Quinoproteins are oxidoreductases that posses one of
the four different o-quinone cofactor family, including pyrroloquinoline
quinine (PQQ), tryptophan tryptophylquinone (TTQ), trihydroxyphenylalanyl
quinone (topaquinone or TPQ), lysine tyrosylquinone (LTQ) and cysteine
tryptophylquinone (CTQ) instead of nicotinamide or flavine cofactors (Salisbury
et al., 1979; Duine, 1991; Ameyama et al., 1981; Adachi
et al., 2003; Anthony, 1992; Cai et al., 1997).
In many prokaryotic organisms, various simple sugar and
alcohol dehydrogenases have noncovalent PQQ cofactor (Davidson, 1993;
Duine et al., 1990; Goodwin and Anthony, 1998). The enzyme quinoproteins,
have certain properties which make them superior to other dehydrogenases
in vinegar fermentation, 2-keto-L- gulconic acid and 5-ketogluconic acid
production (both of which can be easily converted to vitamin C) (Mutsushita
et al., 2002; Saeki et al., 1997) and analytical applications,
especially in biosensor applications (D`Costa et al., 1986).
There are two major types of PQQ-containing alcohol dehydrogenases
(ADHs) (EC 1.1.99.) with and without heme group. Methanol dehydrogenase
(MDH) in methylotrophs and type I alcohol dehydrogenase (ADH I) in Pseudomonas
species have only PQQ as cofactor (Adachi et al., 1998). In
the case of quinohemoprotein ADH, some is present as a free-form of a
single protein called type II ADH (ADH II), while the other, called type
III ADH(ADH III) is as a complex with a cytochrome c subunit. Type II
ADH has been found in Pseudomonas and related species, while type III
ADH only in acetic acid bacteria including the genera Actobacter and
Gluconobacter (Adachi et al., 1987a, b; Mutsushita et
al., 2002). ADH III is a quinohemoprotein-cytochrom c complex bound
to the periplasmic side of the cytoplasmic membrane and function as the
primary dehydrogenase in ethanol oxidase respiratory chain, where ADH
oxidizes ethanol by transferring electrons to ubiquinon embedded in the
membrane phospholipids (Matsushita et al., 1992; Ameyama et
Coupled with ethanol oxidation, ADH reduces phenazine
methosulfate, dichlorophenolindophenol, or ferricyanide as an artificial
electron acceptor in vitro (Ameyama and Adachi, 1982). Since ferricyanide
reacts with the heme components having a high redox potential, the heme
c sites in ADH complex should reduce ferricyanide (Matsushita et al.,
In this study, a successful example of a complete purification
of a quihemoprotein membrane-bound ethanol dehydrogenase has been described
for Iranian Gluconobacter suboxydans. In addition, some properties
of the purified membrane-bound ethanol dehydrogenase (EtDH) have been
MATERIALS AND METHODS
Chemicals: All chemicals used in this study were
DEAE-Sephadex, Hydroxyapatite and Potassium ferricyanide
were purchased from Sigma Chemical Company. Sodium gluconate and Potato
extract were kind from microbiology lab of Alzahra University, Total Protein
Assay Kit from Chem Enzyme Company.
Microorganism: As the enzyme source of purification
of the PQQ alcohol dehydrogenase, the bacterium, Gluconobacter suboxydans
was purchased from Persian Type Culture Collection at IROST Iran (PTCC).
Medium and cultivation: Basal medium employed
in this study contained 20 g of sodium gluconate, 5 g D-glucose, 3 g of
glycerol, 3 g yeast extract, 2 g polypeptone, 200 mL of potato extract
in liter of tap water (pH 7). The type culture of acetic acid bacteria
grown on the yeast extract slant was inoculated to 100 mL of the medium
in 500 mL shaking flask and the cultivation was carried out at 37°C
for 24 h with reciprocal shaking.
Enzyme assay: EtDH was assayed using potassium
ferricyanide as an electron acceptor. The rate of reduction of ferricyanide
to ferrocyanide gives a quantitative amount of ethanol oxidation. The
reaction mixture contained 0.1 mL potassium ferricyanide 0.1 M, 0.5 mL
McIlvaine buffer 0.1 M, pH 5.5, 0.1 mL Triton X-100 10%, 0.1 mL ethyl
alcohol 1 M and enzyme solution in total volume of 1 mL. The reaction
started by the addition of ethanol solution at 25°C and stopped by
adding 0.5 mL of the ferric-dopanol reagent after 5 min. Then, 3.5 mL
of water was further added to the last mixture and well mixed. The resulting
stabilized Prussian blue color formed was measured by spectrophotometer
at 660 nm after standing for 20 min at 25°C. One unit of enzyme activity
was defined as amount of enzyme catalyzing the oxidation of 1 μmol
of ethanol per min under these assay conditions and 4.0 absorbance unit
equaled to 1 μmol of ethanol oxidized (Adachi et al., 1987a,
Protein assay: The protein concentration was estimated
by measuring by Total Protein Chem Enzyme Assay Kit.
Protein concentration in sample (g dL-1) =
(Sample observation/standard observation)xstandard concentration
SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
For estimation of purity of enzyme preparations, slab gel electrophoresis
was performed under essentially the same conditions as described by Laemmli
(1970) using 12.5% of polyacrylamide gel and Tris HCL buffer, pH 8.3,
sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis was performed
to determine purity and subunit composition of the enzyme (Laemmli, 1970).
Preparation of cell homogenate: Cells were harvested
by centrifugation at 12,000 x g for 20 min and washed with saline 0.9%.
The cell paste was suspended in 0.01 M potassium phosphate buffer, pH
6.0, (1 g of wet cell/10 mL buffer) and this suspension was sonicated
with sonicator at 100 W for 5 steps (5 min) with intervolves (2 min).
Intact cells was removed by centrifugation at 5000 x g for 5 min. The
resulting supernatant was disintegrated as cell homogenate.
Solubilization of enzyme: The membrane fraction
is suspended in 0.01 M buffer, pH 6.0 and the protein concentration is
adjusted to 30 mg mL-1 Triton X-100 and 2-mercaptoethanol are
added to final concentrations of 1.0% and 1 mM, respectively. The suspension
is gently stirred for 3 h at 0°C and centrifuged at 68,000 x g for
60 min. Supernatant is obtained as the solubilized enzyme.
DEAE-sephadex column chromatography (I): To the
solubilized enzyme solution, polyethylene glycol 6000 is added to 20%
to precipitate the enzyme. After 30 min of stirring in an ice bath, the
enzyme solution is centrifuged at 12,000 x g for 20 min. The precipitate
is suspended in small volume of 0.01 M buffer and the thick suspension
is dialyzed overnight against 0.002 M buffer containing 0.1% Triton X-100.
The dialyzed solution is applied to a DEAE-Sephadex A50 column (5x30)
that has been equilibrated with 0.002 M buffer, pH 6.0, containing 0.1%
Triton X-100. The column is washed with 500 mL of the same buffer to remove
nonadsorbable materials. The enzyme is eluted from the column with 0.1
M buffer, pH 6.0, containing 1% Triton X-100. Pooled enzyme fraction in
dialyzing tubing is concentrated by dehydration by embedding the enzyme
in dry polyethylene glycol 6000. The concentrated faction is then dialyzed
thoroughly against 0.002 M buffer, pH 6.0, containing 0.1% Triton X-100.
The insoluble material is removed by centrifugation at 12,000 g for 20
min. Chromatography on DEAE-Sephadex A-50(II) was repeated (Fig.
DEAE-Sephadex A50 (I) ADH solution from proceeding step was adsorbed
onto column of DEAE-Sephadex A50 (I) (5x30) that has been equilibrated
with 0.002M buffer, pH 6.0, containing 0.1% Triton X-100. The enzyme
is eluted from the column with 0.1 M buffer, pH 6.0, containing
1% Triton X-100. (___) was enzyme activity and ( - - - ) was protein
DEAE-Sephadex A50 (II) ADH solution from DEAE-Sephadex A50 (I) was
adsorbed onto column of DEAE-Sephadex A50 (II) (1.5x20), which has
been equilibrated with the 0.015M buffer, pH 6.0, containing 0.05%
Triton X-100 and elution of the enzyme was performed by a linear
gradient elution made between 0.015M and 0.1M of phosphate buffer.
(___) was enzyme activity and ( - - -) was protein content
graph of oxidation of ethanol by EtDH. Enzyme activity was measured
at various concentration of ethanol as indicated
A summary of the
purification steps of the enzymes
eae-sephadex column chromatography (II): The dialyzed
enzyme is applied to the second column DEAE-Sephadex A50 (1.5x20), which
has been equilibrated with the 0.015 M buffer, pH 6.0, containing 0.05%
Triton X-100 and elution of the enzyme was performed by a linear gradient
elution made between 0.015 and 0.1 M of phosphate buffer. Each buffer
reservoir contained 500 mL and 0.05% Triton X-100, pH 6.0, was present
throughout this step. This second step of DEAE-Sephadex A-50 chromatography
was found convenient to bring about in removing an impurity which was
fractionated in the next step. Pooled enzyme fraction was dialyzed against
0.01 M buffer containing 0.05% Triton X-100 overnight (Fig.
Hydroxyapatite fractionation: The dialyzed enzyme
from preceding step was applied to a fractionation with Hydroxyapatite,
which had been equilibrated with 0.01 M buffer, pH 6.0, containing 0.05%
Triton X-100. We can pour the solution on the Hydroxyapatite, after gently
mixing, the mixture was stayed to 5 h for adsorption the enzyme to the
beads of Hydroxyapatite. Elution of the enzyme was made stepwise with
0.02, 0.05 and 0.1 M buffer, pH 6.0, containing 0.1% Triton X-100. Pooled
enzyme solution was dialyzed 0.002 M buffer thoroughly (2 days). In Table
1, the steps of purification is summarized.
Kinetic analysis of enzyme activity: A steady-state
kinetic analysis of the ADH reaction was performed in 100 mM KPB (pH 6.0).
To determine the apparent Km value for ethyl alcohol,
its concentration was varied from 10 to 100 μM. An apparent Michaelis
constant for ethanol were examined to be 1.7x10-3 M (Fig.
Substrate specificity: The substrate specificity
of the enzyme was determined using the same enzyme assay method as described
above, except that various substrate solutions (100 mM) include methanol,
ethanol, isopropanol, n-butanol, formaldehyde, benzaldehyde, glycerol,
D-glucose, D-fructose, lactate. The data have been shown in Table
Effects of metal ions and EDTA: The effects of
metal ions and inhibitors on the activity of the enzyme were examined
by measuring the activity using the same assay method as described above.
Each compound solution was
of the purified enzyme from Gluconobacter suboxydans. The
reaction rate with ethanol is expressed as 100
Effect of EDTA and
metals on the activity of the purified enzyme. The reaction rate
without any additive is expressed as 100
Optimum pH of EtDH.
Enzyme activity was assayed under standard conditions except that
pH of the buffer (MacIlvain buffer) was varied as indicated above.
The enzyme shows maximum activity at pH 5.5
Stability pH graph
of EtDH. Enzyme solution was diluted with various pH of MacIlvaine
buffer from 2.5 to 8 as indicated and stored for 24 h at 4°C.
Thereafter, an aliquot of stored enzyme solution was picked up for
the standard assay of enzyme activity performed at pH 5.5. EtDH
was in maximum stability in pH 5-8
of ADH. 20 microliter of the last step purified enzyme solution
was loaded on the top of the gel (right). Standard marker solution
(left) was contained bovine serum albumin, ovalbumine, trypsine,
lysoyzme and ribonuclease A from top to bottom, respectively
tirred into the reaction mixture and the reaction was
started with the addition of the enzyme. Each compound was added to the
reaction mixture at a concentration of 1.0 mM, except that the concentration
of EDTA was 5.0 mM. The data have been shown in Table 3.
Optimal pH and pH stability: The correlation between
the reaction rate of the ADH and pH values of the reaction mixture was
determined by the same assay method as described above, except that various
pHs and buffers were used.
The enzyme optimal pH graph was showed in Fig.
4 and enzyme pH stability graph was showed in Fig. 5.
Electrophoretic analysis: Dissociation into subunits
was observed by SDS gel electrophoresis in the determination of molecular
weight of the enzyme. In the present of SDS the enzyme was dissociated
into three subunits with a molecular weight 44, 14.3 and 12.5 kD from
the top to bottom of the gel column as shown in Fig. 6.
The sum of molecular weight of each band gave 70.8 kD of total molecular
Alcohol Dehydrogenase (ADH) of acetic acid bacteria,
consisting of the genera Actobacter and Gluconobacter catalyzes
the first step of acetic acid production, oxidation of ethanol to acetaldehyde
(Duine and Frank, 1981; Olsthoorn and Duine, 1996; Salisbury et al.,
This study was an attempt to purify and characterize
membrane bound EtDH from Iranian Gluconobacter suboxydans. In PQQ
ADH, PQQ bound noncovalent but tightly to the enzyme, whereas in NAD-dependent
ADH, NAD serves as a noncovalent cofactor that loosely bound to the enzyme.
So, PQQ ADH is more suitable and cheaper than NAD-dependent ADH for industrial
and biotechnological applications.
Purified enzyme has been shown to possess substrate specificity
for primary aliphatic alcohol. Primary aliphatic alcohol was rapidly oxidized
but not methanol. Ethanol is best substrate for ADH. With concern the
tertiary structure of methanol dehydrogenase (MDH) and (EtDH), the volume
of active site cavity, where substrate bind and react on the top of MDH,
EtDH, yielding 18 and 62 A°, respectively. These numbers are well
correlated with the substrate specificity. MDH have rather narrow substrate
specificity, while EtDH react well with a relatively larger alcohol as
well as ethanol. In addition above, the substrate would probably enter
through the hydrophobic mouth of a channel leading to the active site
cavity and located between PQQ and heme-domains in the case of EtDH. In
the case of EtDH, one amino acid residue that helps to form a hydrophobic
wall for the active site cavity is located in the heme-domain. Thus, we
can say methanol can not properly enter the hydrophobic mouth of a channel
leading to the active site cavity of EtDH (Hirohida et al., 2004).
So, the enzyme is suitable for acetic acid industrial
production. Secondary and tertiary alcohols and cyclic alcohol could not
ADH activity has been shown to increase in the presence
of 1 mM Ca+2 and decrease in presence the same concentration
of ethylenediamine tetra acetic acid (EDTA). Experiments clearly indicate
that calcium is required for catalysis in PQQ containing enzymes. In agreement
with a catalytic role, calcium does not seem to be involved in the binding
of the substrate. Instead, may polarize the PQQ C5-O5 bond together with
the active site Arg residue, resulting in a partial negative charge on
the O5 atom and a partial positive charge on the C5 atom (Anthony, 1996)
Because the proper structure and function of the enzyme is related to
structural Ca+2 and EDTA can chelate Ca+2. The enzyme
has a pH optimum at 5-6.5 and enzyme activity was decreased at pH<5
and pH>6.5. That`s reasons may be substrate or enzyme or both not suitable
ionic form or enzyme inactivation or all of them. The enzyme stability
pH was at pH 5-8.5. Therefore, pH 5-6.5 may be used for the enzyme activity