do Minho, Braga,Portugal. (2) CNC- Laboratório de Biologia Molecular e Biotecnologia, Univers. Coimbra, Coimbra, Portugal. The production and secretion of killer (K) toxins is a widespread phenomenon in yeasts, although not uniformly distributed within certain genera or species. Several halotolerant yeast strains, displaying a broader spectrum of K activity in the presence of salt, were identified in a previous survey. The possibility that these zymocins could be more stable than the ones described so far, lead to the selection of the extremely halotolerant yeast C. nodaensis for further work. Preliminary experiments performed to characterize C. nodaensis K factor showed that, besides keeping its biological activity at high NaCl concentrations, is stable after incubation in a relatively broad range of temperature and pH values, whether or not in the presence of salt. These results renewed the interest on this zymocin for the development of yeast-based biocontrol strategies in several biotechnological applications. The isolation of this K toxin was already achieved by a experimental protocol involving ethanol precipitation of C. nodaensis culture supernatants. Several different approaches are now in progress in order to achieve its purification and further molecular and biochemical characterisation. The identification of the cell wall receptor for this K toxin is also under study, to perform toxin purification by affinity chromatography. The study of this zymocin mode of action is under way and will certainly contribute to evaluate its biotechnological potentialities for several applications. 534 C The production of antimycotically active toxins, so-called killer (K) toxins or zymocins, is a widespread phenomenon in various yeast genera, although the most intensively studied killer systems are still those of Saccharomyces cerevisiae and Kluyveromyces lactis (for reviews see 1-2). During the last two decades, killer toxins and killer yeasts were found to have several potential applications; for instance in the food and fermentation industries, in the bio-typing of medically important microorganisms, in the development of novel antimycotic agents for the treatment of fungal infections and in the field of recombinant DNA technology. This increasingly interest in killer toxins for all the mentioned applications requires undoubtedly a detailed knowledge and understanding of the biology of killer yeasts, which will provide important insights relevant for its use as antimicrobial agents. In a previous survey, we studied several halotolerant yeasts which killer activity was expressed, even stimulated, under heavy salt-stress conditions (3). From this research, the halotolerant yeast Candida nodaensis was identified as one of the strongest salt-stimulated K phenotypes, being selected for further studies. Results obtained so far, in what concerns C. nodaensis zymocin activity/stability under temperature, pH and ionic strength, showed that this is in fact a very stable zymocin. Presently, several strategies are under way to achieve the isolation and purification of this zymocin, in order to enable further evaluation of its biotechnological potentialities, namely in the high-salt food products preservation from spoilage by other yeasts. YPD (pH 4) YPD (pH 4) Growth at 25°C, 160 rpm Early stationary phase Centrifugation 2’, 7000 rpm, 4°C Filtration of culture supernatant 0.45 µm Precipitation of culture filtrate Adition of 3/4 vol. ethanol Centrifugation Liophilization Ressuspension in 10 mM CPB,200mM NaCl pH 4.0 Ethanol precipitation 10’, 7000 rpm, 0°C Isolation of C. nodaensis killer factor 0 20 40 60 80 100 lost activity (%) S-1 S-2 P-1 P-2 Chitin Fig.5. Toxin K activity after adsorption to P. guillermondii cell walls components. C- Cell wall receptor for Killer toxin (1) Magliani, W. et al. (1997) Yeast killer systems. Clin. Microbiol. Rev., 10 (3): 369-400. (2) Schmitt, M. And Breinig, F. (2002) The viral killer system in yeast: from molecular biology to application. FEMS Microbiol. Rev. 26: 257-276. (3) Aguiar, C. and Lucas, C. (2000) Killer/sensitivity phenotypes and halotolerance, Food Technol. and Biotechnol., 38: 39-46. (4) Manners, D.J. et al. (1973) Biochem. J.135, 19-30. (5) Fleet, G. H.. (1991). Cell walls, p. 200-277. In A. H. Rose and J. S. Harrison (ed.). The ,yeast vol. 4. Yeast organelles. A. B. Fig.1. A- Elution profile of K toxin through a Superdex 200 column (Pharmacia) equilibrated and eluted with CPB 200 Mm, pH 4.0 ( fractions with K activity; fractions without K activity. B and C -assay for killer activity detection. C. Partial purification Molecular Exclusion Chromatography 0 20 40 60 80 100 0 30 60 90 120 150 Time (min.) % viable cells Fig.2. Assay for killer activity quantification (Influence of partially purified K toxin on P. guilliermondii viability ( fractions with K activity; fractions without K activity). 1 2 3 17,0 32,8 43,07 82,6 221,9 Fig.3. Silver-stained SDS-PAGE (12% gel). Lane 1 fractions with K activity Lane 2 fractions without K activity Lane 3 wt marker polypeptides. B- Affinity Chromatography with Concanavalin A 4 221,9 32 29 17 82,6 43 1 3 2 B A Fig. 4. Silver stained (A) and Schiff-stained (B) SDS-PAGE (12% gel) after affinity chromatography with concanavalin A. Lane 1, wt marker polypeptides; Lane 2, fraction with K activity; Lane 3, fraction which displayed k activity after affinity chromatography; Lane 4, fraction with k activity after Schiff stained. Strategies towards further purification of C. nodaensis K toxin (A, B, C) A - Ion Exchange Chromatography Table 1. Killer activity assay after Ion Exchanger Chromatography. Strong anion Strong cation Type of Exchanger Q Sepharose Fast Flow Sp Sepharose Fast Flow Ion Exchange Chromatography Killer activity 0 20 40 60 80 100 0 15 30 45 60 75 90 Time (min.) % viable cells Fig.6. Influence of P-2 fraction on toxin mediated cell death ( cells with K toxin ; cells with K toxin after previous incubation with P-2 fraction). 0 25 50 75 100 Lost activity (%) Glucan xylan Laminarin Others Fig. 7. Toxin K activity after adsorption to different commercial carbohydrates. (others: Amylopectin, Amylose, Chitin, Polygalacturonic acid and Pullan). _ Glucan by yeast (1-4)-β Xylan (1-6)- α / (1-4)-α Pullan (1-4)-α Polygalacturonic acid (1-3)-β Laminarin (1-4)-β Chitin (1-4)-α Amylose (1-4)-α Amylopectin Main glucosidic linkage Carbohydrate Table 3. Different commercial carbohydrates. Ionic (A) and/or affinity (B,C) chromatography will be further explored to achieve the purification of C. nodaensis K toxin. The main purpose is to identify, in SDS-PAGE, protein band(s) associated with K activity phenotype.
Transcript of 534 C PARTIAL PURIFICATION AND CHARACTERISATION OF · (1) CB-Departamento de Biologia, Universidade...
29,8800Chitin 5281150S-24
9450S-14
1,684P-24
12,8640P-14
%Wt (mg)Fraction
Table 2: Fractionation of P. guillermondii cell walls
4) Glucans were extracted and partially purified according to Manners et al.
5) Chitin were extracted and partially purified as described by Fleet.
PARTIAL PURIFICATION AND CHARACTERISATION OFPARTIAL PURIFICATION AND CHARACTERISATION OF
CANDIDA NODAENSISCANDIDA NODAENSIS KILLER TOXIN KILLER TOXINda Silva S.(1), Aguiar, C. (1), Veríssimo, P. (2), Pires, E. (2) and Lucas, C.
(1) CB-Departamento de Biologia, Universidade do Minho, Braga,Portugal.(2) CNC- Laboratório de Biologia Molecular e Biotecnologia, Univers. Coimbra, Coimbra, Portugal.
The production and secretion of killer (K) toxins isa widespread phenomenon in yeasts, although not uniformlydistributed within certain genera or species. Severalhalotolerant yeast strains, displaying a broader spectrum of Kactivity in the presence of salt, were identified in a previoussurvey. The possibility that these zymocins could be morestable than the ones described so far, lead to the selection ofthe extremely halotolerant yeast C. nodaensis for further work.Preliminary experiments performed to characterize C.nodaensis K factor showed that, besides keeping its biologicalactivity at high NaCl concentrations, is stable after incubationin a relatively broad range of temperature and pH values,whether or not in the presence of salt. These results renewedthe interest on this zymocin for the development of yeast-basedbiocontrol strategies in several biotechnological applications.The isolation of this K toxin was already achieved by aexperimental protocol involving ethanol precipitation of C.nodaensis culture supernatants. Several different approachesare now in progress in order to achieve its purification andfurther molecular and biochemical characterisation. Theidentification of the cell wall receptor for this K toxin is alsounder study, to perform toxin purification by affinitychromatography. The study of this zymocin mode of action isunder way and will certainly contribute to evaluate itsbiotechnological potentialities for several applications.
534 C
The production of antimycotically active toxins, so-called killer (K) toxins or zymocins, is a widespread phenomenon in various yeast genera, although the most intensively studied killer systems arestill those of Saccharomyces cerevisiae and Kluyveromyces lactis (for reviews see 1-2). During the last two decades, killer toxins and killer yeasts were found to have several potential applications; forinstance in the food and fermentation industries, in the bio-typing of medically important microorganisms, in the development of novel antimycotic agents for the treatment of fungal infections and inthe field of recombinant DNA technology. This increasingly interest in killer toxins for all the mentioned applications requires undoubtedly a detailed knowledge and understanding of the biology of killeryeasts, which will provide important insights relevant for its use as antimicrobial agents.
In a previous survey, we studied several halotolerant yeasts which killer activity was expressed, even stimulated, under heavy salt-stress conditions (3). From this research, the halotolerantyeast Candida nodaensis was identified as one of the strongest salt-stimulated K phenotypes, being selected for further studies. Results obtained so far, in what concerns C. nodaensis zymocinactivity/stability under temperature, pH and ionic strength, showed that this is in fact a very stable zymocin. Presently, several strategies are under way to achieve the isolation and purification of thiszymocin, in order to enable further evaluation of its biotechnological potentialities, namely in the high-salt food products preservation from spoilage by other yeasts.
YPD (pH 4)YPD (pH 4)
Growth at 25°C, 160 rpmEarly stationary phase
Centrifugation
2’, 7000 rpm, 4°C
Filtration of culture supernatant
0.45 µm
Precipitation of culture filtrate
Adition of 3/4 vol. ethanol
Centrifugation
Liophilization
Ressuspension in10 mM CPB,200mM NaCl pH 4.0
Ethanol precipitation
10’, 7000 rpm, 0°C
Isolationof C. nodaensis
killer factor
0
20
40
60
80
100
lost
act
ivity
(%
)
S-1 S-2 P-1 P-2 Chitin
Fig.5. Toxin K activity after adsorption to P. guillermondiicell walls components.
C- Cell wall receptor for Killer toxin
(1) Magliani, W. et al. (1997) Yeast killer systems. Clin.Microbiol. Rev., 10 (3): 369-400.(2) Schmitt, M. And Breinig, F. (2002) The viral killersystem in yeast: from molecular biology to application.FEMS Microbiol. Rev. 26: 257-276.(3) Aguiar, C. and Lucas, C. (2000) Killer/sensitivityphenotypes and halotolerance, Food Technol. andBiotechnol., 38: 39-46.(4) Manners, D.J. et al. (1973) Biochem. J.135, 19-30.(5) Fleet, G. H.. (1991). Cell walls, p. 200-277. In A. H.Rose and J. S. Harrison (ed.). The ,yeast vol. 4. Yeastorganelles.
A.
B.
Fig.1. A- Elution profile of K toxin through a Superdex 200 column(Pharmacia) equilibrated and eluted with CPB 200 Mm, pH 4.0 (fractions with K activity; fractions without K activity. B and C -assay forkiller activity detection.
C.
Partial purificationMolecular Exclusion
Chromatography
0
20
40
60
80
100
0 30 60 90 120 150
Time (min.)
% v
iabl
e ce
lls
Fig.2. Assay for killer activity quantification (Influence ofpartially purified K toxin on P. guilliermondii viability ( fractionswith K activity; fractions without K activity).
1 2 3
17,0
32,8
43,07
82,6
221,9
Fig.3. Silver-stained SDS-PAGE (12% gel).
Lane 1 fractions with K activity
Lane 2 fractions without K activity
Lane 3 wt marker polypeptides.
B- Affinity Chromatography with Concanavalin A
4221,9
32
29
17
82,6
43
1 32
BA
Fig. 4. Silver stained (A) and Schiff-stained (B) SDS-PAGE (12% gel) afteraffinity chromatography with concanavalin A.
Lane 1, wt marker polypeptides;
Lane 2, fraction with K activity;
Lane 3, fraction which displayed k activity after affinity chromatography;
Lane 4, fraction with k activity after Schiff stained.
Strategies towards further purification of C. nodaensis K toxin (A, B, C)
A - Ion Exchange Chromatography
Table 1. Killer activity assay after Ion Exchanger Chromatography.
Strong anionStrong cationType of Exchanger
Q Sepharose Fast FlowSp Sepharose Fast FlowIon Exchange Chromatography
Killer activity
0
20
40
60
80
100
0 15 30 45 60 75 90
Time (min.)
% v
iab
le c
ell
s
Fig.6. Influence of P-2 fraction on toxin mediated cell death
( cells with K toxin ; cells with K toxin after previous incubation
with P-2 fraction).
0
25
50
75
100
Lo
st
activity (
%)
Glucan xylan Laminarin
Others
Fig. 7. Toxin K activity after adsorption to different commercial carbohydrates.(others: Amylopectin, Amylose, Chitin, Polygalacturonic acid and Pullan).
_Glucan by yeast(1-4)-βXylan
(1-6)- α / (1-4)-αPullan
(1-4)-αPolygalacturonicacid
(1-3)-βLaminarin(1-4)-βChitin(1-4)-αAmylose(1-4)-αAmylopectin
Main glucosidic linkageCarbohydrate
Table 3. Different commercial carbohydrates.
Ionic (A) and/or affinity (B,C) chromatography will be further explored to achieve the purification of C. nodaensis K toxin.
The main purpose is to identify, in SDS-PAGE, protein band(s) associated with K activity phenotype.
