Solubilization, purification, and properties of membrane-bound D-glucono-δ-lactone hydrolase from Gluconobacter oxydans

Article abstract of DOI:10.1271/bbb.80554

Membrane-bound glucono-δ-lactonase (MGL) was purified to homogeneity from the membrane fraction of Gluconobacter oxydans IFO 3244. After solubilization with 1 M CaCl₂, MGL was purified in the presence of Ca²⁺ and detergent. A single

Full text of DOI:10.1271/bbb.80554

Biosci. Biotechnol. Biochem., 73 (1), 241–244, 2009
Communication
Solubilization, Purification, and Properties of Membrane-Bound
D-Glucono-ꢀ-lactone Hydrolase from Gluconobacter oxydans
y
Emiko SHINAGAWA,1; Yoshitaka ANO,2 Toshiharu YAKUSHI,2
2
Osao ADACHI,2 and Kazunobu MATSUSHITA
1Department of Chemical and Biological Engineering, Ube National College of Technology,
Tokiwadai, Ube 755-8555, Japan
2Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
Received August 8, 2008; Accepted November 15, 2008; Online Publication, January 7, 2009
[doi:10.1271/bbb.80554]
Membrane-bound glucono-ꢀ-lactonase (MGL) was
purified to homogeneity from the membrane fraction
of Gluconobacter oxydans IFO 3244. After solubilization
with 1 M CaCl2, MGL was purified in the presence of
Ca2þ and detergent. A single band corresponding to
60 kDa appeared in SDS–PAGE. The molecular weight
of MGL was judged to be 120k. Differently from
cytoplasmic lactonases, MGL showed optimum pH in
an acidic range of 5–5.5. It was highly sensitive to metal-
chelating agents such as EDTA, and the lost MGL
activity was restored to the original level by the addition
of divalent cations such as Ca2þ or Mg2þ. The purified
MGL was strictly dependent on Ca2þ and underwent
rapid denaturing precipitation on Ca2þ depletion even
in the presence of detergent. This communication can be
the first one dealing with the solubilization, purification
and properties of MGL.
enzymatic spontaneous reaction or by enzymatic reac-
tion employing D-glucono-ꢀ-lactonase. In oxidative
fermentation, besides D-aldonate production, 2-keto-D-
gluconate, 5-keto-D-gluconate, and 2,5-diketo-D-gluco-
nate are formed from D-glucose. Other aldonates, such
as D-galactonate, D-mannonate, and D-xylonate, are also
oxidation products of the respective aldoses catalyzed by
QGDH or other dehydrogenases that are membrane-
bound. Half a century ago, King and Cheldelin predicted
the existence of D-glucono-ꢀ-lactonase in the membrane
fraction of acetic acid bacteria.16) In 1962, the accumu-
lation of lactones in culture media of acetic acid bacteria
and other aerobic organisms including eukaryotes was
indicated by Takahashi and Mitsumoto.17) In 1988,
Buchert and Viikari confirmed the existence of mem-
brane-bound D-glucono-ꢀ-lactonase in the membrane
fraction of G. oxydans ATCC 621 in their study of
D-xylose metabolism,18) but no useful information
on MGL, including solubilization, purification, and
characterization, has been accumulated in the last five
decades.
Key words: acetic acid bacteria; gluconate production;
Gluconobacter; membrane-bound glucono-
ꢀ-lactonase; oxidative fermentation
The use of detergent, a common strategy employed in
membrane-bound enzyme purification, resulted in suc-
cessful solubilization of MGL. However, at the same
time, abundant major species of membrane-bound
dehydrogenases in Gluconobacter strains, such as
QGDH, quinoprotein alcohol dehydrogenase, and qui-
noprotein glycerol dehydrogenase, were also solubi-
lized. Further purification of MGL from such a chaotic
mixture was unsuccessful, and this was discouraging
after many repeats of trial and error. Moreover, storage
of solubilized enzyme solution in a deep freezer caused
irreversible denaturation of MGL, while no appreciable
loss was observed in the enzyme activities of co-
solubilized major species of membrane-bound dehydro-
genases. Finally, MGL was solubilized stably from the
membrane fraction of G. oxydans IFO 3244 in the
presence of CaCl2 at a concentration higher than 1 M.
The hydrophobic nature of solubilized MGL was its
striking feature, and the enzyme was denatured quickly
unless the hydrophobic region of the enzyme was
protected by the addition of a suitable detergent, such
as dodecyl-ꢂ-maltoside (Dojindo, Kumamoto, Japan) or
Mydol 10 (Kao, Tokyo). Enzyme activity was measured
by assaying decreased D-glucono-ꢀ-lactone by the
method of Hestrin.19)
Many different cytoplasmic soluble lactonases (SGLs)
hydrolyzing lactones such as D-glucono-ꢀ-lactone, 6-
phospho-D-glucono-ꢀ-lactone, xylonolactone, L-rhamno-
no-ꢁ-lactone, aldonic lactones, and aromatic lactones,
are known as important enzymes in the cytoplasmic me-
tabolism of eukaryotes and prokaryotes.1–13) When
D-glucose or D-glucose-6-phosphate is oxidized in
cytoplasm by NAD(P)-dependent D-glucose dehydro-
genase or NADP-dependent D-glucose-6-phosphate
dehydrogenase, the corresponding lactone is formed
and subsequently hydrolyzed to D-gluconic acid or
6-phospho-D-gluconic acid by the action of SGL.
Acetic acid bacteria and other aerobic bacteria
participating in oxidative fermentation are equipped
with a highly developed periplasmic oxidase system
yielding bioenergy formation through rapid substrate
oxidation on the outer surface of the cytoplasmic mem-
brane. Quinoprotein D-glucose dehydrogenase (QGDH),
a representative membrane-bound dehydrogenase in
oxidative fermentation, oxidizes D-glucose rapidly,
and subsequently D-gluconate is accumulated in the
culture medium.14,15) In this process, D-glucono-ꢀ-
lactone is the direct oxidation product of D-glucose,
which in turn is converted to D-gluconate either by non-
y
To whom correspondence should be addressed. Tel: +81-836-35-6839; Fax: +81-836-21-7117; E-mail: emiko@ube-k.ac.jp
Abbreviations: MGL, membrane-bound D-glucono-ꢀ-lactonase; QGDH, quinoprotein D-glucose dehydrogenase; SGL, soluble cytoplasmic D-
glucono-ꢀ-lactonase

242
E. SHINAGAWA et al.
Table 1. Summary of Purification of MGL from G. oxydans
IFO 3244
A
B
C
Specific
activity
(units/mg)
kDa
Protein Activity
(mg) (units)
Purification Recovery
Step
30 min
(fold)
(%)
100
103
103
Membrane
fraction
3,000
500
240
248
0.08
0.45
1.0
77
60 min
90 min
Solubilized
fraction
PEG ppt
5.6
MGL
(60 kDa)
50
98
28
130
70
1.32
2.50
16.7
31.2
54
30
0.1
Sephadex G-75
34
28
For instance, only a small portion of the membrane
fraction (3.0 g protein), from which soluble cytoplasmic
proteins and some peripheral proteins had been removed
by washing the membrane with 1 M KCl, was suspended
in 5 mM acetate buffer, pH 6.0, containing 1 M CaCl2
and stirred overnight in a cold room. Major species
of membrane-bound dehydrogenase remained in the
residual membrane precipitated after centrifugation at
68k ꢀ g for 60 min. Almost 100% recovery of MGL was
found in the supernatant. To the supernatant, Mydol 10
was added to 0.3% (v/v) to protect the hydrophobic
region of MGL. Polyethyleneglycol 6,000 (PEG) was
added to 40% (w/v), and the solution was stirred for
60 min in a cold room. The precipitate was collected by
centrifugation at 68k ꢀ g for 60 min. The enzyme
solution was concentrated by PEG precipitation and
the PEG precipitate was dissolved in a minimal volume
of 5 mM acetate buffer, pH 6.0, containing 1 M CaCl2
and 0.3% Mydol 10. The enzyme solution was applied
to a Sephadex G-75 column (1:5 ꢀ 130 cm) prepared
with the same buffer. MGL was eluted as a sharp peak,
and the specific activity of the peak fraction increased to
2.5 units/mg, resulting in 30-fold purification of the
membrane fraction with an overall yield of 30%, as
summarized in Table 1. The final preparation was
composed of 70 units of the total MGL activity and
28 mg of total protein, when protein concentration
measurement was done by a modification of the Lowry
method.20) Only a few steps were sufficient for purifi-
cation of a typical membrane-bound enzyme, if the
respective enzyme was solubilized with high recovery.
The abundance of MGL in the membrane frac-
tion reflected a typical profile of a membrane-bound
enzyme to cytoplasmic enzymes in acetic acid
bacteria.14,21–23) Regarding SGL in the same G. oxydans
IFO 3244, the enzyme activity was too weak to purify,
resulting in poor yield after many steps of purification,
as seen in some cases of SGL purification.10,13) Thus,
purification of SGL from G. oxydans remains to be done
in future.
120 min
500 600
300 400
20
Wavelength (nm)
Fig. 1. Physicochemical Properties of Purified MGL.
A, Sedimentation pattern in analytical ultracentrifugation. MGL,
10.5 mg/ml in 5 mM acetate buffer, pH 6.0, containing 1 M CaCl2
and 0.3% Mydol 10, was analyzed at 20 ꢁC. Photographs were taken
at 30 min intervals, as indicated after reaching 60 k rpm. B,
Absorption spectrum. MGL (0.4 mg/ml) in the same buffer as in
A was used. C, SDS–PAGE of MGL. MGL (5 mg) was analyzed by
SDS–PAGE. The pre-stained standard marker proteins (Bio-Rad,
Hercules, CA) were used in the following molecular sizes: 103 kDa,
phosphorylase B; 77 kDa, bovine serum albumin; 50 kDa, ovalbu-
min; 34 kDa, carbonic anhydrase; 28 kDa, soybean trypsin inhibitor;
20 kDa, lysozyme.
estimate the purity of a purified membrane-bound
enzyme. The absorption spectrum showed a low
absorption ranging from 320 to 500 nm in the visible
region (Fig. 1B). The nature of the absorption band
should be examined in the future, as to whether it comes
from Ca2þ. PAGE in the presence of SDS (SDS–
PAGE)25) showed an apparent molecular mass of 60 kDa
(Fig. 1C). The molecular size of the MGL purified in
this study was inconsistent with the genomic informa-
tion deduced, that gene gnlA carrying D-glucono-ꢀ-
lactonase in the periplasm was composed of 342 amino
acid residues.26) Gel filtration with Sephadex columns27)
confirmed that MGL might have the molecular weight of
120k, implying that MGL might be composed of two
identical subunits.
MGL showed a clear contrast to SGL when the
physicochemical and catalytic properties were compared
(Table 2). It showed optimum pH at 5.0–5.5, while most
SGLs of eukaryotes to prokaryotes show optimum pH at
7.0–7.5. The catalytic properties of MGL were exam-
ined mainly with D-glucono-ꢀ-lactone, a sole substrate
commercially available. To determine the substrate
specificity, we prepared and purified D-galactono-ꢀ-
lactone, D-mannono-ꢀ-lactone, D-xylono-ꢁ-lactone, and
D-arabono-ꢁ-lactone after aldose oxidation with a
membrane fraction involving QGDH, although the final
concentrations of the individual substrates prepared
were unclear. Only D-glucono-ꢀ-lactone and D-xylono-
ꢁ-lactone were hydrolyzed at almost the same relative
reaction rate. D-Galactono-ꢀ-lactone, D-mannono-ꢀ-
lactone, and D-arabono-ꢁ-lactone appeared not to be
hydrolyzed by the purified MGL. It is apparent that
QGDH oxidized D-xylose to D-xylono-ꢁ-lactone in vitro,
and thus there is no MGL specific to D-xylono-ꢁ-lactone.
D-Xylose could not be used as the sole carbon source for
the growth of acetic acid bacteria. The Km value for
D-glucono-ꢀ-lactone was estimated to be 25 mM.
The high hydrophobicity of MGL prevented it from
entering the separation gel in native polyarcylamide gel
electrophoresis (native PAGE),24) and one stained band
appeared at the interface between the stacking gel and
the separation gel (data not shown). There is no critical
method to determine the purity of a highly hydrophobic
protein that is not tolerated in native PAGE. The purified
MGL showed a single symmetrical sedimentation peak
on analytical ultracentrifugation (Fig. 1A). Due to the
presence of PEG in the enzyme solution, the viscosity of
the solution should have affected the sedimentation
coefficient, and thus no measurement of an apparent
sedimentation coefficient was done. However, analytical
ultracentrifugation is still the most powerful way to

Membrane-Bound D-Glucono-ꢀ-lactonase from Gluconobacter oxydans
243
Table 2. Comparison of Properties of MGL from G. oxydans IFO 3244 with SGLs of Various Sources
MGL
SGL
Mol. Wt.
120k
mammalian: 223k4) 233k5)
bacterial: 90k7) 62k10) 33k11) 79k12)
fungal: 125k9) 68k13)
Subunit (kDa)
Opt. pH
60 ꢀ 2
5.0–5.5
mammalian: 37.24) 39.45) 37.2–39:4 ꢀ 64,5)
bacterial: 33 ꢀ 2–44 ꢀ 27) 26:5 ꢀ 210) 3811) 4112)
fungal: 609)
mammalian: 7.03) 7.54) 7.15)
bacterial: 7.07) 7–810) 8–8.511) 6.5–7.512)
fungal: 7.0–8.09)
aldonolactones and aromatic lactones9)
L-pantoyl lactone and aromatic lactones but not for aldonolactones10)
synthetic lactones but not for aldonolactones11)
L-ꢃ-hydroxyglutaric acid-ꢁ-lactone12)
(R)-5-oxo-2-tetrahydrofran carboxylic acid and ꢁ-butyrolactone13)
1,440 D-galactonolactone9)
Substrate
D-glucono-ꢀ-lactone (100%)
D-xylono-ꢁ-lactone (90%)
specificity
Vmax (mmol/mg/min)
2.5 D-glucono-ꢀ-lactone
25 D-glucono-ꢀ-lactone
2,800 dihydrocumarin9)
13.7 L-pantolactone10)
230 (R)-5-oxo-2-tetrahydrofran carboxylic acid11)
95 (S)-5-oxo-2-tetahydrofran carboxylic acid11)
39.3 (S)-5-oxo-2-tetahydrofran carboxylic acid12)
6.2–18 D-glucono-ꢀ-lactone4,5)
Km (mM)
23.1 D-glucono-ꢁ-lactone5)
0.4–0.6 D-glucono-ꢀ-lactone7)
3.6 D-galactonolactone9)
6.3 dihydrocumarin9)
3.59 L-pantoyl lactone10)
1.9 (R)-5-oxo-2-tetrahydrofran carboxylic acid11)
39.8 (S)-5-oxo-2-tetahydrofran carboxylic acid11)
53 (S)-5-oxo-2-tetahydrofran carboxylic acid12)
inhibited7,9,10) not inhibited11,12)
EDTA/EGTA
Restoration of lost
enzyme activity with
inhibitedꢂ
Ca = Mg>Zn>Fe = Co,
Mn, Ni, Cu>Moꢂ
Ca, Mn, Zn7)
Ca9)
*Examined with membrane fraction.
D-Xylose
D-Xylono-γ-lactone
D-Xylonate
D-Glucose D-Glucono-δ-lactone
D-Gluconate
2-KGA
5-KGA 2,5-DKGA
Periplasm
GLDH
QGDH
MGL
GADH
2KGDH
e-
e-
e-
e-
Cytoplasmic
membrane
UQ
UQH2
D-Glucose
2H+
2H+ + O
2-KGR
SGL
2-KGA
5-KGR
NAD(P)-
GDH
D-Glucono
-δ-lactone
D-Gluconate
2H+
5-KGA
Cyt bo3
2H+
Pentose phosphate pathway
H2O
Cytoplasm
Energy
generation
Fig. 2. Schematic Model of MGL and SGL in Acetic Acid Bacteria.
GLDH, quinoprotein glycerol dehydrogenase; GADH, D-gluconate dehydrogenase; 2KGDH, 2-keto-D-gluconate dehydrogenase; 5-KGA,
5-keto-D-gluconate; 2-KGA, 2-keto-D-gluconate; 2,5-DKGA, 2,5-diketo-D-gluconate; 2KGR, 2-keto-D-gluconate reductase; 5KGR, 5-keto-D-
gluconate reductase; NAD(P)-GDH, NAD- or NADP-dependent D-glucose dehydrogenase.
The enzyme activity of MGL was inhibitory with
metal chelating agents, such as EDTA or EGTA,
similarly to many other SGLs. The lost enzyme activity
of the metal chelator-inactivated MGL in membrane
fraction was restored to the original level with 10 mM of
Ca2þ and Mg2þ. Other species of metallic ions, such as
Zn2þ, Fe2þ, Co2þ, Mn2þ, and Cu2þ, also showed
positive effects. The purified MGL was strictly depend-
ent on Ca2þ and it allowed rapid denaturing precipita-
tion on Ca2þ depletion even in the presence of the
detergent. Hence, all the experiments on EDTA inacti-
vation and reactivation with metallic ions were done
with the membrane fraction. A high Ca2þ concentration,
of more than 0.2 M, appeared to be essential to keep the
purified MGL stable. MGL solubilized with MgCl2
became more labile while MGL solubilized with CaCl2
was stable to the end. Taking all the data presented here
and other fundamental information on oxidative fer-
mentation14) together, a schematic model of MGL and
SGL in acetic acid bacteria might help the reader
(Fig. 2).

244
E. SHINAGAWA et al.
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The authors are indebted to Mr. S. Shiraishi for his
skillful performance of SDS–PAGE analysis. Critical
reading the manuscript was kindly done by Dr. G.
Mustafa.
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