purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol...

298 Biochimica et Biophysica A cta, 716 (1982) 298- 307 Elsevier Biomedical Press

BBA 21126

P U R I F I C A T I O N AND C H A R A C T E R I Z A T I O N OF A N I C O T I N A M I D E ADENINE D I N U C L E O T I D E - D E P E N D E N T SECONDARY A L C O H O L D E H Y D R O G E N A S E F R O M C A N D I D A

B O I D I N I I

HORST SCHOTTE, WERNER HUMMEL and MARIA-REGINA KULA *

Gesellschaft fiir Biotechnologische Forschung GmbH, Mascheroder Weg 1, D-3300 Braunschweig-StOckheim (F.R.G.)

(Received September 30th, 1981)

Key words: Alcohol Dehydrogenase; NAD + -dependence," (C. boidiniO

From the yeast CmMida boidinii grown on glucose a new secondary alcohol dehydrogenase was purified 426-foid by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CI-6b, and gel filtration on Sephacryl S-300. The purified enzyme was imnmgeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150000 by sedimentation equilibrium as well as by gel filtration. The enzyme appears to be composed of four identical stdmits (M r =38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD + as an electron acceptor. The K m values were found to be 0.099 mM for isopropanol and 0.14 mM for NAD +. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-oi, cyclobutanol, cyclopentanoi, and cyclobexanoi served as a substrate and were oxidized to the corresponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45°C. The isoelectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhthitor. Zinc ions appear necessary for enzyme production.

Introduction

Lamed et al. [1,2] detected an NADP-specific alcohol-aldehyde/ketone oxidoreductase in cell extracts of thermophilic ethanologenic bacteria. The extreme thermostable enzyme processes a broad range of substrates acting preferentially on linear secondary alcohols. Other NAD(P)-dependent alcohol dehydrogenases have been reported in Pseudomonas [3,4], E.coli [5], and Leuconostoc [6]. However, these enzymes were active only on long-chain primary alcohols or hydroxy fatty acids. Oxidation of primary alcohols in yeasts and liver

* To whom correspondence should be addressed.

0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

has been shown to be catalyzed by a N A D +- dependent alcohol dehydrogenase [7-12].

Hou et al. [13,14] first reported on a N A D +- linked alcohol dehydrogenase, specific for secondary alcohols from methane- or methanol-grown bacteria. This enzyme oxidizes secondary alcohols to their corresponding methyl ketones. Patel et al. [15,16] discovered that cell suspensions of yeasts grown on various C~- compounds, especially on methanol, catalyzed the oxidation of various secondary alcohols. The present paper is concerned with the characterization and purification of a secondary alcohol dehydrogenase from Candida boidinii grown on glucose. The enzyme catalyzes the oxidation of various linear and cyclic sec-

299

ondary alcohols to their corresponding ketones, but does not act on primary alcohols. The molecular properties of this enzyme are distinctly different from the secondary alcohol dehydrogenase purified to homogeneity by Patel et al. [16] from Pichia sp. and by Lamed et al. [1] from Thermo- anaerobicum brockii.

Materials and Methods

Materials. DEAE-Sephacel, Blue Sepharose C1- 6b, Sephacryl S-300 Superfine, and Gel Filtration Calibration Kit Proteins were purchased from De- utsche Pharmacia GmbH. (Freiburg, F.R.G.). U1- trafilter PM10 and Hollowfiber Cartridges Type H1X50 were obtained from Amicon GmbH (Wit- ten, F.R.G.). Coenzymes were purchased from Boehringer (Mannheim, F.R.G.), alcohols, ketones and other chemicals were purchased from Merck- Schuchardt (Darmstadt, F.R.G.). Malt extract, yeast extract and salts for cultivation were purchased from Merck also. Optically active stereoisomers of (S)-(+)-butan-2-ol and ( R ) - ( - ) - butan-2-ol were purchased from Fluka AG (Switzerland). All chemicals and solvents were of the highest quality available and used without further purification.

Microorganisms. Yeast strains for screening were obtained from the German Collection of Micro- organisms (DSM, GOttingen, F.R.G.). The strain used for enzyme production, Candida boidinii, was originally isolated by H. Sahm.

Media and growth conditions. All strains cultivated during the screening procedure were grown on a complex medium containing per 1:10 g glucose, 10g malt extract, 4g yeast extract. Flasks containing 125 ml medium were sterilized at 121 ° C for 15 min. Flasks were inoculated from slant cultures with the same medium, incubated at 30°C and 100 rpm for about 30 h. Cells were harvested by centrifugation, washed and disrupted by ultra- sonic treatment. After separation of cell debris by centrifugation the crude extract was used for the assay of isopropanol dehydrogenase. For isopropanol dehydrogenase production, C. boidinii was grown in a 10 l-bioreactor on a synthetic medium containing per 1 :10 g glucose, 2 g (NH4)2SO4, 3.75 g KH2PO 4, 2.5 g Na2HPO 4, 0.2 g MgSO 4 • 7 H20, 5 mg ZnSO 4 • 7 H20, l0 mg

FeSO 4 • 7 H20, 100/~g biotin. Incubation was performed with an aeration rate of 1 vvm, using a flat blade turbine stirrer at 150 rpm. Cells were harvested after exhaustion of glucose and stored frozen at -20°C.

Analytical methods. The isopropanol dehydrogenase was measured spectrophotometrically at 340 nm using a Zeiss PM4 photometer and a Vogel laboratory dialog calculator LDC 277. The cuvettes were thermostated and activity was measured at 30°C. The assay mixture contained in 50 mM potassium phosphate buffer, pH 7.5, 2.5 /~mols N A D +, 3 #mols isopropanol and limiting amounts of enzyme in a total volume of 3.00 ml. For the other secondary alcohols the substrate concentration in the assay was 10-fold higher than the K m values. One unit of the isopropanol dehydrogenase was defined as the amount of enzyme required to form 1 #mol of N A D H per min under the standard assay conditions.

Protein concentrations were determined according to Lowry et al. [17] using bovine serum albumin as a standard. Proteins were precipitated with 10% trichloroacetic acid before measurement in order to remove thiol reagents. During the screening for enzyme activity and medium optimi- zation the protein concentration was estimated by the method of Bradford et al. [18].

Gel electrophoresis. Analytical gel electrophoresis was carried out according to Jovin et al. [19] at room temperature in Tris-glycine buffer. The gels were stained with Coomassie brilliant blue [20]. Molecular weight determinations in SDS gels were done according to Shapiro et al. [21] using haemoglobin, chymotrypsinogen, ovalbumin and bovine serum albumin as marker proteins. The gels were fixed with 10% trichloroacetic acid and stained with Coomassie brilliant blue.

Isoelectric focusing. Analytical isoelectric focusing [22] was performed in cylindrical 5% polyacrylamide gels with 2% carrier ampholytes (LKB, Bromma, Sweden), in the pH range from 4.0 to 6.0. The electrofocusing was run at 4°C with a constant current of 1-2 mA/gel tube, setting a maximum voltage of 300 volts. Under these conditions focusing will take approx. 2.5-3 h in an 8 cm tube. 0.2 M H2SO 4 and 0.4M ethylene diamine served as electrode solutions. For calibration the following proteins were used: ovalbumin (pI 4.65),

300

bovine serum albumin (pI 4.80), and fl-lacto- globulin (pI 5.1). The colour change of methyl red (pH 4.9) was used to follow the progress of the focusing. Some gels were sliced in about 2.5 mm thick slices, eluted in buffer and the activity determined as a function of the distance. Gels run in parallel were either sliced and the pH of individual slices measured or fixed with 10% trichloroacetic acid and stained with Coomassie brilliant blue for comparison.

Gel filtration. The molecular weight of isopropanol dehydrogenase was determined according to the method of Andrews [23] by gel filtration on Sephacryl S-300 Superfine. The column (2.5 × 85 cm) was equilibrated with 50 mM Tris-HCl buffer, pH 8.0, containing 0.1% (v /v ) 2- mercaptoethanol and developed at a flow rate of 17.5 ml /h . The following marker proteins were used for calibration: chymotrypsinogen, ovalbumin, bovine serum albumin, aldolase, catalase, ferritin, thyroglobulin, and Dextran blue 2000.

Ultracentrifuge. A Beckman Spinco model E analytical ul tracentrifuge together with an improved collimatic optic [24] was used equipped with RTIC temperature control unit, monochro- mator and photoelectric scanner for the determination of the molecular weight of isopropanol dehydrogenase. All measurements were done at 280 nm. The enzyme was dialyzed against 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM dithiothreitol. Experiments were performed at 9000 rev/min. Centrifugation was carried out in a four-place rotor AN-F using Kel-F double sector cells (12 mm) with sapphire windows.

Results

Screening for isopropanol dehydrogenase activity After we noticed the presence of isopropanol

dehydrogenase activity in C. boidinii extracts a limited screening for the enzyme was carried out with several yeast strains. Yeasts were grown on complex liquid medium and enzyme activity was measured in the crude cell extract, the results are summarized in Table I. Among the yeasts tested enzyme activity seems to be limited to Candida strains, only one Hansenula strain exhibited enzyme activity too. Little or no activity (<0.01

TABLE I

SCREENING FOR ISOPROPANOL DEHYDROGENASE ACTIVITY IN YEASTS

Strain Isopropanol dehydrogenase (U/rag)

c. boidinii SAHM 0.92 C. boidinii DSM 70026 0.55 C. boidinii DSM 70033 0.75 C. humicola DSM 70067 0.08 C. intermedia DSM 70053 0.23 C. kefyr DSM 70073 0.78 C. krusei DSM 70086 1.06 C. tropicalis DSM 70151 0.13 C. utilis DSM 70167 1.11 H. wickerhamii DSM 70280 0.70

U / m g ) could be found in Cryptococcusflavus DSM 70227, Kloeckera apiculata D S M 70285, Metschnikowia pulcherrima DSM 70336, Sac- charomyces delbri~ckii DSM 70483, Rhodotorula macerans DSM 70822, and Kluyveromyces lactis DSM 70 799. C. boidinii (Sahm) was selected for further experiments since the cultivation and metabolism of this organism is well known in our laboratory.

Effect of different carbon sources on isopropanol dehydrogenase activity

Patel reported that another facultative methyl- otrophic strain of Candida (Candida utilis ATCC 26387) contains a secondary alcohol dehydrogenase when cultivated on methanol as sole carbon source. To determine whether the production of isopropanol dehydrogenase in C. boidinii depends on methanol metabolism, the yeast was grown on a synthetic medium with different carbon sources added in amounts of 1%. As Table II shows, glucose, fructose, and especially dihydroxyacetone give high yields of specific and volume activity of isopropanol dehydrogenase. No growth was observed after 3 days incubation on the substrate isopropanol as carbon source, or with the product acetone (also tested at 0.5 and 1.5% levels), and also not on n-propanol, glycerol or ethyleneglycol as carbon source. For economic reasons the cultivation of C. boidinii for isopropanol dehydrogenase production was carried out with glucose as carbon and energy source.

TABLE II

EFFECT OF DIFFERENT C-SOURCES ON ISOPRO- PANOL DEHYDROGENASE ACTIVITY.

C-source A~,~ am Isopropanol dehydrogenase (U /mg)

Glucose 26 0.93 Fructose 22 0.84 Ethanol 26 0.43 Sodium pyruvate 7 1.01 Dihydroxy acetone 28 2.05 Methanol l0 0.26

Production of isopropanol dehydrogenase during growth

Fig. 1 illustrates the enzyme production by C. boidinii during growth in a 10 1-bioreactor. Highest specific activity is observed after exhaustion of glucose during the stationary growth phase. Cells were harvested by centrifugation and stored frozen until use.

Purification of isopropanol dehydrogenase All operations were performed at 4°C. 10 mM

Tris-HC1 buffer, pH 8.0, containing 0.1% (v/v) 2-mercaptoethanol ,was used in the purification procedure, unless otherwise noted. Tris-HC1 buffer was chosen, because the enzyme shows the best stability in this media (Fig. 4). All chromatography buffers contained in addition 10% glycerol. The purification is summarized in Table III.

Step 1." crude extract 280 g of frozen C. boidinii cells were thawed at

4°C and then suspended in Tris-HC1 buffer, pH

301

0 1.0-

0,~1-

0 6 - ~

o -i: t Q2-

o -

I0 20 30

TIME (hours)

- tO0

E ao

60

~0

20

Fig. 1. Production of isopropanol dehydrogenase during growth in a 101 bioreactor. Incubation was performed at 30°C with an aeration rate of 1 w m , using a flat blade turbine stirrer at 150 rpm. • • , absorbance at 546 nm; O O, concentration of glucose in the medium; /x A, specific enzyme activity.

9.0, using a Waring blendor. The cell suspension was at least 35%. The cells were disrupted in a Dyno-Mill disintegrator (Type KDL; 0.61 con- tainer) [25]. The mill was operated at a rotational speed of 3000 rpm using glass beads with a diame- .ter of 0.25-0.5 mm. The cell suspension was pumped three times through the mill in single pass mode with a flow rate of 51/h. The pH was checked in the outgoing suspension and adjusted to 8.0 if necessary. The homogenate was centrifuged at 25000 × g for I h and the pellet dis- carded.

Step 2: heat treatment The still turbid crude extract was heated for 10

min in a water bath at 54°C and immediately

TABLE III

PURIFICATION OF ISOPROPANOL DEHYDROGENASE

Fraction Volume Protein Total act. Spec. act. Yield Purification factor (ml) (m8) (U) (U /mg) (%) (-fold)

Crude extract 540 4 212 2 268 0.54 100 I Heat treatment 445 ,2 403 2 051 0.85 90 1.6 DEAE-Sephacel 890 896 1976 2.21 87 4. I Blue Sepharose CI 6b 21 3.4 494 145.3 22 269 Sephacryl S-300 Superfine 32 2.0 460 230.0 20 426

302

cooled in ice and centrifuged at 25 000 x g for 30 min to remove residual cell debris and denatured protein. The supernatant was dialyzed against standard buffer containing 10% glycerol.

Step 3: DEAE-Sephacel column chromatography The dialyzed enzyme solution was applied to a

DEAE-Sephacel column (5 X 40 cm) which had been equilibrated with standard buffer. After washing with standard buffer the enzyme was eluted applying a 41 linear gradient from 0 to 0.8 M sodium chloride in standard buffer. Frac- tions of 18 ml were collected. The flow rate was 72 ml/h. Isopropanol dehydrogenase eluted between 250 and 400 mM sodium chloride. The active factions were combined, concentrated by ultra- filtration and dialyzed against 20 mM Tris-HC1 buffer, pH 7.0.

Step 4: affinity chromatography on Blue Sepharose CI-6b

The enzyme solution was placed on a column of Blue Sepharose C1-6b (2.5 X 20 cm) equilibrated with 20 mM Tris-HCl buffer, pH 7.0. A large amount of inactive protein eluted with starting buffer. A 5 mM NAD + solution in starting buffer was used to elute the isopropanol dehydrogenase. The flow rate of the column was 24 ml/h, fractions of 2.4 ml were collected and assayed for enzymatic activity. The enzyme containing fractions were combined and concentrated by ultra- filtration.

Step 5: Sephacryl S-300 Superfine column chromatography

The enzyme solution obtained in the preceding step was subjected to Sephacryl S-300 Superfine gel filtration. The gel was packed into a column (2.5 x 87 cm) and equilibrated with 50 mM Tris- HC1 buffer. The buffer was allowed to flow at a rate of 16 ml /h and 3.2 ml fractions were collected. The enzyme was located in the effluent, combined and stored at 4°C. For long time storage the enzyme solution was made 50% (v/v) in glycerol and kept at -20°C.

Molecular weight and subunit structure of isopropanol dehydrogenase

The molecular weight of isopropanol dehydro-

genase was determined by the sedimentation equilibrium method of Ypantis [26]. Assuming a par- tial specific volume of 0.750 cma/g a molecular weight of 150 000- 5000 was calculated from the data. The value did not depend on protein concentration over the range tested. Using the gel filtration method with Sephacryl S-300 Superfine the molecular weight of isopropanol dehydrogenase was estimated to be 155 000. The position of markers used are indicated in Fig. 2.

The calculated Kay values for each marker protein are plotted against the logarithms of molecular weight according to the equation:

Ve -Vo Ka~ V t - Vo '

where Ve = elution volume for the protein; Vo = column void volume; Vt = total bed volume.

Kav is the partition coefficient between the liquid phase and the gel phase. The value of the molecular weight determined by gel filtration corresponds well with the results of the equilibrium sedimentation. The native enzyme appears to be a tetramer as under denaturing conditions the molecular weight of the protein chain was found as 38 000 (gel electrophoresis in the presence of sodium dodecyl sulphate).

Kov

10.

0 8

0~,

Ok,

02 -

104

2 3

10

105 106 Log 'MOLECULAR WEIGHT

Fig. 2. Estimation of molecular weight of the isopropanol dehydrogenase b, b' gel filtration on Sephacryl S-300 Superfine. I, chymotrypsinogen; 2, ovalbumin; 3, bovine serum album; 4, ovatbumin dimer; 5, bovine serum album dimer; 6, isopropahol dehydrogenase; 7, aldolase; 8, catalase; 9, ferritin; 10, thyroglobulin.

Homogeneity All studies directed towards determination of

the molecular weight and subunit structure of isopropanol dehydrogenase indicated a homogeneous protein preparation. In addition the homogeneity of the final enzyme preparation was checked by disc electrophoresis. The enzyme migrated as a single band on the gel as revealed by staining with Coomassie blue. The isoelectric point of the enzyme was found to be at pH 4.9 by isoelectric focusing as described in Methods.

Substrate specifity of isopropanol dehydrogenase The K m values and the apparent Vm~ , of the

oxidation for various secondary alcohols by the isopropanol dehydrogenase are summarized in Ta- ble IV. The Michaelis constants were calculated by non-linear regression analysis of the Michaelis- Menten equation. The values indicate that isopropanol is the best substrate with a K m = 0.099 mM. With cyclobutanol and cyclopentanol as substrate

TABLE IV

SUBSTRATE SPECIFITY OF ISOPROPANOL DEHYDRO- GENASE

The reaction mixtures (3 ml) contained 0.05 M potassium phosphate buffer, pH 7.5, 0.83 mM NAD + and 0.17 #g nearly purified isopropanol dehydrogenase. The substrate concentration of the indicated secondary alcohol was 10-fold higher than the K m value. In the case that. the K m value was not known the reaction mixture contained 50 mM of the indicated substrate. The enzyme activity was measured as described in Methods.

Substrate Apparent Vma x K m value (~tmol/min per mg (mM) protein at 30°C)

I sopropanol 96 Butan-2-ol 88 Pentan-2-ol 26 Pentan-3-ol 28 Hexan-2-ol 5 Cyclobutanol 98 Cyclopentanol 98 Cyclohexanol 3 1,2-Propanediol 0 1,3-Propanediol 0 Glycerol 0

Stereoisomers (S)-(+)-butan-2-ol 16 ( R )-( - )-butan-2-ol 103

0.099 O.40 3.7 I.I 7.8 0.17 0.62 9.0

0.42 0.099

303

the fastest reaction rate is observed. The reactivity decreases as the chain length of the secondary aliphatic alcohols increases. The isopropanol dehydrogenase does not oxidize primary alcohols, tests included methanol, ethanol, propan-l-ol, butan-l- ol, and pentan-l-ol at various concentration levels. Surprisingly 1,2-propanediol and glycerol were also not oxidized demonstrating the pronounced influence of the hydrophobic and hydrophilic neighbouring groups of the secondary alcoholic hydroxyl-group in the substrate on the enzymatic activity. Remarkable is the different reactivity of the enzyme with cyclopentanol and cyclohexanol, the first is a good substrate with high Vm~ , values while the second is oxidized only very slowly. Isopropanol dehydrogenase activity depends strictly on NAD + . N A D P was tested over a range of concentrations and found totaly inactive as a coenzyme. The K m value for NAD + was calculated from non-linear regression analysis of the Michaelis-Menten equation using 15 data points and found to be 0.14 mM. The concentration of N A D + in the standard assay was set at 0.83 mM, because higher concentrations of NAD + inhibited already the reaction.

Stereospecifity of isopropanol dehydrogenase The isopropanol dehydrogenase catalyzes the

oxidation of (S)-(+)-butan-2-ol and ( R ) - ( - ) - butan-2-ol, but it reacts not equally on both enantiomers. The reaction rate with the (R)-( - - ) - butan-2-ol is approx. 10-times higher than the reaction rate with (S)-(+)-butan-2-ol when the substrate concentration in the standard assay was set at 1 mM. At a substrate concentration of 10 mM the reaction rate with (R)- ( - ) -butan-2-o l is approx. 6-times higher as with the ( + ) enantiomer. The apparent Vm~ , and the g m values are listed in Table IV.

Effect of temperature and pH on the enzymatic activity of isopropanoi dehydrogenase

The reaction rate of the isopropanol dehydrogenase was measured at various temperatures in 75 mM phosphate buffer. The reaction rate increases with temperature up to approx. 45°C. At still higher temperatures thermal denaturation of the enzyme dominates and the reaction rate declines. The stability of isopropanol dehydrogenase was

20.

tested by incubating the enzyme in 75 mM potassium phosphate buffer, pH 7.5, containing 0.1% (v /v ) 2-mercaptoethanol for 1 h at various temperatures. Then the enzyme solution was cooled and immediately assayed under standard conditions at 30°C. All samples were finally measured at a concentration of 0.1 mM 2-mercaptoethanol. The enzyme was found to be quite stable under this conditions up to 40°C; at 50°C75% of the activity was preserved. In crude extracts the enzyme was found to be quite stable for 15 min up to 55°C in Tris-HC1 buffer. The dependence of the enzymatic activity on the pH was studied in the range pH 5-11 using different buffers. As shown in Fig. 3 the enzyme exhibited a rather broad pH opt imum at values between 8 and 10. The results also dem- onstrate a rather strong influence of the buffer ions on the enzymatic acitivity. There is a remarkable difference in the initial rate in the presence of phosphate or Tris-HC1 buffer. The low reaction rate in Tris-HC1 buffer is dominated by changes in Vma x. The enzyme was found most

stable in Tris-HCl buffers at the pH range 8.5-9.5 at 30°C for l h (Fig. 4). Enzymes stability gener- ally decreases in the presence of other buffers especially below p H 6.5 and above p H 9.0. The opt imum p H of the enzyme in the reverse reaction (reduction of acetone) was found to be 7.0 as shown in Fig. 5.

Effect of various metal ions and inhibitors on the activity of isopropanol dehydrogenase

The effect of various metal ions on the oxidation of isopropanol by the enzyme was studied. The heavy metals Hg 2+ , Zn 2+ , and Cd 2+ inhibit the enzyme at low concentrations (0.1 mM). In a concentration of 10 mM the metal ions Cu 2+ , Co 2÷ and Mn 2÷ were also strongly inhibitory to the enzyme. Reagents reacting with sulfhydryl groups (iodoacetate, C N - ) have a strong inhibitory effect, p-chloromercuribenzoate inactivated the enzyme at a concentration of 40 #M com- pletely, indicating an essential sulfhydryl group(s). The inhibitory effect of chelating agents is shown

/ ,,-15

Lu10

20

pH Fig. 3. Effect of pH on the activity of isopropanol dehydrogenase. The reaction rate was measured in a mixture containing standard concentrations of NAD + and isopropanoi and various buffer ions in 50 mM concentration at the pH value indicated. O, sodium citrate; Q, potassium phosphate; A . triethanolamine-HCl; A, Tris-HC1; I , glycine.

100 -

-~ 8O

80

~ ~o

,oH

304

Fig. 4. The pH stability of isopropanol dehydrogenase was tested by preincubating the isopropanol dehydrogenase at 30°C for 60 min in 75 mM buffers at the pH values indicated. The assay was carried out under standard conditions. ©, sodium citrate; 0, potassium phosphate; A, triethanolamine-HCl: A, Tris-HCl; I1, glycine.

5

(o

t o 3

t~

; ; ; ,b ,,

pH

Fig. 5. Effect of pH on the activity of isopropanol dehydrogenase, in the reverse reaction. The assay mixture for the reverse reaction contained in 50 mM buffer, 0.45 #mol NADH, 0.018 #mol acetone and limiting amounts of enzyme in a total volume of 3.00 ml. Activity was measured by the decrease in absorption of NADH at 340 nm. O, sodium citrate; • , potassium phosphate; A, triethanolamine-HC1; A, Tris-HC1; l , glycine.

in Fig. 6. The strong influence of 1,10- phenanthroline and 2,2'-bipyridyl points towards a possible role of a metal ion in enzyme activity or stability. Therefore we looked for the influence of metal ions especially iron and zinc during growth of C. boidinii on the production of isopropanol dehydrogenase. By the addition of FeSO 4 • 7 H 2 0 in the range of 1 0 - 5 - 1 0 - 3 M enzyme yield was not significantly altered. Growth of C. boidinii in the presence of Zn 2 + however shows a remarkable influence; volume and specific activity of isopropanol dehydrogenase rise by increasing the concentration of Zn 2+ in the medium as shown in Fig. 7.

Influence of sulfhydryl group protecting agents It is also important to notice the rather strong

305

fo

t. ~5 Lu

u j

...0

~4 5

i i , , ,

fO 20 30 0.5 1.0 mM mM

Fig. 6. Effect of chelating agents on the activity of isopropanol dehydrogcnase. The rate of reaction was determined at various concentrations of citrate (O O); EDTA ( • • ) ; 2,2'-bipyridyl ( D - - D ) , and 1,10 phenanthroline (A A) indicated. The remaining enzyme activity was measured under standard conditions.

~ 3

to

A 50

5 2O

1 1

~0

q l , , fO'8 10-7 I0-6 10-5 I0-4 10-3

ZINC CONCENTRATION (M)

Fig. 7. Influence of various concentrations of Zn 2+ during growth on volume and specific activity of isopropanol dehydrogenase. Metal ions were added as ZnSO4-7 H20 to the synthetic medium without zinc. Incubations were carried out in shaking flasks with 100 ml medium. Cells were harvested after 24 h of cultivation; at this time all cultures had reached the stationary growth phase. • • , As46 nm; O O, enzyme activity; A A, specific activity.

306

120

100

~ 4O.

oc 20-

• ,=1

J ~ b ~ lb mM

Fig. 8. Effect of various concentrations of 2-mercaptoethanol (I) dithiothreitol (2) and reduced glutathione (3) on the activity of isopropanol dehydrogenase. Various concentrations of the reagents were included in the standard assay mixture and activity determined as usual. Reaction was started by the addition of enzyme.

inhibition of the enzyme by sulfhydryl group protecting agents like 2-mercaptoethanol or Clelands reagent (Fig. 8). Before analysis especially at low enzyme concentrations 2-mercaptoethanol must be removed. For storage and during preparation the presence of such agents is necessary for the enzyme stability. Storage of purified enzyme at 4°C for 3 days resulted in 80% loss of enzymatic activity, in the presence of 0.1% ( v / v ) 2- mercaptoethanol however the enzyme shows no significant decrease in activity. The inhibition by 2-mercaptoethanol was checked further in detail and found to be of a noncompetitive type. So a higher substrate concentration in the assay mixture does not compensate for the inhibitory effect of 2-mercaptoethanol. Very low concentrations of Clelands reagent or glutathione seem to activate the enzyme, which again points to the importance of a sulfhydryl group(s) in the function or stability of the enzyme.

Discussion

Sahm and Wagner [10] reported on a NAD +- dependent alcohol dehydrogenase from the yeast

C. boidinii. This enzyme catalyzes the oxidation of primary aliphatic alcohols only but does not oxidize methanol. This NAD+-specific alcohol dehydrogenase was found to be very similar to the same enzyme obtained from bakers' yeast and is also present in C. boidinii grown on glucose.

In the light of our present findings the observed [4] low activity towards isopropanol may be attri- buted to a small contamination of the ADH with isopropanol dehydrogenase. The present paper describes the isolation and characterization of a NAD+-dependent secondary alcohol dehydrogenase in C. boidinii grown on glucose. The bulk of the NAD+-dependent primary alcohol dehydrogenase is removed during the initial heat treatment. The newly purified enzyme does not act on primary alcohols. It catalyzes the oxidation of isopropanol to acetone. In addition other linear and cyclic secondary alcohols can also serve as a substrate and were oxidized to the corresponding ketones. We call the enzyme isopropanol dehydrogenase because isopropanol appears to be the best substrate. Inhibition studies with metal ions and mercaptide forming agents point towards special SH-groups involved in catalysis or to maintain the proper structure of the enzyme. Growth experiments with various concentrations of Zn 2+ suggest that isopropanol dehydrogenase is a metallo enzyme. This conclusion is supported by the strong inhibition with chelating agents like 1,10- phenanthroline and 2,2'-bipyridyl. In this respect the isopropanol dehydrogenase seems to resemble the primary alcohol dehydrogenase, which contains Zn 2 + as an integral part of the protein complex [27]. This statement requires further investiga- tions and will be studied in detail, when a larger amount of purified enzyme is available.

The isopropanol dehydrogenase was purified 426-fold to homogeneity by conventional methods and exhibited a specific activity of 230 U/mg'. A molecular weight of 150 000 has been determined for the enzyme, which is probably composed of four identical subunits of a molecular weight of 38000. These molecular properties are quite simi- liar to a secondary alcohol dehydrogenase isolated from Thermoanaerobicurn brockii [1] but different as described for another secondary alcohol dehydrogenase isolated from Pichia species grown on methanol by Patel et al. [ 15]. For this enzyme the

307

specific activity of the homogeneous protein is reported as 6 U/mg and the molecular weight was estimated to be 98 000 with two identical subunits of 48 000. The relative activity for several substrates, cofactor requirements, and the kinetic parameters so far published seem also to be different for these three enzymes. Studies of the oxidation of the (R) - ( - ) and (S)-(+) enantiomers of butan-2-ol by the isopropanol dehydrogenase from C. boidinii showed, that the (R) - ( - ) form is a better substrate and exhibits faster reaction rates as the corresponding (S)-(+) form. The stereo- selectivity of the reactions appears to be kinetically controlled. Further studies to clarify this point are in progress. Hou et al. [14] reported also that the secondary alcohol dehydrogenase from methanol grown yeasts preferentially reacts with (R)-(- ) - butan-2-ol, while an alcohol dehydrogenase from bakers' yeast [28] favours the oxidation of the other enantiomer and the liver alcohol dehydrogenase oxidizes both enantiomers equally well. The reverse reaction provides an interesting approach to prepare optically active solvents from ketones in conjunction with a NADH regenerating system [2]. Since Patel et al. [12] reported the secondary alcohol dehydrogenase of several facultative methyl- otrophic yeasts grown on C 1 compounds, we checked different carbon sources for C. boidinii which is also a facultative methanol utilizer. How- ever, the content of isopropanol dehydrogenase in the cell was much lower when the strain was grown on methanol (Table III). Isopropanol dehydrogenase was isolated by us also from methanol grown cells of C. boidinii and exhibited identical properties as described above for the enzyme obtained from glucose grown cells and purified as a single peak. We conclude therefore that the isopropanol dehydrogenase in C. boidinii is not part of a specific C I metabolism. The role of the enzyme in the metabolism of C. boidinii needs further clarifi- cation. Since neither isopropanol nor acetone sup- port growth of the yeast a simple assimilatory pathway can be excluded.

Acknowledgements

The expert technical assistance of Mrs. P. Schliephake-Ruess and Mr. R. Kraume-Fltigel is gratefully acknowledged. We thank Dr. J. Floss-

dorf and Mr. H. Schillig for the ultracentrifugation studies of the purified enzyme.

References

1 Lamed, R.J. and Zeikus, J.G. (1981) Biochem. J. 195, 183-190

2 Lamed, R.J., Keinan, E. and Zeikus, J.G. (1981) Enzyme Microbiol. Technoi. 3, 144-148

3 Tassin, J.P. and Vandecasteele, J.P. (1972) Biochim. Bio- phys. Acta 276, 31-42

4 Niehaus, W.G. jr., Frielle, T. and Kingsley, E.A. Jr. (1968) J. Bacteriol. 134, 177-183

5 Hatanaka, A., Adachi, O., Chiyonobu, T. and Ameyama, M. (1971)Agric. Biol. Chem. 35, 1142-1143

6 Hatanaka, A., Adachi, O., Chiyonobu, T. and Ameyama, M. (1971) Agric. Biol. Chem. 35, 1304-1306

7 Fugii, T. and Tonomura, K. (1972) Agric. Biol. Chem. 36, 2297-23O6

8 Kato, N., Omori, Y., Tani, Y. and Ogata, K. (1976) Eur. J. Biochem. 64, 341-350

9 Tani, Y., Miya, T., Nishikawa, H. and Ogata, K. (1972) Agric. Biol. Chem. 36, 68-75

10 Sahm, H. and Wagner, F. (1973) Eur. J. Biochem. 36, 250-256

11 Tani, Y., Miya, T. and Ogata, K. (1972) Agric. Biol. Chem. 36, 76-83

12 Branden, C., Jornvall, H., Eklund, H. and Furugren, B. (1975) in The Enzymes (Boyer, P.D. ed.), II, 103-190, Academic Press, New York

13 Hou, C., Patel, R., Laskin, A., Barnabe, N. and Marczak, J. (1979) FEBS letters 101, 179-183

14 Hou, C., Patel, R., Laskin, A., Barnabe, N. and Marczak, J. ( 1981) Appl. Environ. Microbiol. 41,829-832

15 Patel, R., Hou, C., Laskin, A., Derelanko, P. and Felix, A. (1979) Appl. Environ. Microbiol. 38, 219-223

16 Patel, R., Hou, C., Laskin, A., Derelanko, P. and Felix, A. (1979) Eur. J. Biochem. 101,401-406

17 Lowry, D.H., Rosebrough, N.A., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275

18 Bradford, M. (1976) Anal. Biochem. 72, 248 19 Jovin, T., Chrambach, A. and Naughton, M.A. (1964) Anal.

Biochem. 9, 351-369 20 Chrambach, A., Reisfeld, R.A., Wyckhoff, M. and Zaccari,

J. (1967) Anal. Biochem. 20, 150-154 21 Shapiro, A.L., Vinuela, E. and Maizel, J.V. (1967) Biochem.

Biophys. Res. Commun. 28, 815-820 22 Doerr, P. and Chrambach, A. (1971) Anal. Biochem. 42,

96-107 23 Andrews, P. (1965) Biochem. J. 96, 595-606 24 Flossdorf, J. (1980) Makromolekulare Chemie 181,715-724 25 Marffy, F. and Kula, M.-R. (1974) Biotech. Bioeng. 16,

623 -634 26 Yphantis, D.A. (1964) Biochemistry 3, 297-317 27 K~igi, J.H.R. and Vallee, B.L. (1960) J. Biol. Chem. 235,

3188-3192 28 Dickinson, F.M. and Dalziel, K. (1967) Nature (London)

214, 31-33

purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol...

Documents