- Email: [email protected]
[26] d -Glucose dehydrogenase from Gluconobacter su☐ydans
D-GLUCOSEDEHYDROGENASEFROMGluconobacter
[26]
159
becomes labile and less active when the detergent is removed from the enzyme solution. Catalytic Properties. The D-fructose dehydrogenase can be assayed in vitro in the presence of any one of the following dyes as an electron acceptor: potassium ferricyanide, phenazine methosulfate, nitro blue tetrazolium, or 2,6-dichlorophenolindophenol, NAD, NADP, or oxygen are completely inactive as electron acceptors. This finding applies to other membrane-bound dehydrogenases (see this volume [24], [31], [76]). Of tested substrates, only D-fructose is oxidized. When D-fructose (100 /xmol) is oxidized in the presence of substrate analogs (100/zmol each), such as o-glucose, D-mannose, o-fructose 6-phosphate, o-glucose 1-phosphate, D-gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate, the reaction rate is not affected. An apparent Michaelis constant for D-fructose at pH 4.5 is 10 mM. The pH optimum is pH 4.0, and optimum temperature is about 25° . The reaction product of D-fructose oxidation is identified as 5-keto-o-fructose by paper chromatography. The reaction product is specifically reduced to D-fructose in the presence of NADPH and 5-keto-D-fructose reductase, which can be crystallized from the cytosol of G. industrius. ~ 5 M. Ameyama, (1981).
K . M a t s u s h i t a , E . S h i n a g a w a , a n d O . A d a c h i , Agric. Biol. Chem. 45, 863
[26] D - G l u c o s e D e h y d r o g e n a s e f r o m G l u c o n o b a c t e r suboxydans
By OSAO ADACHI and M I i o a u AMEYAMA D-Glucose + NADP ~
D-glucono-8-1actone + NADPH
Assay Method
Principle. D-Glucose dehydrogenase (EC I.I.I.47) is measurcd spectrophotometrically by following the rate of N A D P H formation at 340 nm. Reagents NADE 1.5 mM, in distilled water; stored at - 2 0 ° until use D-Glucose, 1 M, in distilled water Tris-HCl buffer, 1 M, pH 8.0 Enzyme, dissolved in 0.01 M potassium phosphate, pH 7.5, containing 1 mM 2-mercaptoethanol
METHODS IN ENZYMOLOGY. VOL. 89
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2
160
OXIDATION--REDUCTION ENZYMES
[26]
Procedure. The enzyme activity is measured by reading the increase of absorbance at 340 nm in a recording spectrophotometer thermostatted at 25° in a thermostatted room (25°). Prior to the assay, the buffer solution and distilled water are warmed to 25° . The complete reaction mixture contains 0.1 ml of NADP, 0.1 ml of o-glucose, 0.3 ml of Tris-HCl and enzyme solution in a total volume of 3.0 ml. The reaction is initiated by the addition of enzyme. Definition of Unit and Specific Activity. One enzyme unit causes the formation of 1 /xmol of NADPH per minute under the assay conditions described. Specific activity (units per milligram of protein) is based on the spectrophotometric protein estimation by measuring at 280 nm; ~em~'l%value of 15.0 is used throughout. Source of Enzyme
Microorganism and Culture. Gluconobacter suboxydans IFO 12528 can be obtained from the Institute for Fermentation, Osaka (17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan). This is the same strain as that used for preparation of crystalline 6-phospho-o-gluconate dehydrogenase (see this volume [50]). Cultivation of the organism is also performed under the same conditions as mentioned for 6-phosphoo-gluconate dehydrogenase [50]. Purification Procedure ~ All operations are carried out at 0-5 ° , unless otherwise stated. Potassium phosphate buffer containing 1 mM 2-mercaptoethanol is used throughout. The procedure reported is for 150 g of wet cells ofG. suboxydans. Centrifugations are performed at 12,000 g for 20 min. Step 1. Cell-Free Extract. Cell paste of G. suboxydans harvested from 30 liters of a 24-hr culture in a 50-liter jar fermentor is washed twice with cold water and then suspended in 0.01 M buffer, pH 7.5. After cell disruption by a French press (American Instrument Co.) at I000 kg/cm 2, the cell homogenate is centrifuged at 68,000 g for 90 min; o-glucose dehydrogenase is present in the supernatant (1600 ml). Step 2. DEAE-Sephadex Column Chromatography (I). The enzyme solution is applied to a DEAE-Sephadex A-50 column (5 x 35 cm) that has been equilibrated with 0.01 M buffer, pH 6.0. About 50% of the total 10. Adachi, K. Matsushita, E. Shinagawa, and M. Ameyama, Agric. Biol. Chem. 44, 301 (1980).
[26]
D-GLUCOSEDEHYDROGENASEFROMGluconobacter
161
enzyme activity passes through the column, and a large amount of impure protein is adsorbed. Step 3. DEAE-Sephadex Column Chromatography (H). The enzyme solution from step 2 is adjusted to pH 7.5 with ammonia and applied to a second DEAE-Sephadex A-50 column (2 x 20 cm), which has been equilibrated with 0.01 M buffer, pH 7.5. Nonadsorbed proteins are washed through with the same buffer. Elution of the enzyme is affected by a linear gradient of NaC1 formed from 500 ml of 0.01 M buffer, pH 7.5, and 500 ml of the same buffer containing 0.4 M NaCI. The dehydrogenase is eluted at about 0.15 M concentration of the salt. To the combined enzyme solution, ammonium sulfate is added to 3.9 M (51.5 g/100 ml). The precipitated enzyme is collected by centrifugation and dissolved in 0.01 M buffer, pH 7.5, and dialyzed thoroughly against the same buffer. Step 4. Affinity Chromatography on Blue-Dextran Sepharose. After removal of insoluble materials by centrifugation, the dialyzed enzyme is applied to a Blue-Dextran Sepharose 4B column (2 x 20 cm) equilibrated with 0.01 M buffer, pH 7.5. The column is treated with the same buffer until the absorbance of the eluate at 280 nm decreases to 0.05. Elution of the enzyme is made with 0.01 M buffer containing 0.35M KCI, and a sharp protein peak corresponding to the enzyme activity is eluted. The pooled fractions are dialyzed overnight against 0.01 M buffer, pH 7.5, containing 0.1 M NaC1. Step 5. DEAE-Sephadex Column Chromatography (III). The dialyzed enzyme is applied to a DEAE-Sephadex A-50-column (1.5 x 10 cm) equilibrated with 0.01 M buffer, pH 7.5, containing 0.1 M NaCI. The column is washed with the same buffer and eluted by a linear gradient of NaCI formed by 200 ml of 0.01 M buffer, pH 7.5, containing 0.1 M NaC1 and 200 ml of 0.01 M buffer, pH 7.5, containing 0.3 M NaC1. A major protein peak elutes around at 0.15 M NaCI is coincident with enzyme activity. Peak fractions with specific activities over 110 units per milligram of protein are combined and dialyzed against saturated ammonium sulfate containing 0.1 M D-glucose, pH 7.0, until the enzyme precipitates in the dialysis bag. Step 6. Crystallization. The precipitate is collected by centrifugation and dissolved in a minimum volume of 0.1 M buffer, pH 7.5. After standing for a few hours in the cold, insoluble materials are removed by centrifugation. A few grains of solid ammonium sulfate are added to the enzyme solution, and the solution is kept in a refrigerator. Crystals of the enzyme appear as fine rods or needles. The enzyme is purified about 1800-fold with a yield of 30% as shown in the table.
162
OXIDATION--REDUCTION ENZYMES
[26]
PURIFICATION OF D-GLuCOSE DEHYDROGENASE FROM Gluconobacter suboxydans
Step
Total protein (mg)
Cell-free extract DEAE-Sephadex A-50 (I) DEAE-Sephadex A-50 (II) Blue-Dextran Sepharose DEAE-Sephadex A-50 (III) Crystallization
14,640 5,640 1,290 15 4 2.5
Total activity (units) 1160 570 435 403 390 350
Specific activity (units/rag protein)
Yield (%)
0.08 0.10 0.34 26.62 97.50 140.00
100 50 38 35 34 30
Properties
Homogeneity. The sedimentation pattern of the enzyme shows a single symmetrical peak with an apparent sedimentation constant of 5.8 S. Upon gel electrophoresis, the enzyme shows a single protein band coincident with enzyme activity. Molecular Properties. The absorption maximum is at 280 nm, and the Elm ~° value is estimated to be 4.0 on the basis of absorbance and dry weight determinations. The molecular weight of the enzyme is determined to be 153,000 by gel filtration on Sephadex G-200 column. The enzyme dissociates into four identical subunits having a molecular weight of about 40,000 each. Catalytic Properties. General catalytic properties of D-glucose dehydrogenase have been extensively studied with partially purified enzyme preparations, z'3 A crystalline preparation as a suspension in ammonium sulfate is quite stable at 5° for several months. Even in the absence of ammonium sulfate, appreciable loss of activity is not observed after storage at 5° for 2 weeks. The optimum pH for D-glucose oxidation is 8.5-9.0, and a higher reaction rate is usually observed in Tris-HCl than in potassium phosphate. The optimum temperature is 50°. D-Glucose dehydrogenase is specific to NADP and completely inactive with NAD. The activity is potently inhibited by sulfhydryl reagents and divalent heavy metals such as p-chloromercuribenzoate, Hg 2+, Cu z+, and Ni z+. The enzyme possesses broad substrate specificity in the order D-glucose, 100%, D-mannose, 88%, mannose, 6%, D-glucose 6-phosphate, 1%, and 2 K. Okamoto, J. Biochem. 53, 348 (1963). 3 G. Avigad, Y. Alroy, and S. Englard, J. Biol. Chem. 243, 1936 (1968).
[27]
GALACTOSEOXIDASEFROMDactylium
163
Ouco e GDH
/
~
~
?O Catalas e
SCHEME 1. GDH, D-glucose dehydrogenase; DYE, old yellow enzyme.
D-galactose, 1%. The enzyme is inert to D-fructose, D-arabinose, D-xylose, and L-sorbose. The reaction product of the enzyme is D-glucono-8-1actone. The apparent Michaelis constants for D-glucose and NADP are 5 × 10 -a M and 1 × 10 -5 M, respectively. In the reverse direction, only 4% of the reaction occurs at neutral pH. Participation of Old Yellow Enzyme in Regeneration of NADP. It is indicated that NADPH formed in D-glucose oxidation is spontaneously oxidized to NADP by an enzyme, NADPH dehydrogenase, that exists predominantly in the cytoplasma of Gluconobacter species. The enzyme is similar to the old yellow enzyme in yeast. 4 An experiment similar to that described for 6-phospho-D-gluconate dehydrogenase (see this volume [50]) is conducted in a conventional Warburg flask: D-glucose dehydrogenase, old yellow enzyme, excess amounts of D-glucose, and limited amounts of NADP or NADPH are incubated in the presence of catalase. A linear oxygen uptake is observed showing that a cyclic coupling system is functioning to regenerate NADPH, as shown in Scheme 1. 40. Adachi, K. Matsushita, E. Shinagawa, and M. Ameyama, J. Biochem. 86, 699 (1979).
[27] G a l a c t o s e O x i d a s e f r o m Dactylium dendroides By PAUL S. TRESSEL a n d DANIEL J. KOSMAN
Galactose oxidase (EC 1.1.3.9) is a Cu(II)-containing extracellular fungal enzyme that catalyzes the reaction (1). 1-6 RCH,OH + O2---~ RCHO + n,o2
(1)
Although the enzyme exhibits some activity toward a variety of primary alcohols 7-12 (and only primary alcohols10, among hexoses it is nearly specific for the Ca-OH of galactose. 3aa,14 Because of this high degree of J. A. D. Cooper, W. Smith, M. Bacila, and H. Medina, J. Biol. Chem. 234, 445 (1959).
METHODSIN ENZYMOLOGY,VOL.89
Copyright© 1982byAcademicPress,Inc. Allrightsofreproductioninanyformreserved. ISBN0-12-181989-2
[26]
159
becomes labile and less active when the detergent is removed from the enzyme solution. Catalytic Properties. The D-fructose dehydrogenase can be assayed in vitro in the presence of any one of the following dyes as an electron acceptor: potassium ferricyanide, phenazine methosulfate, nitro blue tetrazolium, or 2,6-dichlorophenolindophenol, NAD, NADP, or oxygen are completely inactive as electron acceptors. This finding applies to other membrane-bound dehydrogenases (see this volume [24], [31], [76]). Of tested substrates, only D-fructose is oxidized. When D-fructose (100 /xmol) is oxidized in the presence of substrate analogs (100/zmol each), such as o-glucose, D-mannose, o-fructose 6-phosphate, o-glucose 1-phosphate, D-gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate, the reaction rate is not affected. An apparent Michaelis constant for D-fructose at pH 4.5 is 10 mM. The pH optimum is pH 4.0, and optimum temperature is about 25° . The reaction product of D-fructose oxidation is identified as 5-keto-o-fructose by paper chromatography. The reaction product is specifically reduced to D-fructose in the presence of NADPH and 5-keto-D-fructose reductase, which can be crystallized from the cytosol of G. industrius. ~ 5 M. Ameyama, (1981).
K . M a t s u s h i t a , E . S h i n a g a w a , a n d O . A d a c h i , Agric. Biol. Chem. 45, 863
[26] D - G l u c o s e D e h y d r o g e n a s e f r o m G l u c o n o b a c t e r suboxydans
By OSAO ADACHI and M I i o a u AMEYAMA D-Glucose + NADP ~
D-glucono-8-1actone + NADPH
Assay Method
Principle. D-Glucose dehydrogenase (EC I.I.I.47) is measurcd spectrophotometrically by following the rate of N A D P H formation at 340 nm. Reagents NADE 1.5 mM, in distilled water; stored at - 2 0 ° until use D-Glucose, 1 M, in distilled water Tris-HCl buffer, 1 M, pH 8.0 Enzyme, dissolved in 0.01 M potassium phosphate, pH 7.5, containing 1 mM 2-mercaptoethanol
METHODS IN ENZYMOLOGY. VOL. 89
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2
160
OXIDATION--REDUCTION ENZYMES
[26]
Procedure. The enzyme activity is measured by reading the increase of absorbance at 340 nm in a recording spectrophotometer thermostatted at 25° in a thermostatted room (25°). Prior to the assay, the buffer solution and distilled water are warmed to 25° . The complete reaction mixture contains 0.1 ml of NADP, 0.1 ml of o-glucose, 0.3 ml of Tris-HCl and enzyme solution in a total volume of 3.0 ml. The reaction is initiated by the addition of enzyme. Definition of Unit and Specific Activity. One enzyme unit causes the formation of 1 /xmol of NADPH per minute under the assay conditions described. Specific activity (units per milligram of protein) is based on the spectrophotometric protein estimation by measuring at 280 nm; ~em~'l%value of 15.0 is used throughout. Source of Enzyme
Microorganism and Culture. Gluconobacter suboxydans IFO 12528 can be obtained from the Institute for Fermentation, Osaka (17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan). This is the same strain as that used for preparation of crystalline 6-phospho-o-gluconate dehydrogenase (see this volume [50]). Cultivation of the organism is also performed under the same conditions as mentioned for 6-phosphoo-gluconate dehydrogenase [50]. Purification Procedure ~ All operations are carried out at 0-5 ° , unless otherwise stated. Potassium phosphate buffer containing 1 mM 2-mercaptoethanol is used throughout. The procedure reported is for 150 g of wet cells ofG. suboxydans. Centrifugations are performed at 12,000 g for 20 min. Step 1. Cell-Free Extract. Cell paste of G. suboxydans harvested from 30 liters of a 24-hr culture in a 50-liter jar fermentor is washed twice with cold water and then suspended in 0.01 M buffer, pH 7.5. After cell disruption by a French press (American Instrument Co.) at I000 kg/cm 2, the cell homogenate is centrifuged at 68,000 g for 90 min; o-glucose dehydrogenase is present in the supernatant (1600 ml). Step 2. DEAE-Sephadex Column Chromatography (I). The enzyme solution is applied to a DEAE-Sephadex A-50 column (5 x 35 cm) that has been equilibrated with 0.01 M buffer, pH 6.0. About 50% of the total 10. Adachi, K. Matsushita, E. Shinagawa, and M. Ameyama, Agric. Biol. Chem. 44, 301 (1980).
[26]
D-GLUCOSEDEHYDROGENASEFROMGluconobacter
161
enzyme activity passes through the column, and a large amount of impure protein is adsorbed. Step 3. DEAE-Sephadex Column Chromatography (H). The enzyme solution from step 2 is adjusted to pH 7.5 with ammonia and applied to a second DEAE-Sephadex A-50 column (2 x 20 cm), which has been equilibrated with 0.01 M buffer, pH 7.5. Nonadsorbed proteins are washed through with the same buffer. Elution of the enzyme is affected by a linear gradient of NaC1 formed from 500 ml of 0.01 M buffer, pH 7.5, and 500 ml of the same buffer containing 0.4 M NaCI. The dehydrogenase is eluted at about 0.15 M concentration of the salt. To the combined enzyme solution, ammonium sulfate is added to 3.9 M (51.5 g/100 ml). The precipitated enzyme is collected by centrifugation and dissolved in 0.01 M buffer, pH 7.5, and dialyzed thoroughly against the same buffer. Step 4. Affinity Chromatography on Blue-Dextran Sepharose. After removal of insoluble materials by centrifugation, the dialyzed enzyme is applied to a Blue-Dextran Sepharose 4B column (2 x 20 cm) equilibrated with 0.01 M buffer, pH 7.5. The column is treated with the same buffer until the absorbance of the eluate at 280 nm decreases to 0.05. Elution of the enzyme is made with 0.01 M buffer containing 0.35M KCI, and a sharp protein peak corresponding to the enzyme activity is eluted. The pooled fractions are dialyzed overnight against 0.01 M buffer, pH 7.5, containing 0.1 M NaC1. Step 5. DEAE-Sephadex Column Chromatography (III). The dialyzed enzyme is applied to a DEAE-Sephadex A-50-column (1.5 x 10 cm) equilibrated with 0.01 M buffer, pH 7.5, containing 0.1 M NaCI. The column is washed with the same buffer and eluted by a linear gradient of NaCI formed by 200 ml of 0.01 M buffer, pH 7.5, containing 0.1 M NaC1 and 200 ml of 0.01 M buffer, pH 7.5, containing 0.3 M NaC1. A major protein peak elutes around at 0.15 M NaCI is coincident with enzyme activity. Peak fractions with specific activities over 110 units per milligram of protein are combined and dialyzed against saturated ammonium sulfate containing 0.1 M D-glucose, pH 7.0, until the enzyme precipitates in the dialysis bag. Step 6. Crystallization. The precipitate is collected by centrifugation and dissolved in a minimum volume of 0.1 M buffer, pH 7.5. After standing for a few hours in the cold, insoluble materials are removed by centrifugation. A few grains of solid ammonium sulfate are added to the enzyme solution, and the solution is kept in a refrigerator. Crystals of the enzyme appear as fine rods or needles. The enzyme is purified about 1800-fold with a yield of 30% as shown in the table.
162
OXIDATION--REDUCTION ENZYMES
[26]
PURIFICATION OF D-GLuCOSE DEHYDROGENASE FROM Gluconobacter suboxydans
Step
Total protein (mg)
Cell-free extract DEAE-Sephadex A-50 (I) DEAE-Sephadex A-50 (II) Blue-Dextran Sepharose DEAE-Sephadex A-50 (III) Crystallization
14,640 5,640 1,290 15 4 2.5
Total activity (units) 1160 570 435 403 390 350
Specific activity (units/rag protein)
Yield (%)
0.08 0.10 0.34 26.62 97.50 140.00
100 50 38 35 34 30
Properties
Homogeneity. The sedimentation pattern of the enzyme shows a single symmetrical peak with an apparent sedimentation constant of 5.8 S. Upon gel electrophoresis, the enzyme shows a single protein band coincident with enzyme activity. Molecular Properties. The absorption maximum is at 280 nm, and the Elm ~° value is estimated to be 4.0 on the basis of absorbance and dry weight determinations. The molecular weight of the enzyme is determined to be 153,000 by gel filtration on Sephadex G-200 column. The enzyme dissociates into four identical subunits having a molecular weight of about 40,000 each. Catalytic Properties. General catalytic properties of D-glucose dehydrogenase have been extensively studied with partially purified enzyme preparations, z'3 A crystalline preparation as a suspension in ammonium sulfate is quite stable at 5° for several months. Even in the absence of ammonium sulfate, appreciable loss of activity is not observed after storage at 5° for 2 weeks. The optimum pH for D-glucose oxidation is 8.5-9.0, and a higher reaction rate is usually observed in Tris-HCl than in potassium phosphate. The optimum temperature is 50°. D-Glucose dehydrogenase is specific to NADP and completely inactive with NAD. The activity is potently inhibited by sulfhydryl reagents and divalent heavy metals such as p-chloromercuribenzoate, Hg 2+, Cu z+, and Ni z+. The enzyme possesses broad substrate specificity in the order D-glucose, 100%, D-mannose, 88%, mannose, 6%, D-glucose 6-phosphate, 1%, and 2 K. Okamoto, J. Biochem. 53, 348 (1963). 3 G. Avigad, Y. Alroy, and S. Englard, J. Biol. Chem. 243, 1936 (1968).
[27]
GALACTOSEOXIDASEFROMDactylium
163
Ouco e GDH
/
~
~
?O Catalas e
SCHEME 1. GDH, D-glucose dehydrogenase; DYE, old yellow enzyme.
D-galactose, 1%. The enzyme is inert to D-fructose, D-arabinose, D-xylose, and L-sorbose. The reaction product of the enzyme is D-glucono-8-1actone. The apparent Michaelis constants for D-glucose and NADP are 5 × 10 -a M and 1 × 10 -5 M, respectively. In the reverse direction, only 4% of the reaction occurs at neutral pH. Participation of Old Yellow Enzyme in Regeneration of NADP. It is indicated that NADPH formed in D-glucose oxidation is spontaneously oxidized to NADP by an enzyme, NADPH dehydrogenase, that exists predominantly in the cytoplasma of Gluconobacter species. The enzyme is similar to the old yellow enzyme in yeast. 4 An experiment similar to that described for 6-phospho-D-gluconate dehydrogenase (see this volume [50]) is conducted in a conventional Warburg flask: D-glucose dehydrogenase, old yellow enzyme, excess amounts of D-glucose, and limited amounts of NADP or NADPH are incubated in the presence of catalase. A linear oxygen uptake is observed showing that a cyclic coupling system is functioning to regenerate NADPH, as shown in Scheme 1. 40. Adachi, K. Matsushita, E. Shinagawa, and M. Ameyama, J. Biochem. 86, 699 (1979).
[27] G a l a c t o s e O x i d a s e f r o m Dactylium dendroides By PAUL S. TRESSEL a n d DANIEL J. KOSMAN
Galactose oxidase (EC 1.1.3.9) is a Cu(II)-containing extracellular fungal enzyme that catalyzes the reaction (1). 1-6 RCH,OH + O2---~ RCHO + n,o2
(1)
Although the enzyme exhibits some activity toward a variety of primary alcohols 7-12 (and only primary alcohols10, among hexoses it is nearly specific for the Ca-OH of galactose. 3aa,14 Because of this high degree of J. A. D. Cooper, W. Smith, M. Bacila, and H. Medina, J. Biol. Chem. 234, 445 (1959).
METHODSIN ENZYMOLOGY,VOL.89
Copyright© 1982byAcademicPress,Inc. Allrightsofreproductioninanyformreserved. ISBN0-12-181989-2