THERMOSTABILITY OF SUPEROXIDE DISMUTASES FROM MONASCUS PURPUREUS VAR. ALBINUS

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1 THERMOSTABILITY OF SUPEROXIDE DISMUTASES FROM MONASCUS PURPUREUS VAR. ALBINUS E. Pisareva 1, A. Kujumdzieva 2 National Bank for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria 1 University of Sofia "St. Kliment Ohridski", Faculty of Biology, Department of Microbiology, Sofia, Bulgaria 2 ABSTRACT Monascus purpureus var. albinus was used to study thermostability of the superoxide dismutase in crude enzyme preparation. The tested culture was prepared through submerged cultivation in laboratory fermentor on medium Chapec-Dox. Samples were collected from the middle and the end of the logarithmic and from stationary phases. Thermostability of the crude extract obtained was investigated at 5 ºC, ºC, 7 ºC and ºC. The samples from the middle logarithmic phase preserve its total superoxide dismutase activity between 33% and % after treatment at ºC for 3 utes. The samples from stationary phase preserve almost % activity at the same conditions. Critical point for inactivation of total SOD activity is 5 utes after the temperature shock. The SOD enzymes in Monascus purpureus var. albinus are represented by three izoemzimic forms - one for Cu/Zn type of SOD and two for Mn type of SOD with relative mobility.1,.43 and.45 respectively. The Mn type of SOD was inactivated completely after treatment at ºC for 3 utes. The thermostability of enzyme was detered by Cu/Zn type SOD. Introduction Superoxide dismutase (SOD) is one of the crucial enzyme of the cell defense against high reactive oxygen radicals, arising through single electron reduction of the oxygen. This enzyme, together with catalase protects oxygenation of the lipids, proteins and nucleic acids by reactive oxygen species. The eucariotic cells posses two type of SOD Mn SOD and Cu/Zn SOD (16, 13, 1, 15). There are a lot of investigations about antioxidant system and its thermostability in bacteria (9, 12), but this question is not well clarified in fungi. There is some data about this property of SOD from Neurospora crassa, which preserve 74% of its Cu/Zn SOD and % of its Mn SOD during 1 utes exposure at 75 ºC (6). The strains of thermotolerant yeasts Kluyveromyes lactis 9-3 and 9-4 posses a high thermo-resistance of SOD with half-life between 8 and 22 utes at 75 º respectively (11). Fungi belonging to genus Monascus are well known as producers of primary and secondary metabolites for biotechnology and of natural red pigments, chemically related to polyketides. These pigments can react with oxygen and take a part in cell antioxigen protection. The studying of this protection is very interesting because the pigment biosynthesis depend on oxygen supply (7). This is the reason to investigate the antioxidant defense system and their properties, but independently of pigments production in an albino strain Monascus purpureus var. albinus. Materials and Methods Microorganism and cultivation: The strain Monascus purpureus var. albinus, lacking possibility to produce pigments, was used in present investigation. A submerged cultivation of the strain was per- Biotechnol. & Biotechnol. Eq. 19/5/1 98

2 formed in laboratory fermentor Galencamp with volume 4.5l on modified medium Chapec Dox (g/l): glucose, NH 4 Cl 3, yeast extract 1, KH 2 PO 4 1, MgSO 4.7H 2 O.5, NaCl.5, FeSO 4.7H 2 O.1, ZnSO 4.1, ph was adjusted to 6.. The bioreactor was inoculated with 1% from total volume with 48h submerged shaken culture on the same medium. The cultivation was carried out 48 hours, till the carbon source was exhausted under the following conditions: temperature 3 ºC, ph=4.5, agitation 3 rpm, air l/l/. Samples were collected at 24, 28, 32, 36 and 48 hours. Analytical and biochemistry analyses Cell-free extract was prepared as was described by Rasheva et al. (14) SOD activity was detered by the method of Bauchamp and Fridovich (2). Protein concentration was analyzed by the method of Lowry et al. (8). Thermostability assay of the SOD was expressed as residual total enzyme activity after treatment of the samples of cell-free extracts at 5 ºC, ºC, 7 ºC and ºC at every 5 utes during half an hour. Inhibition analyses were performed by 2mM KCN and 2mM NaN 3 according to Borders and Fridovich (3). Electrophoretic analyses were performed on native PAGE as was described by Zamfirova et al. (17). Results and Discussion It is known that there is no method for prediction of thermostability of an enzyme. It is supposed that this property is characteristic for microorganisms growing at elevated temperatures. However, some mesophiles are also able to produce enzymes which are stable at temperatures significantly higher than the organism s optimal growth ones. Monascus purpureus var. albinus is a temperature sensitive mutant strain isolated from thermotolerant parent strain Monascus purpureus CBS. The maximal growth temperature is detected as TABLE Specific SOD activity in cell-free extracts form samples taken at different hours after the beginning of cultivation Time, h SOD activity (U/mg protein) ºC. It is a matter of particular interest to study the thermostability of SOD enzymes and to detere its regarding to different type of SOD (Mn and Cu/Zn containing ones). Specific activity and native electrophoretic profile of SOD The specific activity of SOD enzymes of each cell-free extract of samples varied between 1.1 U/mg protein and U/mg protein (Table). Maximal enzyme activity was observed during the end of the logarithmic phase (about 36 hour). SOD enzymes are occurred in three different types - Cu/Zn SOD, Mn SOD or by Fe SOD (16, 6, 3). The types of isoenzimic SOD forms in investigated strain were detered by native PAGE of the cell-free extract of the sample from the 48 th hour of the beginning of cultivation (line 1, control) and after inhibition with NaN 3 (line 2) and with KCN (line 3). The obtined results are presented on Fig. 5. SOD enzymes in Monascus purpureus var. albinus were presented by 3 izoenzimic forms, which have relative electrophoretic mobility (R m ).1,.43 and.45 respectively. After inhibition with 2 mm NaN 3 there are no difference between the control and tested sample, which means that no one of these forms is Fe SOD. After inhibition with 2mM KCN the bands with R m.1 disappeared. This form is presented by Cu/Zn type of SOD and another two by Mn type. 99 Biotechnol. & Biotechnol. Eq. 19/5/1

3 residual SOD activiti, % native cell-free exract (not treated) 5C C 7C C Fig. 1. Residual SOD activity of the samples taken at 24 hour. residual SOD, % native cell-free exract (not treated) 5C C 7C C Fig. 2. Residual SOD activity of the samples taken at 28 hour. Thermostability of SOD The free-cell extracts obtained from the samples collected at 24, 28, 32, 36 and 48 hours from the beginning of the fermentation process were treated with different temperatures: 5 ºC, ºC, 7 ºC and ºC. As a control was used not treated cell-free extract of samples, which activity was considered as %. The residual enzyme activity of the SOD was measured at every 5 utes for 3 utes and was presented as percentage of residual activity in comparison to those of the control. The obtained results are shown on Figs The inactivation of SOD in different samples was influenced differently by high temperatures. The sample from 24 hour after 3 utes, preserves its activity at 5 ºC, but strong decrease is observed after ºC and 7 ºC treatment (respectively 45% and 53% losses of the activity). At ºC the residual SOD activity was only 33%. The sample from 28 hour preserves 55% of the activity at ºC, and was inactivated with 55% and % at 7 ºC and ºC respectively. The crude extracts samples obtained from the cells collected after 32, 36 and 48 hours cultivation, presented high thermostability and retained their activity almost at % for 3 utes at ºC. The kinetics of the inactivation of the SOD in the samples taken at 24 and 28 hours at ºC has a non-linear character (Fig. 1 and 2). The 5 th ute of the treatment was a critical for SOD activity and after then the inactivation of the enzyme was preserved almost the same lines. Inhibition analysis To clarify which type of SOD is responsi- Biotechnol. & Biotechnol. Eq. 19/5/1

4 residual SOD activity, % 5C C 7C C temperature cell-free exract (not treated) sample taken at 32 hour sample taken at 36 hour sample taken at 48 hour Fig. 3. Residual SOD acivity of the cell-free exracts after exposure at high temperature for 3 ues. residual SOD activity, % NaN3 KCN Fig. 4. Residual SOD activity of the sample taken at 48 hour after inhibition with KCN and NaN 3. ble for the thermostability of the enzyme, the inhibition experiments with the cellfree extract from the sample taken at 48 hour from the beginning of the fermentation, were performed. This sample shown the highest thermostability at C. After the temperature treatment the sample was inhibited with two different inhibitors KCN and NaN 3 with the same concentration of 2mM. KCN inhibited Cu/Zn SOD, while NaN 3 inhibited Fe SOD and partly Mn SOD (1, 5). The results of the experiments are shown on Fig. 4. After inhibition of the cell-free extracts of the samples with KCN, they shown only 15% residual SOD activity, while these treated with NaN 3 did not shown changes in their activity and preserved % of its. This results lead to conclusion that the high thermostability of the enzyme is due to SOD of Cu/Zn type, while Mn type SOD is thermolabilе and is inactivated at the high temperature. Electrophoretic analysis The results from the inhibition analysis were confirmed from the electrophoretic ones. The native PAGE was carried out with the cell-free extract of the sample taken at 48 hour native cell-free extract (line1), native cell-free extract treated with C for 3 utes (line 4) and native cell-free extract treated with high temperature and inhibited with 2mM KCN (line 5). The obtained results are schematically presented on Fig. 5. In accordance to the previous results received from the inhibition analyses in the sample treated with high temperature was preserved only the isoensimic forms, which have lower electrophoretic mobility. After inhibition with KCN this isoenzimic form was not observed too, which demonstrated that this form, responsible for thermostability is Cu/Zn type of SOD. 11 Biotechnol. & Biotechnol. Eq. 19/5/1

5 Cu/Zn SOD Mn SOD Fig. 5. Native PAGE - Electrophoretic mobility of the SOD types. 1 cell-free extract; 2 - cell-free extract inhibited with NaN 3 ; 3 - cell-free extract inhibited with KCN; 4 - cell-free extract treated at ºC for 3 utes; 5 - cell-free extract treated at ºC for 3 utes and inhibited with KCN. As a conclusion of the obtained results it can be supposed that the higher thermostability of the Cu/Zn SOD from the samples from the end of logarithmic phase and stationary phase than the samples taken at the middle of the logarithmic phase, is a result from the production of the stabilization factor, which conjugate with Cu/Zn SOD and increase its thermostability. The established thermostability of the SOD from Monascus purpureus var. albinus is a field for farther investigation of the properties of this enzyme in fungi belonging to genus Monascus and possibility for their industrial production and application. REFERENCES 1. Asada K., Yoshikawa K., Takanashi M. (1975) J. Biol. Chem., 379, p Beaucchamp C., Fridovich I. (1971) Anal. Biochem., 44, Borders C. J., Fridovich I. (1985) Arch. Biochem. Biophys., 241, Dunlap P., Steinman H. (1986) J. Bacteriol., 165(2), Hafner D. (1973) J. Biol. Chem., 248, Henry L.E., Hall D.O. (1977) Plant Physiol., Kujumdzieva A., Rasheva T., Savov V. (1994) Elsevier Science, Lowry H.O., Rosebrough N.J., Farr O.L., Randle R.J. (1951) J. Biol. Chem., 193, Lumsden J. (1974) Biochem. Biophys., 58, p Misra H., Fridovich I. (1972) J. Biol. Chem., 241, p Nedeva T., Savov V., Kujumdzieva-Savova A., Davidov E. (1993) FEMS Microbiol. Letters, 17, Puget K., Michelson A. (1974) Biochem, Biophys. Res. Comun., 58, p Ravindranath S., Fridovich I. (1975) J. Biol. Chem., 25, p Rasheva T., Nedeva T., Hallet J.-N., Kujumdzieva A. (3) Antonie van Leewenhoek, 83, Shatsman A., Kosman D. (1979) J. Bacteriol., 137, p Weser U., Fritzdorff A., Prinz R. (1972) FEBS Lett., 27, p Zamfirova D., Todorova T., Hristozova Ts., Nedeva T., Michailova L. (3) Biotech. Biotechnol. Equipment, 17(2), Biotechnol. & Biotechnol. Eq. 19/5/1 12