Effect of cations on the activity of glutamate dehydrogenase in Acinetobacter calcoaceticus IMV, Rhodococcus erythropolis IMV and Nocardia vaccinii IMV grown on industrial waste

Introduction. It is studied the activity of NADP⁺-dependent glutamate dehydrogenase in the presence of mono- and divalent cations (potential activators of this key enzyme of surface-active aminolipids biosynthesis) in A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 during cultivation on waste of biodiesel production and sunflower oil waste.

Materials and methods. Cultivation of strains was performed in liquid mineral media using as substrates: refined and waste (after frying potato) sunflower oil, refined glycerol and waste of biodiesel production. NADP+-dependent (EC 1.4.1.4) glutamate dehydrogenase activity in cell-free extracts was analyzed for glutamate formation during oxidation of NADPH at 340 nm. Monovalent (Na+, K+) and divalent (Mg²+, Ca²+, Zn²+) cations in the form of salts of NaCl, KCl, MgSO4 ^x 7H2O, CaCl2 and ZnSO4 ^x 7H2O were added to the reaction mixture, as well as into the medium for strains cultivation.

Results and discussion. Calcium cations were found to be activators of NADP+-dependent glutamate dehydrogenase activity in R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 grown on refined and waste sunflower oil: in the presence of 1-5 mmol Ca²+ in the mixture, the activity of the enzyme increased 1.3-2 times compared with that without these cations. The increase in the concentration of CaCl2 to 0.2-0.4 g/l in oil-containing medium of strains IMV Ac-5017 and IMV B-7405 cultivation was accompanied by an increase in NADP+-dependent glutamate dehydrogenase activity by 1.3-1.5 times compared with that on basic medium. When additional quantity of CaCl2 (0.1-0.2 g/l) was introduced into the medium with purified glycerol for the cultivation of A. calcoaceticus IMV B-7241, an increase in NADP⁺-dependent glutamate dehydrogenase activity was observed by almost 2.5-3 times compared with those for strain IMV B- 7241 on the basic medium. There was no impact of activating cations magnesium, zinc, potassium and sodium on NADP⁺-dependent glutamate dehydrogenase activity of all strains grown on oil-containing substrates and glycerol of different degrees of purification.

Conclusion. The results demonstrate the possibility to increase activity of key enzymes of the biosynthesis of the desired product: the composition of the medium should be modified by changing the content of enzymes' activators.

Keywords: Glutamate dehydrogenase Activators Waste Biosurfactants

Introduction

Microbial surfactants are products of multifunctional purpose because they not only reduce the surface tension at the interface and emulsify different substrates, but also exhibit antimicrobial and antiadhesive activity (e.g. ability to destruct biofilms) [1-4]. However, under different conditions of producers cultivation, the composition of surfactants and their properties may change. This is due to the fact that microbial surfactants are secondary metabolites that are synthesized as a complex of similar compounds, the composition and ratio of which may vary depending on the growing conditions of the producer [5], which in turn will change the properties of the final product.

Earlier [5] we showed that the detection of potential activators and / or inhibitors of key enzymes (defining biosynthesis of components of the microbial surfactant complex responsible for certain properties) allows to regulate the composition and therefore the properties of the final product. This could be achieved by following modification of the nutrient composition.

A key enzyme of biosynthesis of surface-active aminolipid, responsible for the antimicrobial activity in Acinetobacter calcoaceticus IMV B-7241, Nocardia vaccinii IMV B-7405 and Rhodococcus erythropolis IMV Ac-5017 is NADP+-dependent glutamate dehydrogenase. Its activators in IMV В-7241 strain are cations of calcium, magnesium and zinc, in IMV Ac-5017 - calcium, in IMV B-7405 - calcium, sodium and potassium [6]. Further increase of the content of enzyme's activators in the medium cultivation was accompanied by an increase of NADP+ dependent glutamate dehydrogenase by 1.5-3 times compared to a basic medium.

Further experiments showed that the additional introduction of CaCh (0.1 g/l) into the cultivation medium of R. erythropolis IMV Ac-5017, increasing the concentration of this salt to 0.4 g/l in the medium for growing N. vaccinii IMV -7405, as well as the adding of CaCh (0.1 g/l), increasing the content of MgSO4-7H2O to 0.2 g/l or the adding of Zn²+ (38 pm) in the medium of A. calcoaceticus IMV B-7241 cultivation was accompanied by the synthesis of surfactants, which minimal inhibitory concentrations (MICs)relative to bacterial and yeast test cultures were 1.2-13 times lower, their adhesion on abiotic surfaces treated with the surfactants was on average 10-40% lower, and the degree of biofilm destruction was 7 -20% higher compared to the indicators for surfactants obtained on the base medium [6].

In publication [6] A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 were grown on ethanol, while N. vaccinii IMV B-7405 - on purified glycerol. One of the approaches to reducing the cost of the final product is the use of industrial waste as substrates for their production. In our previous studies [7], we have established the possibility of surfactant synthesis under cultivation of strains IMV B-7241, IMV Ac-5017 and IMV B- 7405 on waste oil and waste of biodiesel production. However, the antimicrobial activity of surfactants synthesized by N. vaccinii IMV B-7405 on the oil-based substrates depended on the quality of waste oil [8]. The biological activity of surfactants synthesized by A. calcoaceticus IMV B-7241 on waste of biodiesel production was lower than the surfactants obtained on purified glycerol [9].

We assumed that antimicrobial and antiadhesive activity of surfactants synthesized on industrial waste can be increased by adding of activators of NADP⁺-dependent glutamate dehydrogenase into the medium cultivation. However, the presence of toxic substances in such substrates [7-9] may cause inhibition of the activity of this key enzyme.

The aim of this work was to determine activity of NADP⁺-dependent glutamate dehydrogenase in the presence of mono- and divalent cations (potential activators of this key enzyme of surface-active aminolipids biosynthesis) in A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 during cultivation on waste of biodiesel production and sunflower oil waste.

Materials and methods

The objects of research were strains of oil-oxidizing bacteria, identified as Nocardia vaccinii K-8, Acinetobacter calcoaceticus K-4 and Rhodococcus erythropolis EK-1. Strains K-8, K-4 and EK-1 are registered in the Depository of Microorganisms of D.K. Zabolotnyi Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine with the numbers IMV B-7405, IMV B-7241 and IMV Ac-5017, respectively.

Medium composition and conditions of

A liquid medium of the following composition (g/l) was used for the cultivation of N. vaccinii IMV B-7405: NaNOs - 0.5; MgSЬ4 x 7H2O - 0.1; CaCh - 0.1; KH2PO4 - 0.1; FeSЬ4 x 7H2O - 0.01; yeast autolysate - 0.5% (v/v).

Modifications of basic medium:

1. increasing the content of CaCh to 0.2 g/l;

2. increasing the content of CaChto 0.4 g/l;

3. adding NaCl (0.5 g/L);

4. adding KCl (0.5 g/l);

5. adding NaCl (0.5 g/l) and KCl (0.5 g/l).

A. calcoaceticus IMV B-7241 strain was cultivated in the following medium (g/l): (NH2)2CO - 0.35; MgSO4 x 7H2O - 0.1; NaCl - 1.0; Na2HPO4 - 0.6; KH2PO4 - 0.14; pH 6.8-7.0. Yeast autolysate - 0.5% (v/v) and a solution of microelements - 0.1% (v/v) were additionally included into the medium. The solution of microelements contained (g/100 ml): ZnSO4 x 7H2O - 1,1; MnSO4 x H2O - 0.6; FeSO4 x 7H2O - 0.1; CuSO4 x 5H2O - 0.004; CoSO4 x 7H2O - 0.03; H3BO3 - 0.006; KI - 0.0001; EDTA (Trilon B) - 0.5.

Modifications of the basic medium: 1) adding CaCh (0.1 g/l), 2) adding CaCh (0.2 g/l), 3) adding Zn²+ (38 pmol), 4) adding CaCh (0.1 g/l) and Zn²+ (38 pmol), 5) CaCh (0.2 g/l) and Zn²+ (38 pmol).

R. erythropolis IMV Ac-5017 strain was grown in the following medium (g/l): NaNO3 - 1,3; NaCl - 1.0; Na2HPO4 x 12H2O - 0,6; KH2PO4 - 0.14; MgSO4 x 7H2O - 0.1; FeSO4 x 7H2O - 0.001.

Modifications of the basic medium: 1) adding CaCh (0.1 g/l).

Refined and waste sunflower oil (after frying potato at McDonald's restaurant chain, Kyiv, Ukraine), purified glycerol and waste of biodiesel production (biofuel plant, Poltava region, Ukraine) were used as carbon sources. The concentration of substrates was 1% (v/v).

Cultures in the exponential growth phase, grown in media of the above mentioned composition, containing 0.5% (v/v) of the corresponding substrate were used as the inoculum. The amount of inoculum (10^-4-10^-5 cells/ml) was 5-10% from the volume of the nutrient medium. Cultivation of bacteria was carried out in 750 ml flasks with the volume of medium equal to 100 ml on a shaker (220 rpm) at 28-30° C until mid exponential growth phase (24-48 hours).

Enzymatic analyses

Glutamate dehydrogenase is responsible for reductive amination of 2-oxoglutarate with the formation of glutamate (donor of amino groups in the subsequent biosynthesis of amino lipids) [10]. Therefore, the higher the activity of this enzyme in the cells producing surfactants, the higher the content in the surfactant complex of amino lipids responsible for the antimicrobial activity of the target product.

Activity of NADP +-glutamate dehydrogenase depending on the concentration of cations in the reaction mixture.

Table 1 presents the data on NADP+-dependent glutamate dehydrogenase activity in the presence of different concentrations of monovalent cations in the reaction mixture in A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 grown on refined and waste sunflower oil.

As shown in Table 1 calcium cations at a concentration of 1-5 mmol triggered NADP+- dependent glutamate dehydrogenase activity in R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 cells grown on both oil containing substrates. Other divalent cations (magnesium and zinc) either inhibited the activity of this enzyme in both strains, or did not influence the activity of glutamate dehydrogenase in the mixture (e.g. the same as in the control without cations). At the same time, all studied divalent cations did not have any effect on the NADP+-dependent glutamate dehydrogenase activity in A. calcoaceticus IMVB-7241. Such trends were observed during the cultivation of the strain on both refined and spent sunflower oil. glutamate dehydrogenase waste biodiesel

Table 1 Effect of cations on NADP+-dependent glutamate dehydrogenase activity in cell-free extracts A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405

Note: N.d. - not determined

Monovalent cations did not affect the activity of NADP+-dependent glutamate dehydrogenase during of A. calcoaceticus IMV B-7241 cultivation on refined oil and inhibited the activity when culturing the strain in waste oil. In the presence of potassium and sodium cations, a decrease in the activity of this enzyme was observed in R. erythropolis IMV Ac-5017 and N. vaccinii IMV-7405 cells grown on refined oil. However, underg the cultivation on waste oil monovalent cations activated NADP+-dependent glutamate dehydrogenase in R. erythropolis IMV Ac-5017 (see Table 1).

NADP+-glutamate dehydrogenase activity depending on the concentration of cations in the culture medium of the strains.

At the next step, the activity of NADP⁺-dependent glutamate dehydrogenase was determined during cultivation of A. calcoaceticus IMV B-7241, R. erythropolis IMV Ac- 5017 and N. vaccinii IMV B-7405 cells in basic and modified oil containing media. Mono- and divalent cations - potential enzyme activators (Table 2) - were added to the media. The choice of cations for the modification of the cultivation media of the studied strains was based on their effect on the activity of the enzyme according to the table. 1. Also the results of previous studies [6] were considered: triggers of NADP+-dependent glutamate dehydrogenase were identified during cultivation of A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 on ethanol, N. vaccinii IMV B-7405 - on purified glycerol.

The research results given in the Table 2, confirmed the data in Table 1: there are no positive effects of calcium and zinc cations on the enzyme activity of strain A. calcoaceticus IMV B-7241; calcium cations triggered NADP+-dependent glutamate dehydrogenase activity in R. erythropolis IMV Ac-5017 and N. vaccinii IMV-7405 cells grown on both refined and waste oil.

At the same time adding of potassium chloride and sodium to the oil containing medium of R. erythropolis IMV Ac-5017 did not increase NADP+-dependent glutamate dehydrogenase activity (see the Table 2). The lack of correlation between the monovalent cations' effect on the activity of the enzyme in the reaction mixture (see the Table 1) and in the culture medium (see the Table 2) can be explained as follows. Actual content of cations in bacterial cells and culture medium differs. Values of enzymatic activity in cell-free extracts do not always correspond to the speed of real process in intact cells. This speed depends not only on the content of the enzyme but also on the pool of substrates, enzyme regulations and other factors.

Previously, [6] we showed that cations of calcium and zinc were activators of NADP- dependent glutamate dehydrogenase when A. calcoaceticus IMV B-7241 cultivated on ethanol. While sodium and potassium cations were activators when N. vaccinii IMV-7405 cultivated on purified glycerol

There were no activating effects observed if oil-containing media used (see Table1 and 2). We can assume that A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 have several glutamate dehydrogenases, which operate during cultivation on various substrates.

For example, two NAD+-dependent glutamate dehydrogenases were found in the extremely halophilic strain Salinibacter ruber M31 (DSM 13855T) [12]. The activity of one of the enzymes increased 1.4 and 67 times in the presence of 3 M sodium chloride and 3 M potassium chloride, respectively, and the activity of the other, under similar conditions, decreased 8.8 and 4 times, respectively.

It is known from the literature that monovalent and divalent cations can be both inhibitors and activators of glutamate dehydrogenase in microorganisms [13-24].

The activity of this enzyme in the archaea Thermococcussp. increased by 135, 104 and 250% in the presence of 5 mmol CaCh, MgCh and MnCh, respectively [15]. Later [18] it was found that calcium and magnesium cations are also activators of NADP+-dependent glutamate dehydrogenase in archaea Thermococcus waiotapuensis: the presence of 10 mmol CaCl2 and 10 mmol MgSO4 increased an activity by 1.3 times compared to the control (without cations of metals). Divalent iron cations at a concentration of 1 mmol increased the activity of NADP+- glutamate dehydrogenase in Klebsiella pneumoniae F-5-2 by 10% [19]. It was found in [19] that silver cations were potent inhibitors of this enzyme in the K. pneumoniae F-5-2 strain.

Table 2 Influence of cations in media for cultivation of A. calcoaceticus IMV B-7241, R. erythropolis IMV AC-5017 and N. vaccinii IMV B-7405 on NADP+-dependent glutamate dehydrogenase activity

At the next step it was analyzed how activators impact NADP+-dependent glutamate dehydrogenase during the cultivation of A. calcoaceticus IMV B-7241 on purified glycerol and waste of biodiesel production. Data in Table 3 show that after adding calcium cations into the medium with refined glycerol for A. calcoaceticus IMV B-7241 cultivation activity of NADP+-glutamate dehydrogenase increased almost 2.5-3 times comparing to the basic medium.

However, after adding CaCh into the media with waste of biodiesel production NADP+- dependent glutamate dehydrogenase activity remained almost the same as in the basic medium. Also under cultivating A. calcoaceticus IMV B-7241 on oil containing substrates adding zinc cations into glycerol media did not increase activity of the enzyme (see Table 2 and 3).

Table 3 NADP+-dependent glutamate dehydrogenase activity during cultivation of A. calcoaceticus IMV B-7241 on glycerol of various purity

The results of this study confirm the earlier data on possibility to regulate the activity of key enzymes of the biosynthesis of the final product. This can be achieved by modifying the composition of the medium, e.g. by changing a content of activators (inhibitors) of these enzymes.

The obtained data suggest that adding CaCh to the oil-containing media of R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405, as well as to the medium with purified glycerol for growing A. calcoaceticus IMV B-7241 will result in synthesis of surfactants with increased antimicrobial and antiadhesive activity.

References

1. Jimoh A.A., Lin J. (2019), Biosurfactant: A new frontier for greener technology and environmental sustainability, Ecotoxicology and Environmental Safety, 184, 109607, DOI: 10.1016/j.ecoenv.2019.109607.

2. Mazeyrat-Gourbeyre F., Baillieul F., Clement C., Ongena M., Dorey S. (2020),

3. Biosurfactants in Plant Protection Against Diseases: Rhamnolipids and Lipopeptides Case Study, Frontiers in Bioengineering and Biotechnology, 8, 1014, DOI: 10.3389/fbioe.2020.01014.

4. Adu S.A., Naughton P.J., Marchant R., Banat I.M. (2020), Microbial Biosurfactants in Cosmetic and Personal Skincare Pharmaceutical Formulations, Pharmaceutics, 12(11), pp. 1099, DOI: 10.3390/pharmaceutics12111099.

5. Ribeiro B.G., Guerra J.M.C., Sarubbo L.A. (2020), Biosurfactants: Production and application prospects in the food industry. Biotechnology Progress, 36(5), e3030, DOI: 10.1002/btpr.3030.

6. Pirog T.P., Kliuchka L.V., Shevchuk T.A., Muchnyk F.V. (2019), Interrelation of Chemical Composition and Biological Properties of Microbial Surfactants, Mikrobiologichny Zhurnal, 81(3), рр. 84-104, DOI: https://doi.org/10.15407/microbiolj81.03.084.

7. Pirog T.P., Shevchuk T.A., Nikituk L.V., Lutsai D.A., Paliichuk O.I. (2018), Influence of cultivation conditions on antimicrobial and anti-adhesive activity of surfactants of bacteria of Acinetobacter, Rhodococcus and Nocardia genera. Proceedings of the National Academy of Sciences of Belarus. Biological series, 63(3), pp. 307-315. https://doi.org/10.29235/1029- 8940-2018-63-3-307-315.

8. Pirog T., Sofilkanych A., Konon A., Shevchuk T., Ivanov S. (2013), Intensification of surfactants' synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium, Food and Bioproducts Processing, 91(2), pp. 149-157. http://dx.doi.org/10.1016/j.fbp.2013.01.001.

9. Pirog T.P., Nikituk L.V., Antonuk S.I., Shevchuk T.A., Iutynska G.O. (2017), Peculiarities of Nocardia vaccinii IMV В-7405 surfactants synthesis on waste oil of different quality and their antimicrobial properties. Mikrobiologichny Zhurnal, 79(2), рр. 13-22, DOI: https://doi.org/10.15407/microbiolj79.02.013.

10. Pirog T.P., Lutsay D.A., Shevchuk T.A., Iutynska G.O., Elperin I.V. (2018) Antimicrobial and antiadhesive activity of surfactants synthesized by Acinetobacter calcoaceticus IIB B- 7241 on technical glycerin. Mikrobiologichny Zhurnal, 80(2), pp. 14-27, DOI: https://doi.org/10.15407/microbiolj80.02.014 .

11. Smith E.L., Austen B.M., Blumenthal K.M., Nyc J.F. (1975), Glutamate dehydrogenases. In: The Enzymes, 3rd ed., vol. 11, New York: Academic Press, pp. 293-367.

12. Bradford M. (1976), A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry, 72 (3), pp. 248-254.

13. Bonete M.J., Perez-Pomares F., D^az S., Ferrer J., Oren A. (2003), Occurrence of two different glutamate dehydrogenase activities in the halophilic bacterium Salinibacter ruber. FEMSMicrobiology Letters, 226(1), рр. 181-186, DOI: 10.1016/S0378-1097(03)00592-5.

14. Sarada K.V., Rao N.A., Venkitasubramanian T.A. (1980), Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46. Biochimica et Biophysica Acta, 615(2), pp. 299-308.

15. Lin H.P.P., Reeves H.C. (1991), Purification and characterization of NADP⁺-specific glutamate dehydrogenase from Escherichia coli. Current Microbiology, 22(6), pp. 371-376.

16. Hudson R.C., Ruttersmith L.D., Daniel R.M. (1993), Glutamate dehydrogenase from the extremely thermophilic archaebacterial isolate AN1. Biochimica et Biophysica Acta, 1202(2), pp. 244-250.

17. Garnier A., Berredjem A., Botton B. (1997), Purification and characterization of the NAD- dependent glutamate dehydrogenase in the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton. Fungal Genetics and Biology, 22(3), pp.168-176, DOI: 10.1006/fgbi.1997.1004.

18. Bhuiya M.W., Sakuraba H., Kujo C., Nunoura-Kominato N., Kawarabayasi Y., Kikuchi H. et al. (2000), Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications, Extremophiles, 4(6), рр. 333-341, DOI: 10.1007/s007920070002.

19. Lee M.K., Gonzalez J.M., Robb F.T. (2002), Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs, Extremophiles, 6(2), рр. 151-159, DOI: 10.1007/s007920100238.

20. Kim Y.J., Yoshizawa M., Takenaka S., Murakami S., Aoki K. (2002), Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase, The Journal of Bioscience and Bioengineering, 93(6), рр. 584-588, DOI: 10.1016/s1389-1723(02)80241-9.

21. Alba-Lois L., Segal C., Rodarte B., Valdes-Lopez V., DeLuna A., Cardenas R. (2004), NADP-glutamate dehydrogenase activity is increased under hyperosmotic conditions in the halotolerant yeast Debaryomyces hansenii, Current Microbiology, 48(1), рр. 68-72, DOI: 10.1007/s00284-003-4076-7.

22. Choudhury R., Punekar N.S. (2009), Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli. Mycological Research, 113(10), pp. 1121-1126, DOI: 10.1016/j.mycres.2009.07.009.

23. Wakamatsu T., Higashi C., Ohmori T., Doi K., Ohshima T. (2013), Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis, Extremophiles, 17(3), 379-389, DOI: 10.1007/s00792-013-0527-7.

24. Zhu J., Lu K., Xu X., Wang X., Shi J. (2017), Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity, AMBExpress, 7(1), 9, DOI: 10.1186/s13568-016-0307-8.

25. van Heeswijk W.C., Westerhoff H.V., Boogerd F.C. (2013), Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective. Microbiology and Molecular Biology Reviews, 77(4), pp. 628-695, dOi: 10.1128/MMBR.00025-13.

Размещено на Allbest.ru