[70]
FORMALDEHYDE DEHYDROGENASE FROM YEASTS
455
assay. The highest activity was found with Mg2+ (Table II). If magnesium sulfate is replaced by cobalt(II) or calcium ions, the reaction rates are only 57.3 and 30.3%, respectively, in relation to the assay with magnesium ions. Manganese chloride in the assay led to a complete loss of activity. Stability. The purified enzyme, in the presence of 60% glycerol, is stable for at least 4 weeks at - 2 0 o. pH Optimum. The enzyme has optimum activity at pH 7.5. Inhibitors 12. Among metabolites of the methanol oxidation sequence, glycolysis, and the tricarboxylic acid cycle as well as intermediates of the cyclic oxidation no inhibitors were found, but the enzyme is influenced by the energy charge. In the presence of 5 m M of ADP the enzyme is inhibited by 60%. 12 K. H. Hofmann and W. Babel, Z. Allg. Mikrobiol. 20, 389 (1980).
[70] F o r m a l d e h y d e D e h y d r o g e n a s e Methylotrophic Yeasts
from
By NoBuo I~ATO Formaldehyde + glutathionc + NAD + ~ S-formylglutathione + NADH + H +
The true substrate of NAD-linked and glutathione-dependent formaldehyde dehyclrogenase (glutathione) (EC 1.2.1.1) is a hemimercaptal, Shydroxymethylglutathione, spontaneously formed from formaldehyde and glutathione, the reaction product being S-formylglutathione. This enzyme catalyzes a key step of the methanol catabolism in yeasts and has so far been purified from several methanol-grown yeasts: Candida boidinii (Kloeckera sp.) 2201,1 Candida boidinii ATCC 32195, 2 Hansenula polymorpha, 3 Pichia NRRL-Y-11328, 4 and Pichia pastoris IFP 206. 5
Assay Method
Principle. Formaldehyde dchydrogenasc is measured spectrophotometrically by following the rate of N A D H
formation at 340 nm.
i N. Kato, T. Tamaoki, Y. Tani, and K. Ogata, Agric. Biol. Chem. 36, 2411 (1972). 2 H. Schutte, J. Flossdorf, H. Sahm, and M. R. Kula, Eur. J. Biochem. 62, 151 (1976). 3 j. p. van Dijken, G. J. Oostra-Demkes, R. Otto, and W. Harder, Arch. Microbiol. 111, 77 (1976). 4 R. Patel, C. T. Hou, and P. Derelanko, Arch. Bioehem. Biophys. 221, 135 (1983). 5 j. j. Allais, A. Louktibi, and J. Baratti, Agric. Biol. Chem. 47, 1509 (1983).
METHODS IN ENZYMOLOGY, VOL 188
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any form rc~rv~l.
456
METHYLOTROPHY
[70]
Reagents Potassium phosphate buffer, 100 raM, pH 7.5 NAD +, 60 mM, in distilled water Glutathione (reduced), 120 raM, in distilled water Formaldehyde, 60 raM, prepared by heating 0.5 g of paraformaldehyde in 5 ml of water at 100 ° in a sealed tube for 15 hr; the formaldehyde solution is standardized with glutathione-independent formaldehyde dehydrogenase6 (Grade II, from Pseudomonas putida; Nacalai Tesque, Inc., Kyoto), and the solution is diluted accordingly Procedure. The enzyme activity is measured by reading the increase in absorbance at 340 nm with a recording spectrophotometer thermostatically maintained at 30 °. The complete assay mixture consists of 1.0 ml of potassium phosphate buffer, 0.2 ml of NAD +, 0.1 ml of glutathione, 0.1 ml of formaldehyde, and a limited amount of the enzyme, in a final volume of 3.0 ml. The reaction is initiated by the addition of enzyme. A blank test, that is, a parallel assay, is performed with a reaction mixture with formaldehyde omitted. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 #mol of NADH per minute at 30 °. Specific activity is defined as the number of units per milligram of protein. Growth of Organism
Candida boidinii 2201 is cultivated at 30 ° for 3 days in a 2-liter shake flask containing 500 ml of growth medium with the following composition (per liter): 10 ml methanol, 5 g (NH4)2SO4, 2 g Na2HPO4, 4 g KH2PO4, 0.6 g MgSO4" 7H20, 0.2 g NaCI, 0.2 g CaC12"2H20, and 0.2 g yeast extract, pH 6.0. Methanol is added aseptically to the medium just before inoculation. The cells are harvested by centrifugation at 6700 g for 15 min and then washed twice with 10 m M potassium phosphate buffer (pH 7.0). The cell paste is stored at - 2 0 o until use. Purification P r o c e d u r e All procedures are performed at 0 to 5 o. The buffer solution comprises 1 m M dithiothreitol, 1 m M EDTA, and 0.5 m M phenylmethylsulfonyl fluoride. Step 1: Preparation of a Cell-Free Extract. Frozen cell paste of C. 6 M. Ando,T. Yoshimoto,S. Ogushi,K. Rikitake,S. Shibata, and D. Tsuru,J. Biochem.85, 1165 (1979).
[70]
FORMALDEHYDE DEHYDROGENASE FROM YEASTS
457
boidinii 2201 (180 g wet weight) is suspended in 180 ml of 10 m M potassium phosphate buffer (pH 7.5). Twenty-milliliter portions of the suspension are disrupted, separately, with a Braun MSK Cell Homogenizer with 20 g of glass beads for 4 min at 0 °, followed by centrifugation at 14,000 g for 20 min, the resultant supernatant being used as the cell-free extract, which is dialyzed against 10 m M potassium phosphate buffer (pH 7.5). Step 2: First DEAE-Toyopearl 650 Chromatography. The dialyzed solution is applied to a DEAE-Toyopearl 650 column (2.6 × 25 cm) previously equilibrated with 10 m M potassium phosphate buffer (pH 7.5). The column is washed with 500 ml of the buffer, and the enzyme is eluted with a linear gradient between 250 ml of 10 m M and 250 ml of 200 m M potassium phosphate buffer (pH 7.5) at a flow rate of 50 cm/hr. The active fractions are concentrated and dialyzed against 10 m M potassium phosphate buffer (pH 7.0). Step 3: Second DEAE-Toyopearl 650 Chromatography. The dialyzed solution is applied to a DEAE-Toyopearl 650 column (1.6 × 50 cm) previously equilibrated with 10 m M potassium phosphate buffer (pH 7.0), then the column is washed with 100 ml of the buffer. The enzyme is eluted with a linear gradient between 250 ml of 10 m M and 250 ml of 150 m M phosphate buffer (pH 7.0) at a flow rate of 20 cm/hr. The peak fractions are pooled, concentrated to about 10 ml by ultrafiltration, using an Amicon YM 10 membrane (Danvers, MA), and dialyzed against 10 m M potassium phosphate buffer (pH 7.5). Step 4: Hydroxylapatite Chromatography. The dialyzed solution is placed on a hydroxylapatite column (2.6 × 20 cm) previously equilibrated with 50 m M potassium phosphate buffer (pH 7.5). Successive elution is carried out with 200-ml volumes of 10, 50, and 100 m M potassium phosphate buffer (pH 7.5). The active peak, which appears in the eluate with the 100 m M buffer, is concentrated to a volume of 5 ml. Step 5: TSK Gel Toyopearl HW55 Gel Filtration. The concentrated enzyme solution is subjected to gel filtration on a TSK gel Toyopearl HW55 column (2.6 × 80 cm). Elution is carried out with 50 m M potassium phosphate buffer (pH 7.0) at a flow rate of 3 cm/hr. The active peak is concentrated to 5 ml. The concentrated enzyme solution is added to an equal volume of cold glycerol and stored at - 200. Under these conditions, the enzyme can be stored for at least 6 months without loss of activity. A typical purification is summarized in Table I. Properties
Purity. The purified enzyme preparation gives a single band on acrylamide (7%) gel electrophoresis at pH 8.3 in the presence of sodium dodecyl sulfate (SDS).
458
METHYLOTROPHY
[70]
TABLE I PURIFICATIONOF FORMALDEHYDEDEHYDROGENASEFROM Candida boidinii 2201
Procedure
Total protein (mg)
Total activity (units)
Specific activity (units/rag protein)
Purification (-fold)
Yield (%)
Cell-free extract First DEAE-Toyopearl Second DEAE-Toyopearl Hydroxylapatite TSK gel Toyopearl HW55
3170 91.5 24.2 14.5 10.8
1010 933 568 458 414
0.32 10.2 23.5 31.6 38.3
1 32 73 99 120
100 92 56 45 41
Molecular Weight and Subunit Structure. The relative molecular weight of formaldehyde dehydrogenase from C. boidinii 2201 is determined to be 82,000 by high-performance liquid chromatography on a column of TSK gel G-3000SW (Tosoh, Tokyo). The molecular weights of the subunits are determined to be 43,000 by SDS gel electrophoresis. The enzyme is assumed to be a dimer. Substrate Specificity. The enzyme catalyzes the oxidation of formaldehyde and methylglyoxal. The relative activity toward methylglyoxal is 89% of that toward formaldehyde. The enzyme does not use other aliphatic or aromatic aldehydes and is inactive toward NADP +. Glutathione cannot be replaced by other thiol compounds such as cysteine, 2-mercaptoethanol, dithiothreitol, or thioglycolate. The K,, values are 0.29 m M for formaldehyde, 2.8 m M for methylglyoxal, and 25 # M for NAD +. Reaction Product and Reverse Reaction. The oxidation product of formaldehyde in the reaction system is S-formylglutathione, the formation of which is followed as the increase in absorbance at 240 m M owing to formation of a thiol ester. When purified S-formylglutathione hydrolase (EC 3.1.2.12) is added to the reaction mixture, formate formation is detected.7 In the reverse reaction, the K., values for S-formylglutathione and NADH are 0.12 and 0.025 raM, respectively.8 Effect of pH. The enzyme exhibits optimum activity at pH 8.0 in potassium phosphate buffer (Tris-HC1 buffer is not suitable for the reaction) and is stable in the pH range of 6.0 to 10 at 20 ° for 1 hr. Inhibition. The enzyme is completely inhibited by 1 mMp-chloromercuribenzoate and several cations (1 m M each), Cd 2+, Cu 2÷, Hg2+, and Ag+. EDTA (1 mM) has no effect on the activity. 7 N. Kato, C. Sakazawa, T. Nishizawa, Y. Tani, and H. Yamada, Biochim. Biophys. Acta 611, 323 (1980). s N. Kato, H. Sahm, and F. Wagner, Biochim. Biophys. Acta 566, 12 (1979).
[71]
FORMATE DEHYDROGENASE FROM YEASTS
459
Kinetics. The results of kinetic studies on formaldehyde dehydrogenase from C. boidinii ATCC 32195 suggest an ordered Bi-Bi mechanism, with NAD + as the first substrate and NADH as the last product) In the forward reaction, NADH is a competitive inhibitor (product inhibition) with respect to NAD + (K~ 1.1 mM), and S-formylglutathione is a noncompetitive inhibitor with respect to NAD+ and formaldehyde (actually, S-hydroxymethylglutathione) (Ki 20-80 gM). Nucleoside phosphates are competitive inhibitors with respect to NAD÷. The ~ values at pH 6.0 are 0.80 m M for ATP, 0.15 m M for ADP, and 6.0 m M for AMP. Others. The general properties of formaldehyde dehydrogenase from methylotrophic yeasts2-5 are very similar to those of that from C. boidinii 2201.
[71] F o r m a t e D e h y d r o g e n a s e f r o m M e t h y l o t r o p h i c Yeasts
By NoBuo KATO Formate + NAD + ~ CO2 + NADH + H +
In methylotrophic yeasts, NAD-linked formate dehydrogenase (EC 1.2.1.2) is the last enzyme in methanol catabolism. This enzyme has so far been purified from several methanol-grown yeasts; Candida boidinii (Kloeckera sp.) 2201,1 Candida boidinii ATCC 32195, 2 Hansenula polymorpha CBS 4732, 3 Pichia pastoris NRRL-Y-75564 and IFP 206, 5 Candida methylica, 6 and Candida methanolica ATCC 26175. 7 Assay Method
Principle. Formate dehydrogenase is measured spectrophotometrically by following the rate of NADH formation at 340 nm. N. Kato, M. Kano, Y. Tani, and K. Ogata, Agric. BioL Chem. 38, 111 (1974). 2 H. Sehutte, J. Flossdoff, H. Sabra, and M. R. Kula, Fur. J. Biochem. 62, 151 (1976). 3 j. p. van Dijken, G. J. Oostra-Demkes, R. Otto, and W. Harder, Arch. Microbiol. 111, 77 (1976). 4 C. T. Hou, R. N. Patel, A. I. Laskin, and N. Barnabe, Arch. Biochem. Biophys. 216, 296 (1982). 5 j. j. Allais, A. Louktibi, and J. Baratti, Agric. BioL Chem. 47, 2547 (1983). 6 T. V. Avilova, O. A. Egorova, L. S. Ioanesyan, and A. M. Egorov, Fur. J. Biochem. 152, 657 (1985). 7 y. Izumi, H. Kanzaki, S. Morita, and H. Yamada, FEMSMicrobiol. Lett. 48, 139 (1987).
METHODS IN ENZYMOLOGY, VOL. 188
Cop,y~ht © 1990 by Academic Press, Inc. All rights of reproduction in any form r~werved.
FORMALDEHYDE DEHYDROGENASE FROM YEASTS
455
assay. The highest activity was found with Mg2+ (Table II). If magnesium sulfate is replaced by cobalt(II) or calcium ions, the reaction rates are only 57.3 and 30.3%, respectively, in relation to the assay with magnesium ions. Manganese chloride in the assay led to a complete loss of activity. Stability. The purified enzyme, in the presence of 60% glycerol, is stable for at least 4 weeks at - 2 0 o. pH Optimum. The enzyme has optimum activity at pH 7.5. Inhibitors 12. Among metabolites of the methanol oxidation sequence, glycolysis, and the tricarboxylic acid cycle as well as intermediates of the cyclic oxidation no inhibitors were found, but the enzyme is influenced by the energy charge. In the presence of 5 m M of ADP the enzyme is inhibited by 60%. 12 K. H. Hofmann and W. Babel, Z. Allg. Mikrobiol. 20, 389 (1980).
[70] F o r m a l d e h y d e D e h y d r o g e n a s e Methylotrophic Yeasts
from
By NoBuo I~ATO Formaldehyde + glutathionc + NAD + ~ S-formylglutathione + NADH + H +
The true substrate of NAD-linked and glutathione-dependent formaldehyde dehyclrogenase (glutathione) (EC 1.2.1.1) is a hemimercaptal, Shydroxymethylglutathione, spontaneously formed from formaldehyde and glutathione, the reaction product being S-formylglutathione. This enzyme catalyzes a key step of the methanol catabolism in yeasts and has so far been purified from several methanol-grown yeasts: Candida boidinii (Kloeckera sp.) 2201,1 Candida boidinii ATCC 32195, 2 Hansenula polymorpha, 3 Pichia NRRL-Y-11328, 4 and Pichia pastoris IFP 206. 5
Assay Method
Principle. Formaldehyde dchydrogenasc is measured spectrophotometrically by following the rate of N A D H
formation at 340 nm.
i N. Kato, T. Tamaoki, Y. Tani, and K. Ogata, Agric. Biol. Chem. 36, 2411 (1972). 2 H. Schutte, J. Flossdorf, H. Sahm, and M. R. Kula, Eur. J. Biochem. 62, 151 (1976). 3 j. p. van Dijken, G. J. Oostra-Demkes, R. Otto, and W. Harder, Arch. Microbiol. 111, 77 (1976). 4 R. Patel, C. T. Hou, and P. Derelanko, Arch. Bioehem. Biophys. 221, 135 (1983). 5 j. j. Allais, A. Louktibi, and J. Baratti, Agric. Biol. Chem. 47, 1509 (1983).
METHODS IN ENZYMOLOGY, VOL 188
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any form rc~rv~l.
456
METHYLOTROPHY
[70]
Reagents Potassium phosphate buffer, 100 raM, pH 7.5 NAD +, 60 mM, in distilled water Glutathione (reduced), 120 raM, in distilled water Formaldehyde, 60 raM, prepared by heating 0.5 g of paraformaldehyde in 5 ml of water at 100 ° in a sealed tube for 15 hr; the formaldehyde solution is standardized with glutathione-independent formaldehyde dehydrogenase6 (Grade II, from Pseudomonas putida; Nacalai Tesque, Inc., Kyoto), and the solution is diluted accordingly Procedure. The enzyme activity is measured by reading the increase in absorbance at 340 nm with a recording spectrophotometer thermostatically maintained at 30 °. The complete assay mixture consists of 1.0 ml of potassium phosphate buffer, 0.2 ml of NAD +, 0.1 ml of glutathione, 0.1 ml of formaldehyde, and a limited amount of the enzyme, in a final volume of 3.0 ml. The reaction is initiated by the addition of enzyme. A blank test, that is, a parallel assay, is performed with a reaction mixture with formaldehyde omitted. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 #mol of NADH per minute at 30 °. Specific activity is defined as the number of units per milligram of protein. Growth of Organism
Candida boidinii 2201 is cultivated at 30 ° for 3 days in a 2-liter shake flask containing 500 ml of growth medium with the following composition (per liter): 10 ml methanol, 5 g (NH4)2SO4, 2 g Na2HPO4, 4 g KH2PO4, 0.6 g MgSO4" 7H20, 0.2 g NaCI, 0.2 g CaC12"2H20, and 0.2 g yeast extract, pH 6.0. Methanol is added aseptically to the medium just before inoculation. The cells are harvested by centrifugation at 6700 g for 15 min and then washed twice with 10 m M potassium phosphate buffer (pH 7.0). The cell paste is stored at - 2 0 o until use. Purification P r o c e d u r e All procedures are performed at 0 to 5 o. The buffer solution comprises 1 m M dithiothreitol, 1 m M EDTA, and 0.5 m M phenylmethylsulfonyl fluoride. Step 1: Preparation of a Cell-Free Extract. Frozen cell paste of C. 6 M. Ando,T. Yoshimoto,S. Ogushi,K. Rikitake,S. Shibata, and D. Tsuru,J. Biochem.85, 1165 (1979).
[70]
FORMALDEHYDE DEHYDROGENASE FROM YEASTS
457
boidinii 2201 (180 g wet weight) is suspended in 180 ml of 10 m M potassium phosphate buffer (pH 7.5). Twenty-milliliter portions of the suspension are disrupted, separately, with a Braun MSK Cell Homogenizer with 20 g of glass beads for 4 min at 0 °, followed by centrifugation at 14,000 g for 20 min, the resultant supernatant being used as the cell-free extract, which is dialyzed against 10 m M potassium phosphate buffer (pH 7.5). Step 2: First DEAE-Toyopearl 650 Chromatography. The dialyzed solution is applied to a DEAE-Toyopearl 650 column (2.6 × 25 cm) previously equilibrated with 10 m M potassium phosphate buffer (pH 7.5). The column is washed with 500 ml of the buffer, and the enzyme is eluted with a linear gradient between 250 ml of 10 m M and 250 ml of 200 m M potassium phosphate buffer (pH 7.5) at a flow rate of 50 cm/hr. The active fractions are concentrated and dialyzed against 10 m M potassium phosphate buffer (pH 7.0). Step 3: Second DEAE-Toyopearl 650 Chromatography. The dialyzed solution is applied to a DEAE-Toyopearl 650 column (1.6 × 50 cm) previously equilibrated with 10 m M potassium phosphate buffer (pH 7.0), then the column is washed with 100 ml of the buffer. The enzyme is eluted with a linear gradient between 250 ml of 10 m M and 250 ml of 150 m M phosphate buffer (pH 7.0) at a flow rate of 20 cm/hr. The peak fractions are pooled, concentrated to about 10 ml by ultrafiltration, using an Amicon YM 10 membrane (Danvers, MA), and dialyzed against 10 m M potassium phosphate buffer (pH 7.5). Step 4: Hydroxylapatite Chromatography. The dialyzed solution is placed on a hydroxylapatite column (2.6 × 20 cm) previously equilibrated with 50 m M potassium phosphate buffer (pH 7.5). Successive elution is carried out with 200-ml volumes of 10, 50, and 100 m M potassium phosphate buffer (pH 7.5). The active peak, which appears in the eluate with the 100 m M buffer, is concentrated to a volume of 5 ml. Step 5: TSK Gel Toyopearl HW55 Gel Filtration. The concentrated enzyme solution is subjected to gel filtration on a TSK gel Toyopearl HW55 column (2.6 × 80 cm). Elution is carried out with 50 m M potassium phosphate buffer (pH 7.0) at a flow rate of 3 cm/hr. The active peak is concentrated to 5 ml. The concentrated enzyme solution is added to an equal volume of cold glycerol and stored at - 200. Under these conditions, the enzyme can be stored for at least 6 months without loss of activity. A typical purification is summarized in Table I. Properties
Purity. The purified enzyme preparation gives a single band on acrylamide (7%) gel electrophoresis at pH 8.3 in the presence of sodium dodecyl sulfate (SDS).
458
METHYLOTROPHY
[70]
TABLE I PURIFICATIONOF FORMALDEHYDEDEHYDROGENASEFROM Candida boidinii 2201
Procedure
Total protein (mg)
Total activity (units)
Specific activity (units/rag protein)
Purification (-fold)
Yield (%)
Cell-free extract First DEAE-Toyopearl Second DEAE-Toyopearl Hydroxylapatite TSK gel Toyopearl HW55
3170 91.5 24.2 14.5 10.8
1010 933 568 458 414
0.32 10.2 23.5 31.6 38.3
1 32 73 99 120
100 92 56 45 41
Molecular Weight and Subunit Structure. The relative molecular weight of formaldehyde dehydrogenase from C. boidinii 2201 is determined to be 82,000 by high-performance liquid chromatography on a column of TSK gel G-3000SW (Tosoh, Tokyo). The molecular weights of the subunits are determined to be 43,000 by SDS gel electrophoresis. The enzyme is assumed to be a dimer. Substrate Specificity. The enzyme catalyzes the oxidation of formaldehyde and methylglyoxal. The relative activity toward methylglyoxal is 89% of that toward formaldehyde. The enzyme does not use other aliphatic or aromatic aldehydes and is inactive toward NADP +. Glutathione cannot be replaced by other thiol compounds such as cysteine, 2-mercaptoethanol, dithiothreitol, or thioglycolate. The K,, values are 0.29 m M for formaldehyde, 2.8 m M for methylglyoxal, and 25 # M for NAD +. Reaction Product and Reverse Reaction. The oxidation product of formaldehyde in the reaction system is S-formylglutathione, the formation of which is followed as the increase in absorbance at 240 m M owing to formation of a thiol ester. When purified S-formylglutathione hydrolase (EC 3.1.2.12) is added to the reaction mixture, formate formation is detected.7 In the reverse reaction, the K., values for S-formylglutathione and NADH are 0.12 and 0.025 raM, respectively.8 Effect of pH. The enzyme exhibits optimum activity at pH 8.0 in potassium phosphate buffer (Tris-HC1 buffer is not suitable for the reaction) and is stable in the pH range of 6.0 to 10 at 20 ° for 1 hr. Inhibition. The enzyme is completely inhibited by 1 mMp-chloromercuribenzoate and several cations (1 m M each), Cd 2+, Cu 2÷, Hg2+, and Ag+. EDTA (1 mM) has no effect on the activity. 7 N. Kato, C. Sakazawa, T. Nishizawa, Y. Tani, and H. Yamada, Biochim. Biophys. Acta 611, 323 (1980). s N. Kato, H. Sahm, and F. Wagner, Biochim. Biophys. Acta 566, 12 (1979).
[71]
FORMATE DEHYDROGENASE FROM YEASTS
459
Kinetics. The results of kinetic studies on formaldehyde dehydrogenase from C. boidinii ATCC 32195 suggest an ordered Bi-Bi mechanism, with NAD + as the first substrate and NADH as the last product) In the forward reaction, NADH is a competitive inhibitor (product inhibition) with respect to NAD + (K~ 1.1 mM), and S-formylglutathione is a noncompetitive inhibitor with respect to NAD+ and formaldehyde (actually, S-hydroxymethylglutathione) (Ki 20-80 gM). Nucleoside phosphates are competitive inhibitors with respect to NAD÷. The ~ values at pH 6.0 are 0.80 m M for ATP, 0.15 m M for ADP, and 6.0 m M for AMP. Others. The general properties of formaldehyde dehydrogenase from methylotrophic yeasts2-5 are very similar to those of that from C. boidinii 2201.
[71] F o r m a t e D e h y d r o g e n a s e f r o m M e t h y l o t r o p h i c Yeasts
By NoBuo KATO Formate + NAD + ~ CO2 + NADH + H +
In methylotrophic yeasts, NAD-linked formate dehydrogenase (EC 1.2.1.2) is the last enzyme in methanol catabolism. This enzyme has so far been purified from several methanol-grown yeasts; Candida boidinii (Kloeckera sp.) 2201,1 Candida boidinii ATCC 32195, 2 Hansenula polymorpha CBS 4732, 3 Pichia pastoris NRRL-Y-75564 and IFP 206, 5 Candida methylica, 6 and Candida methanolica ATCC 26175. 7 Assay Method
Principle. Formate dehydrogenase is measured spectrophotometrically by following the rate of NADH formation at 340 nm. N. Kato, M. Kano, Y. Tani, and K. Ogata, Agric. BioL Chem. 38, 111 (1974). 2 H. Sehutte, J. Flossdoff, H. Sabra, and M. R. Kula, Fur. J. Biochem. 62, 151 (1976). 3 j. p. van Dijken, G. J. Oostra-Demkes, R. Otto, and W. Harder, Arch. Microbiol. 111, 77 (1976). 4 C. T. Hou, R. N. Patel, A. I. Laskin, and N. Barnabe, Arch. Biochem. Biophys. 216, 296 (1982). 5 j. j. Allais, A. Louktibi, and J. Baratti, Agric. BioL Chem. 47, 2547 (1983). 6 T. V. Avilova, O. A. Egorova, L. S. Ioanesyan, and A. M. Egorov, Fur. J. Biochem. 152, 657 (1985). 7 y. Izumi, H. Kanzaki, S. Morita, and H. Yamada, FEMSMicrobiol. Lett. 48, 139 (1987).
METHODS IN ENZYMOLOGY, VOL. 188
Cop,y~ht © 1990 by Academic Press, Inc. All rights of reproduction in any form r~werved.
