Improved purification and properties of glucose dehydrogenase fromBacillus subtilis

Biochimie 70 (1988) 1401-1409 (~ Soci6t6de Chimie biologique/Elsevier, Paris

1401

Research article

Improved purification and properties of glucose dehydrogenase from Bacillus subtilis Amin K A R M A L I and Luisa S E R R A L H E I R O L N E T I / DTIQ-Bioquimica, Estrada das Palmeiras, Queluz de Baixo 2745, Portugal (Received 15-1-1988, acceptedafter revision 11-4-1988)

Summary R Glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was purified about 5240-fold, using an aqueous two-phase system and triazine-dye affinity chromatography. The specific activity of the purified preparation was about 460 units / mg of protein with a final recovery of enzyme activity of about 75%. The affinity column could be regenerated and reused again several times. The purified enzyme appeared to be homogeneous when analyzed both on S D S - P A G E and native PAGE. The protein band on native P A G E coincided with the activity stain. ATP acts apparently as a competitive inhibitor for this enzyme with respect to NAD and protects the enzyme from dissociation into partially inactive dimers. In the absence of either glycerol or ATP, the enzyme dissociates into partially inactive dimers. purification / glucose dehydrogenase / triazine-dye affinity chromatography/ Bacillus subtUis / association-dissociation / properties

Introduction Glucose dehydrogenase (EC 1.1.1.47) is synthesized during sporulation of spore-forming bacteria, such as Bacillus subtilis [1, 2]. Although this enzyme has been used as a marker in bacterial sporulation, its biochemical role in this process is not very clear. Furthermore, the lability of this enzyme, due to its tendency to dissociate into partially inactive subunits under non-denaturing conditions [3, 4], makes it difficult to obtain reasonable amounts of highly purified glucose dehydrogenase for biochemical and genetic studies of bacteral sporulation [5--7]. On the other hand, this enzyme is of great commercial interest, since it is widely used for glucose determination in biological and non-biological fluids either in free or immobilized forms [8, 9]. The published purification procedures for this enzyme are time-consuming, involving several non-specific chromatographic steps which result in low yields of enzyme activity [3, 10]. The present paper reports a markedly improved purification scheme for glucose dehydro-

genase from Bacillus subtilis, using a triazinedye affinity chromatography technique. The effect of ATP on enzyme activity as well as on the association-dissociation of the enzyme is also investigated.

Materials and methods Materials Sepharose 4B was obtained from Pharmacia LKB Biotechnology, Inc. Lysozyme, bovine serum albumin (BSA), ovalbumin, alcohol dehydrogenase (yeast), glucose oxidase, peroxidase (horseradish), chymo-trypsin, NAD, NADH, ATP, phenazine methosulfate and p-iodo-nitrotetrazo!ium blue were purchased from Sigma Chemical Compagny. Cibacron yellow E3 was a generous ~h ~om Ciba Geigy, Portugal. (~ulture media components were obtained from Difco Laboratories. Bacillus subtilis (Marburg 168 strain, wild type) was used for enzyme production. All other reagents used were of analytical grade. Methods Protein determination and aqueous two-phase system were carried out as described previously [11, 12].

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A. Karmali and L. Serralheiro

Native polyacrylamide gel electrophoresis (PAGE) and sodium dod~cyl sulfate ( S D S ) - P A G E of protein samples were carried out as reported earlier [10, 13]. The native gel was stained for protein as well as for enzyme activity as published previously [10]. Sepharose 4B-Cibacron yellow was prepared as described elsewhere [14]. Glucose dehydrogenase activity was determined using an assay mixture containing 0.1 M glucose and 1 mM NAD ÷ in G.1 M Tris/HCl, pH 7.8. One enzyme unit is defined as 1/~mol of D-glucose oxidized to o-glucono-8-1actone in the presence of N A D / m i n at 22oC. Bacillus subtilis was maintained in slants containing nutrient agar and the inoculum was grown overnight in nutrient broth at pH 7.0, 37oC, 170 rpm with orbital st.~king. For enzyme production purposes, a 10% inoculum was used to innoculate 5 1 flasks containing nutrient broth and grown at the same temperature and agitation. The culture was harvested 3 h after the end of exponential growth and cells were collected by centrifugation at 10 000 x g for 30 min. The pellets were washed twice with saline, centrifuged and the resulting pellets stored at -20oC. Frozen cells (15 g wet weight) were thawed in 0.05 M Tris/glycine buffer, pH 6.0, containing 20% (w/v) glycerol and 1 mM mercaptoethanol (45 ml), the pH adjusted to 6.0 with dilute acetic acid and 0.4 mg of lysozyme/ml of suspension was added to the cell extract and incubated at 37 °C for 30 min. The cell extract was fractionated at room temperature using an aqueous two-phase system which contained 18% polyethylene glycol (PEG) ( v / v ) and 7.5% potassium phosphate (v/v), pH 7.0, and a final cell concentration of 35 % (v/v). The mixture was stirred vigorously for 5 min, centrifuged at 2000 x g for 5 min, the upper PEG-rich phase was removed and solid ammonium sulfate was added to a final concentration of 1M. The mixture was stirred and centrifuged as above and the lower salt-rich phase containing the enzyme activity was concentrated and dialyzed against 0.05 M Tris/glycine buffer, pH 6.0, containing 20% (w / v) giy~:erol and 1 mM mercaptoethanol, using pressure dialysis with a P-10 membrane at 4oC. The concentrated enzyme extract was applied to a column (2 x 20 cm) at room temperature packed wah Sepharose 4B-Cibacron yellow previously equilibrated with 0.05 M Tris/glycine, pH 6.0, containing 20% (w/v) glycerol and 1 mM mercaptoethanol at a flow rate of 25 ml / h. The column was washed with the same buffer system at the same flow rate until A280was <0.05. The enzyme was eluted with a linear gradient of ATP (0-20 mM) in the same buffer system and fractions containing glucose dehydrogenas~e activity were pooled and concentrated by pressure dialysis using a P-10 membrane. Initial velocity studies were carried out in the same reaction mixture used for enzyme assays by varying the concentration of NAD and all data are represented as double reciprocal plots. The slopes and infercepts of these lines were estimated by fitting the data to the Michaelis-Menten equation by the least square

method. The inhibition constant (Ki) for ATP was determined from the Dixon plot [15]. Relative molecular mass (Mr) determinations of purified glucose dehydrogenase were carried out by gel-filtration on a column (1 x 100 cm) packed with Sephacryl S-200 using protein markers [16]. The effect of ATP and glycerol on molecular weight changes of glucose dehydrogenase were analyzed by gel-filtration columns packed with Sephacryl S-200 which were equilibrated and eluted with 50 mM Tris/glycine buffer, pH 6.0, containing 1 mM mercaptoethanol and the appropriate ligand. The enzyme activity peak obtained from gel-filtration column corresponding to the dimer form of glucose dehydrogenase was reactivated by incubating the column fractions containing enzyme activity in 50 mM Tris/glycine buffer, pH 6.0, containing 1 mM mercaptoethanol and ATP at a final concentration of 10 mM for 1 h at 37oC.

Results and Discussion The general affinity chromatography technique has been widely used by some investigators for the purification of a number of enzymes, such as dehydrogenases, kinases and t R N A synthetases [17-19]. Glucose dehydrogenase was purified to apparent homogeneity using an aqueous twophase system and triazine dye affinity chromatography as shown in Fig. 1. The activity yield of the purified enzyme was about 7.5% (Table I) with a specific activity of 460 U / m g of protein as opposed to the values of 18% and 368 U / r a g orotein, rest~ectively, reported by Ramaley and Vasantha [10], using a 9 - s t e p purification scheme for this enzyme. The enzyme bound to Sepharose 4 B - C i b a c r o n yellow was specifically eluted with a linear gradient of A T P (Fig. 2), since this ligand acts as a competitive inhibitor for this enzyme (Fig. 3). The aqueous two-phase system was used in this purification scheme to replace the high speed centrifugation step for removal of cell debris and other cellular components. Kroner et al. [20] suggested the use of this separation technique as opposed to the high speed centrifugation one for large scale purification of enzymes, since it is more economical and provides a higher purification factor. The affinity column could be regenerated with 5 vol of 2 M KCI and reused at least 20 times without any loss of binding capacity and resolution (data not shown). The purified enzyme preparation ran apparently as a single protein band when analyzed both by native P A G E and S D S - P A G E (Fig. 1) and it appears to be a tetramer with an Mr of 120 000 which consists of 4 identical sub•

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Table !. Purification of glucose dehydrogenase from Bacillus subtilis. Purification steps

Total protein (mg)

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nnit~ with an AAtr ~ t : "11 ~ a s reported previously [10]. The native gel was also stained for enzyme activity (Fig. 1C) which was found coinciding with the protein band. Initial velocity studies in the presence of A T P revealed that this ligand acts as a competitive inhibitor for the enzyme with respect to N A D increasing the K m value for NAD from 30 -+ 2.7 ~M to 80 _ 6.5/zM without affecting the Vma~ value as shown in Fig. 3. The inhibition constant (Ki) for ATP was found to be 3.2 -+ 0.25 mM from the Dixon plot (Fig. 4). Since this ligand acted as a competitive inhibitor for glucose dehydrogenase, we investigated the effect of ATP on enzyme inactivation in the presence and absence of glycerol. As can be observed in Fig. 5, ATP protects the enzyme against inactivation both in the presence and absence of glycerol, whereas the enzyme solution in the absence of ATP and glycerol loses activity very rapidly. Furthermore, ATP can partially reactivate the enzyme, as shown in Fig. 5. In order . . . . . . . .

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to prove that the observed enzyme inactivation as well as enzyme protection in the presence and absence of ATP and glycerol are related to dissociation- association of the enzyme molecule, we investigated the effect of these ligands on the Mr of the enzyme by gel-filtration chromatography, as suggested by Frieden [21]. As can be seen in Fig. 6 (A and B), both glycerol and ATP prevent the dissociation of the enzyme, since glucose dehy6rogenase runs as a single peak on Sephacryl S-200 column with an Mr of 120 000. However, in the absence of these two ligands, the tetrameric form of the enzyme dissociates partially into partially inactive dimers with an Mr of 63 000, as shown in Fig. 6C. Using the data shown in Fig. 6C and by measuring the protein content of both peaks, the specific activities for the tetrameric and dimeric forms of the enzyme were found to be 365 and 49 U / m g of protein, respectively. It is interesting to note in Fig. 6C that the dissociation of the enzyme is partial, since it is still possible to observe an enzyme ac-

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tivity peak corresponding to the tetrameric form of the enzyme. Ramaley and Vasantha [10] reported the glycerol protection against inactivation of glucose dehydrogenase from Bacillus subtilis, suggesting that this ligand prevents the dissociation of the tetrameric form of the enzyme into partially inactive dimers. In regard to the effect of ATP, it seems that ATP inhibits the enzyme activity by competing with N A D ÷ for the active site of the tetrameric form of glucose dehydrogenase but, on the other hand, it prevents the dissociation of the tetrameric form of the enzyme into partially inactive dimers. Yokota et al. [3] reported the reactivation of glucose dehydrogenase from a mutant strain of Bacillus species by pyridine and adenine nucleotides. However, they did not report either the inhibitory effect of ATP on the kinetics of this enzyme or the A T P protection against dissociation of the tetrameric form of the enzyme into

partially inactive dimers [3]. The physiological significance of the inhibitory effect of ATP on enzyme kinetics as well as on the association-dissociation phenomena observed in vitro is not known. Its elucidation will require measurements of steady state levels of ATP, glucose, N A D and other possible effectors both in vegetative cells and during the sporulation process of Bacillus subtilis.

Acknowledgments We would like to thank Dr. P.R. Brown, Department of Biochemistry, King's College, London, for helpful discussions of the manuscript. We are indebted to Dr. L. Quinta (LNETI/DCEAI) for the gift of B. subtilis strains.

1406

A. Karmali and L. Serralheiro

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Fig. S. Effects of glycerol and ATP on enzyme activity. Purified enzyme (1 ~g) was incubated in 50 mM Tris/glycine buffer, pH 6.0, containing 1 mM mercaptoethanoi and the appropriate ligand at 37oC and aliquots were removed at the time intervals indicated for enzyme activity measurements: e: 20% (v/v) glycerol; o: 10 mM ATP;/x: 20% (v/v) glycerol and 10 mM ATP; A: no ligands. The arrow indicates that a final concentration of 10 mM ATP was added to the partially inactive enzyme solution for reactivation purposes (11).

References 1 Church B.D. & Halvorson H. (1957) J. Bacteriol. 73,470-476 2 Doi R., Halvorson H. & Church B. (1959)J. Bacteriol. 77, 4 3 - 5 4 3 Yokota A., Sasajima K. & Yoneda M. (1979) Agric. Biol. Chem. 43,271-278 4 Fujita Y., Ramaley R. & Freese E. (1977) J. Bacteriol. 132,282-293 5 Setlow P. (1981) in: Sporulation and Germination (Levinson H., Sonershein A.L. & Tipper D.J., eds.), American Society for Microbiology, Washington, D.C., pp. 13-28 6 Piggot P.J., Moir A. & Smith D.A. (1981) in: Sporulation and Germination (Levinson H., Sonershein A.L. & Tipper D.J., eds.), American Society for Microbiology, Washington, D.C., pp. 29-39 7 Szulmajster J. (1975) Trends Biochem. Sci. 4, 18 - 2 2 8 Bisse E. & Vonderschmitt D.J. (1977) FEBS Lett. 8 1 , 3 2 6 - 3 3 0

9 Bisse E. & Vonderschmitt D.J. (1978) FEBS Lett. 93, 102-104 10 Ramaley R.F. & Vasantha N. (1983) J. Bial. Chem. 258, 12558-12565 11 Bradford M.M. (1976) Anal. Biochem. 72, 248-254 12 Kroner K.H., Hustedt H., Granda S. & Kula M.-R. (1978) Biochem. Bioeng. 20, 1967-1988 13 Laemmli U.K. (1970) Nature 227,680-685 14 Baird J.K., Sherwood R.J., Carr R.J.G. & Atkinson A. (1976) FEBS Lett. 70, 61-66 15 Dixon M. (1953) Biochem. J. 55,170-171 16 Andrews P. (1965) Biochem. J. 96, 595-599 17 Atkinson T. & Lowe C.R. (1981) Biochem. Soc. Trans. 9, 290-293 18 Hedman P.O. & Gustafsson J.-G. (1984) Anal. Biochem. 138,411-415 19 Carrillo N. & Vallejos R.H. (1983) Biochim. Biophys. Acta 742, 285-294 20 Kroner K.H., Hustedt H. & Kula M.-R. (1984) Process Biochem. 19, 170-179 21 Frieden C. (1970)J. Biol. Chem. 245, 5788-5797

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