Dextransucrase as an Enzyme Associating with Alkaline Earth Metal Ions

Transcription

1 Agr. Biol. Chem., 39 (6), 1187 `1975 Dextransucrase as an Enzyme Associating with Alkaline Earth Metal Ions Kuniko ITAYA and Takehiko YAMAMOTO Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka Received October 4, 1974 Dextransucrase (EC ) of Leuconostoc mesenteroides was purified from the culture filtrate by precipitation with solid ammonium sulfate in the presence of egg white albumin followed by successively treating with columns of DEAE-cellulose and Bio Gel P-150. The purified enzyme lost the activity upon dialysis against EDTA, and was reactivated by the addition of alkaline arth metal ions. The best reactivation was brought about by calcium ion. The enzyme inactivated by EDTA was unstable and readily denatured ir reversibly. Several other properties of the purified enzyme were also investigated and dis cussed. Though it has been long since dextransucrase (EC ) was discovered in certain Leu conostoc bacilli,1 `12 few papers have been published on its chemical properties. Several papers reported that dextransucrase always contained dextran and this preexisting dextran might act as an acceptor or initiator for dextran synthesis from sucrose.7,9,13) Recently, the present authors found that dextransucrase of Leuconostoc mesenteroides was precipitated in a good yield on salting out the bacterial culture filtrate with solid ammonium sulfate provided that a small amount of egg white albumin was added to the filtrate. Thus, the work of purification of the enzyme was undertaken. The purified preparation contained a small amount of dextran, but it required alkaline earth metallic ions for its activity and stability. The present paper describes the method of purification of dextransucrase of Leuconostoc mesenteroides and several properties of the purified enzyme, especially the experiments on inactivation of the enzyme by EDTA and reactivation by metallic ions. MATERIALS AND METHODS 1. The bacterium employed and its culture. Leu conostoc mesenteroides IAM 1046 which was supplied by the Institute of Applied Microbiology, Tokyo Uni versity, was used and its stock culture was made at 4 Ž on an agar slant medium consisting of 2.0% sucrose, 0.5% each of yeast extract and polypeptone, 0.1% KH2P04, 0.02% NaCl, 0.01% MgSO4 E7H2O, 1.5% each of CaCO3 and agar, ph 7.0. The stock culture was transplanted every one month. For dextransucrase production, the bacterium was cultured in a medium (2 liter medium in a 5 liter volume Fernbach's flask) consisting of 3.0% each of sucrose and corn steep liquor, 0.2% KNO3, 0.02% MgS04 E7H2O buffered at ph 7.4 with 0.1 M phosphate at 28 Ž for 16 `18hr. 2. Preparation of dextransucrase. The bacterial culture broth was centrifuged at 11,000 x g for 20 min, and solid ammonium sulfate was added to the supernatant to 0.75 saturation after adding egg white albumin to a 0.05% concentration. The addition of egg white albumin was effective to result in a good precipitation of dextransucrase. The precipitate obtained after leaving the mixture overnight was collected by centrifugation and dissolved in M acetate buffer, ph 5.2, followed by dialyzing against the same buffer solution. The dialyzed solution was used as a crude enzyme prepara tion and subjected to experiments for further purifi cation. 3. Enzyme assay. A 2.0ml enzyme was added to 4.0ml of 15% sucrose solution containing 0.05 M acetate buffer of ph 5.2 at 30 Ž. After one hr incubation, 5.0ml of Shaffer-Somogyi's reagent14) was added to the incubation mixture and reducing sugar formed was determined as fructose. 5,61 One unit of enzyme activity was defined as the enzyme quantity that converts one mg of sucrose into fructose and dextran in one hr under the condition.3) 4. Analysis of the enzyme preparation. Nitrogen

2 1188 K. ITAYA and T. YAMAMOTO was determined by the micro Kjeldahl method. The total sugar content was estimated by the sulfuric acidphenol method. 15) The specific activity was tentatively expressed by dividing the enzyme activity per m] by absorbance at 280 nm of the enzyme solution. 5. Analysis of dextran synthesized by the enzyme. - cellulose and eluted by the same method as above. As shown in Fig. 1, the elution pattern was symmetrical and the enzyme activity was in parallel with the absorbance at 280 nm of the eluates. The dextran synthesized by the enzyme was isolated and analyzed. To the enzyme-sucrose mixture incubated under the conditions indicated to the respective ex periments was added an equal volume of methanol. After one hr standing at 30 Ž, the mixture was centri fuged at 12,000 ~g for 20 min and, after washing several times with 50% methanol, the precipitate was suspended in a suitable amount of water and subjected to lyo philization to estimate its yield. The total sugar content of dextran preparation was determined by the sulfuric acid-phenol method. The synthetic effeciency of dextran was expressed by divid ing the amount of dextran obtained by that of sucrose consumed. The amount of sucrose remaining in the incubation mixture was determined by measuring the reducing sugar increased on incubation with invertase from Candida utilis.16) The constituting sugars of dextran preparations were analyzed by thin layer chromatography using anisaldehyde-sulfuric acid as a locator17,18) after hydrolyzing with 1.0 N HCl at 100 Ž for 2hr. Linkage homogeneity of dextran was investigated by measuring the hydrolysis degree on incubation with dextranase from Aspergillus carneus.19-21) One unit of dextranase activity was the enzyme quantity that pro duced one ƒêmole of reducing sugar as glucose per min on incubation with dextran at 40 Ž. RESULTS 1. Purification of the dextransucrase by chro matography on columns of DEAE-cellulose and Bio Gel P-150 The crude enzyme preparation obtained as described in MATERIALS AND METHODS was subjected to chromatography on a column of DEAE-cellulose. The enzyme was adsorbed on the column equilibrated with 0.02 M acetate buffer, ph 5.5, and eluted by sodium chloride solution of around 1.0 M. The enzymatically active fractions in the first chromatography with DEAE-cellulose were combined and precipitated by adding ammonium sulfate to 0.75 saturation and, after dialyzing against 0.02 M acetate buffer, ph 5.5, the dialyzed enzyme was reapplied to a column of DEAE FIG. 1. Rechromatography on a DEAE-Cellulose Column of Dextransucrase. Column, 2.5 x 22.5cm; enzyme charged, 164ml, 6.5 units/ml. E280nm=5.5; 0.02M acetate buffer, ph 5.5; elution of enzyme, the linear gradient method of NaCl. \, enzyme activity; œ \ œ, absorbance at 280 nm; -----, NaCl, M. The active effluents obtained by rechromato graphy on DEAE-cellulose were combined and precipitated with ammonium sulfate. After dissolving in a suitable amount of 0.02 M acetate buffer, ph 5.2, the precipitated enzyme was filtered through a column of Bio Gel P-150 equilibrated with 0.02 M acetate buffer, ph 5.2. As shown in Fig. 2, the enzyme was highly purified and the specific activity was increased nearly two hundreds fold that of the crude enzyme preparation. Changes in the specific activity and activity recovery during purification are summarized in Table I. 2. General properties as an enzyme protein 1) Contents of nitrogen and sugars of the purled enzyme. The purified enzyme of which specific activity was 110 (Table I) contained 0.019mg of nitrogen and mg of sugar per ml as determined by the sulfuric acid-phenol method. 2) Isoelectric point. The isoelectric point of dextransucrase was examined by the iso-

3 Dextran Sucrase 1189 FIG. 3. Isoelectric Focusing of the Purified Dex transucrase. Column, 110ml, ph (3 `10)- and density gradient, FIG. 2. Gel Filtration with Bio Gel P-150 of Dex transucrase Obtained in Fig. 1. Column, 4.2 ~90cm; enzyme, 20ml, 200 units/ml, E280nm=92; 0.02M acetate buffer, ph 5.2. \, enzyme activity; œ \ œ, absorbance at 280 nm. electric focusing method and estimated to be 3.9. As can be seen from Fig. 3, however, the specific activity of the enzyme was signi ficantly decreased by the isoelectric focusing test, i.e., the specific activity before the iso electric focusing was 110 while that after the test was only 55 even for the highest activity fraction (Fraction number 25 in Fig. 3). 3. General enzymic properties Similarly to the papers so far publish ed, 4 `9,11,13) the purified enzyme showed its op timum ph at around 5.2 and its Kin on sucrose was around 2.0 ~10-2 M, as far as the activity was determined by the method of measuring fructose liberation power from sucrose. Also, Ampholyte and glycerol; enzyme, 20ml, 30units/ml, E280nm=0.354; 46hr, 4 Ž, 280V (5mA) for initial 4hr, then 700V (1.1mA) for 42hr. \, enzyme activity; œ \ œ, absorbance at 280 nm; ----, ph. the enzyme increased its activity significantly by the addition of maltose, as has been reported by Koepsell et al.,7) and Bailey et al.11.13) The highest activity to release reduc ing sugar from sucrose was at 30 Ž by the test of one hr incubation. However, dextran synthesis by the enzyme was much more active at 10 Ž than at 20 `30 Ž (Fig. 4). The purified enzyme was stable in a ph region 5.0 to 6.4 and temperatures lower than 30 Ž, though the stability was greatly affected by removing calcium ion from the enzyme, as well be described below. 4. Effects of EDTA and alkaline earth metal ions on the purified dextransucrase The purified enzyme was stable on dialysis against deionized water as far as the dialysis was done in the cold. However, contrary to TABLE I. PURIFICATION OF DEXTRANSUCRASE FROM THE CULTURE FILTRATE OF Leuconostoc mesenteroides IAM 1046

4 1190 K. ITAYA and T. YAMAMOTO FIG. 4. Effect of Temperature on Dextransucrase. Left: activity determined by the fructose releasing power, 0.05 M acetate buffer, ph 5.2, 10% sucrose, 1.0hr; right: synthesis of dextran,, and its efficiency,,0.05 M acetate buffer, ph 5.2, 10% sucrose, 1 unit/ml, 48hr. the report by Koepsell et a1.7) the addition of EDTA caused inactivation of the enzyme and the inactivated enzyme was reactivated by the addition of calcium ion (Fig. 5). Other alka line earth metal ions were also effective for reactivation, but calcium ion was the best and caused reactivation of nearly 90% of the activity lost (Table II and Fig. 6). A rela tionship between concentrations of calcium ion added and reactivation degrees of the enzyme which was inactivated by EDTA followed by dialysis against deionized water, is shown in Fig. 6. Other metallic ions than alkaline earth metal ions were quite ineffec tive. The enzyme reversibly inactivated by EDTA FIG. 6. A Relationship between the Reactivation Degree of Dextransucrase and the Concentration of Ca2+ added. The enzyme, 2units/ml, E280nm=0.0204, was incubat ed with 0.02 M EDTA at ph 5.2, 20 Ž for 1.0hr and then, dialyzed against deionized water overnight in the cold. The reactivation degree was expressed on the basis of the activity before treating with EDTA. FIG. 7. Thermal Stability of Dextransucrase, 15 min heating at the temperature indicated. \, native-enzyme; œ \ œ, EDTA-treated enzyme. Activity determined after the addition of 0.1 mm Ca2+. FIG. 5. A Progressive Curve of Inactivation of Dextransucrase by EDTA and Its Reactivation by the Addition of Caz+ The purified enzyme was incubated with 0.02 M EDTA and at intervals, the activity was determined with or without addition of 0.03 M Ca-acetate. œ \ œ, remaining activity; \, activity determined after addition of Ca-acetate. FIG. 8. ph-stability of Dextransucrase. Incubated for 1.0hr at the ph indicated, 20 Ž. \, native-enzyme; œ \ œ, EDTA-treated enzyme. Ac tivity determined after addition of 0.1 mm Ca2+. was sensitive and its reactivation ability was lost on incubation at temperatures higher than 25 Ž and at ph values over 5.5 (Figs. 7

5 Dextran Sucrase 1191 and 8). 5. Effect of calcium ion on dextran synthesis by the enzyme Using the dextransucrase dialyzed against deionized water, the effect of addition of calcium ion on synthesis of dextran and susceptibility to dextranase of the dextran synthesized were investigated. The dextran synthesized by the enzyme was shown to produce only glucose on hydrolysis with 1.0 N HCl at 100 Ž for 2hr and susceptible to dextranase. As given in Table III, however, the addition of calcium ion was rather inhi bitory for synthesis of dextran, and the dextran synthesized in the presence of calcium ion was less susceptible to dextranase, but the degree was slight. TABLE II. REACTIVATION BY VARIOUS METAL IONS OF DEXTRANSUCRASE INACTIVATED BY EDTA The enzyme, 2 units activity per ml, was incubated with 0.02 M EDTA at 20 Ž for 1.0hr and the reacti vation degrees upon addition of various metal ions in an equivalent amount to EDTA were determined. DISCUSSION Several papers have reported that dextran sucrase preparation from Leuconostoc mesen teroides7) and Streptococcus bovis13) contains dextran. Our dextransucrase preparation obtained from Leuconostoc mesenteroides also contained sugar, and its content was estimated to be twice as much as that of protein if taken the data described above into con sideration. Our enzyme preparation, however, behaved as if it was a single component when subjected to gel filtration on Bio Gel P-150 with an elution volume comparable to that of human y-globulin as a reference. An ex periment carried out independently showed that a large part of the sugar component of the enzyme preparation was dextran because it was degraded on incubation with Aspergillus carneus dextranase. Of particular interest in the experiment was the fact that the dextran sucrase incubated with dextranase became unstable and lost its activity. Details of this experiment will be publish elsewhere. Our present paper made clear that dext ransucrase is an alkaline earth metallo enzyme, as seen from Table II and Figs. 5 `8, i.e. the enzyme was not inactivated by dialysis against deionized water, but inactivated by incubation with EDTA and the inactivated enzyme recovered its activity upon addition of alkaline earth metal ions. It is most probable that the enzyme occurs as a Ca enzyme naturally. The addition of a slight excess of Cal I, on the other hand, was shown TABLE III. EFFECT OF CALCIUM ION ON DEXTRAN SYNTHESIS AND SUSCEPTIBILITY TO DEXTRANASE OF THE DEXTRAN SYNTHESIZED Enzyme, 1 unit per ml incubation mixture, 10 Ž, ph 6.5, 72hr. a) See the text. b) 2mg dextran and 0.6 units of dextranase per ml, ph 5.5, 40 Ž, 24hr.

6 1192 K. ITAYA and T. YAMAMOTO to have no significant effects on synthesis of dextran by the enzyme, as given in Table III. The activity of dextransucrase to synthesize dextran was not necessarily consistent with the activity to release fructose from sucrose as seen from Fig. 4. This fact seems to be important for study of kinetics of the enzyme and further experiments will be necessary to solve this problem as well as the role of metal ion in the enzyme action. REFERENCES 1) F. Fowler, F. L. Buckland, I. K. Brauns and H. Hibbert, Can. J. Research, 15B, 486 (1937). 2) E. J. Hehre, Science, 93, 237 (1941). 3) E. J. Hehre and Y. J. Sugg, J. Exptl. Med., 75, 339 (1942). 4) E. J. Hehre, J. Biol. Chem., 168, 221 (1946); Advances in Enzymol., 11, 297 (1951). 5) H. J. Koepsell and H. M. Tsuchiya, J. Bacteriol., 63, 293 (1952). 6) H. M. Tsuchiya, H. J. Koepsell, J. Corman, G. Bryant, M. 0. Bogard, V. H. Feger and R. W. Jackson, ibid., 64, 521 (1952). 7) H. J. Koepsell, H. M. Tsuchiya, N. N. Hellman, A. Kazeno, C. A. Hoffman, E. S. Charpe and R. W. Jackson, J. Biol. Chem., 200, 793 (1953). 8) W. W. Carlson, C. L. Rosano and V. Whiteside Carlson, J. Bacteriol., 65, 136 (1953). 9) R. W. Baily, S. A. Barker, E. J. Bourne and M. Stacey, J. Chem. Soc., 1957, 3530, ) W. B. Neely, Nature, 182, 1007 (1958); J. Am. Chem. Soc., 80, 2010 (1958). 11) C. S. Stringer and H. M. Tsuchiya, ibid., 80, 6620 (1958). 12) W. B. Neely, ibid., 81, 4416 (1959); Arch. Biochem. Biophys., 79, 297 (1959). 13) R. W. Bailey, Biochem. J., 72, 42 (1959). 14) P. A. Schaffer and M. Somogyi, J. Biol. Chem., 100, 695 (1933). 15) M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Robers and F. Smith, Anal. Chem., 28, 350 (1956). 16) M. lizuka and T. Yamamoto, Agr. Biol. Chem., 36, 349 (1972); ibid., 38, 213 (1974). 17) E. Stahl and U. Kallenbach, J. Chromatog., 5,351 (1961). 18) B. P. Lisboa, ibid., 16,136 (1964). 19) D. Tsuru, N. Hiraoka, T. Hirsoe and J. Fuku moto, Agr. Biol. Chem., 35, 1727 (1971). 20) N. Hiraoka, J. Fukumoto and D. Tsuru, J. Bi ochem., 71, 57 (1972). 21) D. Tsuru, N. Hiraoka and J. Fukumoto, ibid., 71, 653 (1972).