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[28] Mannitol dehydrogenase (crystalline) from Lactobacillus brevis
[28]
MANNITOL DEHYDROGENASE
[28]
Mannitol
Dehydrogenase (Crystalline) Lactobacillus brevis 1
By B.
143
from
L. HORV,CKER
D-Mannitol -b DPN+ ~- D-fructose -b DPNHq- H+ Assay Method Principle. A spectrophotometric assay, based on the oxidation of D P N H by fructose is employed. The oxidation of D P N H is followed by the change in absorbance at 340 m#. Reagents
Fructose stock solution, 1 M. Dissolve 1.8 g of fructose (Pfanstiehl) in 10 ml of water and dilute to 10.0 ml. Store in the refrigerator. DPNH, 10-~ M in 0.001 N NaOH Buffer solutions, 0.05 M sodium acetate buffer, pH 5.35, and 0.05 M sodium acetate buffer, pH 6.0. The latter solution is made up to contain 2 X 10-4M mercaptoethanol and 1% bovine serum albumin. Procedure. To 0.5 ml of water and 0.4 ml of 0.05 M acetate buffer, pH 5.35, in a 1.0 ml quartz absorption cell add 0.01 ml of D P N H solution and sufficient diluted enzyme solution to produce an absorbance change of about 0.020 per minute. Start the reaction by addition of 0.1 ml of substrate and read every 30 seconds for 3 minutes. Make the enzyme dilutions in the 0.05 M acetate buffer, pH 6.0, containing albumin and mercaptoethanol. The unit of enzyme activity is defined as the amount required to oxidize 1 micromole of D P N H per minute in the spectrophotometric test carried out at 25 °.
Purification Procedure Growth of Cells. Obtain a tyophilized culture of Lactobacillus brevis (ATCC 367) from the American Type Culture Collection. Prepare growth medium of the following compositionS: 0.5% yeast extract, 1.0% nutrient broth, 1.0% sodium acetate.3 H20, 0.001% NaC1, 0.001% FeS04" 7 H20, 0.02% MgS04-7 H20, 0.001% MnS04.4 H20, and 1.0% fructose. Maint~in on 2% agar stabs of this composition and transfer every 15 days.
G. Martinez, H. A. Barker, and B. L. Horecker, J. Biol. Chem. 238, 5 (1963). H. Gest and J. O. Lampen, J. Biol. Chem. 194, 555 (1952).
144
D:EHYDROGENASES AND OXIDASES
[28]
Grow the cultures in liquid medium 3 by incubation at 32 ° without aeration. For preparation of large amounts of cells for enzyme purification, inoculate 10 ml of a 24-hour liquid culture into 1 liter of fresh medium. This is used 24 hours later as inocnlum for 16 liters of medium in a 20-1tier bottle. Incubate at 32 ° for about 17 hours, at which time nearly all the fructose will have been consumed, and collect the cells by centrifugation in a Sharples supercentrifuge. Wash the harvested cells twice with 150-ml portions of 0.05 M sodium phosphate buffer (pH 6.5) and store the packed cell paste at --16 °. About 2.0g of cells (wet weight) are obtained per liter of culture. Preparation of Extracts. Perform all operations at 0 ° unless otherwise stated. Take about one-third of the cells produced in 16 liters of culture for each preparation. Suspend three 3.5-g portions of frozen cell paste in 7.0 ml of 0.005 M sodium phosphate buffer (pH 6.5) containing 2 X 10-4M mercaptoethanol and shake with 8 g of glass beads in the Nossal tissue disintegrator 4 for four 20-second periods, cooling the cartridges in ice between each treatment. Centrifuge the suspensions for 10 minutes at 10,000 g. Wash the residues twice with a total of 20 ml of the same buffer and combine the washings with the first supernatant solution. Protamine Fraction. Treat the clear extract with 6.0 ml of 2% protamine sulfate solution and centrifuge after 20 minutes at 0 °. Discard the precipitate. Ammonium Sulfate I. To the supernatant solution add 3.0 ml of 0.5 M sodium phosphate buffer (pH 6.5) and 12.85 g of ammonium sulfate. After 10 minutes remove the precipitate by centrifugation and discard it. Treat the clear solution (42 ml) with 2.86 g of ammonium sulfate; collect the precipitate by centrifugation after 10 minutes, and dissolve it in 7.5 ml of 0.05 M sodium phosphate buffer, pH 6.5. Heated Fraction. To the solution add 0.7 ml of 0.5 M sodium phosphate buffer (pH 6.5) containing 2 X 10-4 M mercaptoethanol. Heat the diluted solution in a water bath at 63-65 ° for 5 minutes, centrifuge for 10 minutes at 10,000 g, and discard the precipitate. Ammonium Sulfate H. Determine the amonium sulfate concentration in the supernatant solution (heated fraction, 7.3 ml) with a Barnstead PR2 conductivity meter and adiust to 70% saturation by addition of solid ammonium sulfate. After 10 minutes collect the precipitate by centrifugation at 10,000 g for 5 minutes. Extract this precipitate with 4.0 3 The substitution of glucose for fructose in the growth media does n o t alter the yield of cells b u t reduces the enzymatic activity in the crude extract to about 5% of t h a t found in the fructose medium. 4 p. M. Nossal, Australian J. Exptl. Biol. Med. Sci. 31, 583 (1953).
[28]
MANNITOL DEHYDROGENASE
145
ml of 65% saturated ammonium sulfate (pH 7.0), and after 10 minutes again collect the precipitate by centrifugation. Extract successively, as before, with 3.0 ml and 2.0 ml of 60% saturated ammonium sulfate. Combine the last two extracts. Crystallization. Adjust the ammonium sulfate II solution to pH 6.0 with 0.1 N acetic acid and add a solution of saturated ammonium sulfate until faint turbidity is observed. Allow to stand overnight at 3 ° and collect the crystals by centrifugation. Dissolve them in 1.6 ml of 0.1 M sodium acetate buffer (pH 6.0) containing 2 X 10-4M mercaptoethanol (first crystals). Repeat this procedure (second crystals), and, after centrifugation to separate, repeat again (third crystals). Finally add mercaptoethanol (2 X 10-4 M) and ammonium sulfate until crystallization is begun and store the crystal suspension at 3 ° . The crystals obtained in this manner are very transparent; for the purpose of visualization carry out another crystallization in the presence of 10-6 M methylene blue. Specificity. The enzyme is specific for D-fructose and D-mannitol. It does not catalyze oxidation of the following polyols: glycerol, erythritol, xylitol, D-sorbitol, D-arabitol, L-arabitol, dulcitol, inositol, ribitol, melibiitol, L-glycero-D-glucoheptitol. The enzyme will not catalyze reduction of the following sugars: D-sorbose, D-xylulose, D-ribulose, sedoheptulose, fructose 6-phosphate, D-glucose. T P N H will replace D P N H in the reduction of fructose; with the standard assay conditions the reaction rate is about one-half as fast with this coenzyme. In the reaction between oxidized coenzyme and mannitol at pH 8.6 the rate with TPN is only 2.5% of that with DPN. Distribution. An enzyme with this specificity has thus far been reported only in L-brevis, but it probably occurs in other species of Lactobacillus that accumulate mannitol. Use ]or Analytical Purpose.s. The enzyme can be employed for the determination of D-mannitol and D-fructose. For fructose, prepare a reaction mixture containing 0.1 M sodium acetate buffer, pH 6.0, 0.1 mM DPNH, 0.02 mg of mannitol dehydrogenase, and 0.01-0.05 micromole of D-fructose in a total volume of 1.0 ml. Read the absorbance at 340 m~ before and after addition of D-fructose, and calculate the amount present in the assay cuvette from the change in absorbance2 For mannitol, prepare a reaction mixture (0.30 ml) containing 0.02M Tris buffer, pH 8.0, 7.5 mM pyruvate, 2.0 mM DPN, 0.01 mg of lactic dehydrogenase, 6 and 0.2 mg mannitol dehydrogenase. Add 0.5-1.0 micro5B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). Vol. I [67].
146
DEHYDROGENASES AND OXIDASES
[28]
mole of mannitol, incubate a t room temperature for 3 hours, and measure fructose colorimetrically. 7 D-Mannose can be determined with this procedure after reduction with sodium borohydride. PURIFICATION PROCEDURE
Fraction Extract Protamine fraction Ammonium sulfate I Heated fraction Ammonium sulfate II First crystals Second crystals Third crystals
Total volume (ml)
Units/ml
Total units
34 35 7.5 7.3 5.1 1.6 1.6 1.6
21.3 20.3 59.3 41.8 49.0 90.7 65.7 50.0
725 690 430 305 250 145 105 80
Specific activity Recovery (units/mg) (%) 0.75 2.7 4.9 8.6 16.7 23.7 28.9 27.3
-95 59 42 34 20 14 11
Properties
Stability. The crude extracts show a rapid loss of activity at 2 °, but this loss can be partially restored by incubation with 10-3 M mercaptoethanol at 37% The purified enzyme is most stable in solution at p H 6.0 in a 0 . 0 2 M sodium acetate buffer containing 10- 3 M mercaptoethanol. Such solutions can be stored for weeks at - - 1 6 ° . The enzyme solutions lose no activity when dialyzed at 3 ° against oxygen-free water containing 2 X 10 -4 M mercaptoethanol for a period of 18 hours. Suspensions of the crystalline enzyme preparation k e p t in a m m o n i u m sulfate at 3 ° lose about 20% of their activity in 2 months. This loss is largely reversed by the addition of mercaptoethanol to 2 X 10- 4 M followed by warming to 37 ° for 1 hour. Ef]ect a] pH on Reaction Rate. The optimal p H for the conversion of fructose to mannitol, the reaction utilized in the enzyme assay, is 5.35. The optimal p H for the reverse reaction, the conversion of mannitol to fructose, is 8.6.
~J. H. Roe, J. Biol. Chem. 1@7, 15 (1934).
MANNITOL DEHYDROGENASE
[28]
Mannitol
Dehydrogenase (Crystalline) Lactobacillus brevis 1
By B.
143
from
L. HORV,CKER
D-Mannitol -b DPN+ ~- D-fructose -b DPNHq- H+ Assay Method Principle. A spectrophotometric assay, based on the oxidation of D P N H by fructose is employed. The oxidation of D P N H is followed by the change in absorbance at 340 m#. Reagents
Fructose stock solution, 1 M. Dissolve 1.8 g of fructose (Pfanstiehl) in 10 ml of water and dilute to 10.0 ml. Store in the refrigerator. DPNH, 10-~ M in 0.001 N NaOH Buffer solutions, 0.05 M sodium acetate buffer, pH 5.35, and 0.05 M sodium acetate buffer, pH 6.0. The latter solution is made up to contain 2 X 10-4M mercaptoethanol and 1% bovine serum albumin. Procedure. To 0.5 ml of water and 0.4 ml of 0.05 M acetate buffer, pH 5.35, in a 1.0 ml quartz absorption cell add 0.01 ml of D P N H solution and sufficient diluted enzyme solution to produce an absorbance change of about 0.020 per minute. Start the reaction by addition of 0.1 ml of substrate and read every 30 seconds for 3 minutes. Make the enzyme dilutions in the 0.05 M acetate buffer, pH 6.0, containing albumin and mercaptoethanol. The unit of enzyme activity is defined as the amount required to oxidize 1 micromole of D P N H per minute in the spectrophotometric test carried out at 25 °.
Purification Procedure Growth of Cells. Obtain a tyophilized culture of Lactobacillus brevis (ATCC 367) from the American Type Culture Collection. Prepare growth medium of the following compositionS: 0.5% yeast extract, 1.0% nutrient broth, 1.0% sodium acetate.3 H20, 0.001% NaC1, 0.001% FeS04" 7 H20, 0.02% MgS04-7 H20, 0.001% MnS04.4 H20, and 1.0% fructose. Maint~in on 2% agar stabs of this composition and transfer every 15 days.
G. Martinez, H. A. Barker, and B. L. Horecker, J. Biol. Chem. 238, 5 (1963). H. Gest and J. O. Lampen, J. Biol. Chem. 194, 555 (1952).
144
D:EHYDROGENASES AND OXIDASES
[28]
Grow the cultures in liquid medium 3 by incubation at 32 ° without aeration. For preparation of large amounts of cells for enzyme purification, inoculate 10 ml of a 24-hour liquid culture into 1 liter of fresh medium. This is used 24 hours later as inocnlum for 16 liters of medium in a 20-1tier bottle. Incubate at 32 ° for about 17 hours, at which time nearly all the fructose will have been consumed, and collect the cells by centrifugation in a Sharples supercentrifuge. Wash the harvested cells twice with 150-ml portions of 0.05 M sodium phosphate buffer (pH 6.5) and store the packed cell paste at --16 °. About 2.0g of cells (wet weight) are obtained per liter of culture. Preparation of Extracts. Perform all operations at 0 ° unless otherwise stated. Take about one-third of the cells produced in 16 liters of culture for each preparation. Suspend three 3.5-g portions of frozen cell paste in 7.0 ml of 0.005 M sodium phosphate buffer (pH 6.5) containing 2 X 10-4M mercaptoethanol and shake with 8 g of glass beads in the Nossal tissue disintegrator 4 for four 20-second periods, cooling the cartridges in ice between each treatment. Centrifuge the suspensions for 10 minutes at 10,000 g. Wash the residues twice with a total of 20 ml of the same buffer and combine the washings with the first supernatant solution. Protamine Fraction. Treat the clear extract with 6.0 ml of 2% protamine sulfate solution and centrifuge after 20 minutes at 0 °. Discard the precipitate. Ammonium Sulfate I. To the supernatant solution add 3.0 ml of 0.5 M sodium phosphate buffer (pH 6.5) and 12.85 g of ammonium sulfate. After 10 minutes remove the precipitate by centrifugation and discard it. Treat the clear solution (42 ml) with 2.86 g of ammonium sulfate; collect the precipitate by centrifugation after 10 minutes, and dissolve it in 7.5 ml of 0.05 M sodium phosphate buffer, pH 6.5. Heated Fraction. To the solution add 0.7 ml of 0.5 M sodium phosphate buffer (pH 6.5) containing 2 X 10-4 M mercaptoethanol. Heat the diluted solution in a water bath at 63-65 ° for 5 minutes, centrifuge for 10 minutes at 10,000 g, and discard the precipitate. Ammonium Sulfate H. Determine the amonium sulfate concentration in the supernatant solution (heated fraction, 7.3 ml) with a Barnstead PR2 conductivity meter and adiust to 70% saturation by addition of solid ammonium sulfate. After 10 minutes collect the precipitate by centrifugation at 10,000 g for 5 minutes. Extract this precipitate with 4.0 3 The substitution of glucose for fructose in the growth media does n o t alter the yield of cells b u t reduces the enzymatic activity in the crude extract to about 5% of t h a t found in the fructose medium. 4 p. M. Nossal, Australian J. Exptl. Biol. Med. Sci. 31, 583 (1953).
[28]
MANNITOL DEHYDROGENASE
145
ml of 65% saturated ammonium sulfate (pH 7.0), and after 10 minutes again collect the precipitate by centrifugation. Extract successively, as before, with 3.0 ml and 2.0 ml of 60% saturated ammonium sulfate. Combine the last two extracts. Crystallization. Adjust the ammonium sulfate II solution to pH 6.0 with 0.1 N acetic acid and add a solution of saturated ammonium sulfate until faint turbidity is observed. Allow to stand overnight at 3 ° and collect the crystals by centrifugation. Dissolve them in 1.6 ml of 0.1 M sodium acetate buffer (pH 6.0) containing 2 X 10-4M mercaptoethanol (first crystals). Repeat this procedure (second crystals), and, after centrifugation to separate, repeat again (third crystals). Finally add mercaptoethanol (2 X 10-4 M) and ammonium sulfate until crystallization is begun and store the crystal suspension at 3 ° . The crystals obtained in this manner are very transparent; for the purpose of visualization carry out another crystallization in the presence of 10-6 M methylene blue. Specificity. The enzyme is specific for D-fructose and D-mannitol. It does not catalyze oxidation of the following polyols: glycerol, erythritol, xylitol, D-sorbitol, D-arabitol, L-arabitol, dulcitol, inositol, ribitol, melibiitol, L-glycero-D-glucoheptitol. The enzyme will not catalyze reduction of the following sugars: D-sorbose, D-xylulose, D-ribulose, sedoheptulose, fructose 6-phosphate, D-glucose. T P N H will replace D P N H in the reduction of fructose; with the standard assay conditions the reaction rate is about one-half as fast with this coenzyme. In the reaction between oxidized coenzyme and mannitol at pH 8.6 the rate with TPN is only 2.5% of that with DPN. Distribution. An enzyme with this specificity has thus far been reported only in L-brevis, but it probably occurs in other species of Lactobacillus that accumulate mannitol. Use ]or Analytical Purpose.s. The enzyme can be employed for the determination of D-mannitol and D-fructose. For fructose, prepare a reaction mixture containing 0.1 M sodium acetate buffer, pH 6.0, 0.1 mM DPNH, 0.02 mg of mannitol dehydrogenase, and 0.01-0.05 micromole of D-fructose in a total volume of 1.0 ml. Read the absorbance at 340 m~ before and after addition of D-fructose, and calculate the amount present in the assay cuvette from the change in absorbance2 For mannitol, prepare a reaction mixture (0.30 ml) containing 0.02M Tris buffer, pH 8.0, 7.5 mM pyruvate, 2.0 mM DPN, 0.01 mg of lactic dehydrogenase, 6 and 0.2 mg mannitol dehydrogenase. Add 0.5-1.0 micro5B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). Vol. I [67].
146
DEHYDROGENASES AND OXIDASES
[28]
mole of mannitol, incubate a t room temperature for 3 hours, and measure fructose colorimetrically. 7 D-Mannose can be determined with this procedure after reduction with sodium borohydride. PURIFICATION PROCEDURE
Fraction Extract Protamine fraction Ammonium sulfate I Heated fraction Ammonium sulfate II First crystals Second crystals Third crystals
Total volume (ml)
Units/ml
Total units
34 35 7.5 7.3 5.1 1.6 1.6 1.6
21.3 20.3 59.3 41.8 49.0 90.7 65.7 50.0
725 690 430 305 250 145 105 80
Specific activity Recovery (units/mg) (%) 0.75 2.7 4.9 8.6 16.7 23.7 28.9 27.3
-95 59 42 34 20 14 11
Properties
Stability. The crude extracts show a rapid loss of activity at 2 °, but this loss can be partially restored by incubation with 10-3 M mercaptoethanol at 37% The purified enzyme is most stable in solution at p H 6.0 in a 0 . 0 2 M sodium acetate buffer containing 10- 3 M mercaptoethanol. Such solutions can be stored for weeks at - - 1 6 ° . The enzyme solutions lose no activity when dialyzed at 3 ° against oxygen-free water containing 2 X 10 -4 M mercaptoethanol for a period of 18 hours. Suspensions of the crystalline enzyme preparation k e p t in a m m o n i u m sulfate at 3 ° lose about 20% of their activity in 2 months. This loss is largely reversed by the addition of mercaptoethanol to 2 X 10- 4 M followed by warming to 37 ° for 1 hour. Ef]ect a] pH on Reaction Rate. The optimal p H for the conversion of fructose to mannitol, the reaction utilized in the enzyme assay, is 5.35. The optimal p H for the reverse reaction, the conversion of mannitol to fructose, is 8.6.
~J. H. Roe, J. Biol. Chem. 1@7, 15 (1934).