Probing reactivity of PQQ-dependent carbohydrate dehydrogenases using artificial electron acceptor.
ABSTRACT The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k (ox) = 660 M(-1) s(-1), while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.
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ABSTRACT: A new class of oxidoreductase containing an amino acid-derived o-quinone cofactor, of which the most typical is pyrroloquinoline quinone (PQQ), is called quinoproteins, and has been recognized as the third redox enzyme following pyridine nucleotide- and flavin-dependent dehydrogenases. Some quinoproteins include a heme c moiety in addition to the quinone cofactor in the molecule and are called quinohemoproteins. PQQ-containing quinoproteins and quinohemoproteins have a common structural basis, in which PQQ is deeply embedded in the center of the unique superbarrel structure. Increased evidence for the structure and function of quinoproteins has revealed their unique position within the redox enzymes with respect to catalytic and electron transfer properties, and also to physiological and energetic function. The peculiarities of the quinoproteins, together with their unique substrate specificity, have encouraged their biotechnological application in the fields of biosensing and bioconversion of useful compounds, and also to environmental treatment.Applied Microbiology and Biotechnology 02/2002; 58(1):13-22. · 3.69 Impact Factor
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ABSTRACT: A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The beta-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.Journal of Biological Chemistry 11/2006; 281(41):30650-9. · 4.65 Impact Factor
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ABSTRACT: Quinoprotein glucose dehydrogenase (EC 184.108.40.206) from Acinetobacter calcoaceticus L.M.D. 79.41 was purified to homogeneity. It is a basic protein with an isoelectric point of 9.5 and an Mr of 94,000. Denaturation yields two molecules of PQQ/molecule and a protein with an Mr of 48000, indicating that the enzyme consists of two subunits, which are probably identical because even numbers of aromatic amino acids were found. The oxidized enzyme form has an absorption maximum at 350 nm, and the reduced form, obtained after the addition of glucose, at 338 nm. Since double-reciprocal plots of initial reaction rates with various concentrations of glucose or electron acceptor show parallel lines, and substrate inhibition is observed for glucose as well as for electron acceptor at high concentrations, a ping-pong kinetic behaviour with the two reactants exists. From the plots, Km values for glucose and Wurster's Blue of 22 mM and 0.78 mM respectively, and a Vmax. of 7.730 mumol of glucose oxidized/min per mg of protein were derived. The enzyme shows a broad substrate specificity for aldose sugars. Cationic electron acceptors are active in the assay, anionic acceptors are not. A pH optimum of 9.0 was found with Wurster's Blue and 6.0 with 2,6-dichlorophenol-indophenol. Two types of quinoprotein glucose dehydrogenases seem to exist: type I enzymes are acidic proteins from which PQQ can be removed by dialysis against EDTA-containing buffers (examples are found in Escherichia coli, Klebsiella aerogenes and Pseudomonas sp.); type II enzymes are basic proteins from which PQQ is not removed by dialysis against EDTA-containing buffers (examples are found in A. calcoaceticus and Gluconobacter oxydans).Biochemical Journal 11/1986; 239(1):163-7. · 4.65 Impact Factor
Probing Reactivity of PQQ-Dependent Carbohydrate
Dehydrogenases Using Artificial Electron Acceptor
Lidija Tetianec & Irina Bratkovskaja & Juozas Kulys &
Vida Casaite & Rolandas Meskys
Received: 12 February 2010 /Accepted: 19 July 2010 /
Published online: 9 October 2010
# Springer Science+Business Media, LLC 2010
Abstract The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter
calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH)
and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were deter-
mined using various electron acceptors. The radical cations of organic compounds and 2,6-
dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose.
The reactivity of dioxygen with ASDH is low; the bimolecular constant kox=660 M−1s−1,
while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-
10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of
dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is
higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the
carbohydrate monomer number increase. The specificity of quinoproteins was compared
with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential
enzymes application for lactose oxidation.
Quinoprotein oxidoreductases contain as a cofactor o-quinone derivative produced from an
amino acid. The most typical o-quinone cofactor is pyrroloquinoline quinone (PQQ).
Methanol dehydrogenase and glucose dehydrogenase are classic examples of quinoproteins
containing PQQ as a cofactor . Quinoproteins can be distinguished by their localization:
soluble or membrane bound.
Appl Biochem Biotechnol (2011) 163:404–414
L. Tetianec (*):I. Bratkovskaja:J. Kulys
Department of Enzyme Chemistry, Institute of Biochemistry, Mokslininku 12, 08662 Vilnius, Lithuania
V. Casaite:R. Meskys
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Mokslininku 12,
08662 Vilnius, Lithuania
Glucose dehydrogenase has been found both as soluble and membrane bound [1, 2].
Soluble PQQ-dependent glucose dehydrogenase (GDH), originating from Acinetobacter
calcoaceticus, is a homodimeric enzyme containing one PQQ and three Ca2+ions per
monomer . The enzyme has broad substrate specificity. GDH catalyzes the oxidation of
monosaccharides such as glucose, xylose, and galactose, as well as disaccharides such as
lactose and melibiose. The products of carbohydrates oxidation are corresponding lactones
[2, 4]. GDH acts according to the ping-pong mechanism in which substrate inhibition by
glucose and negative cooperativity effect were demonstrated . The structure of the
enzyme has been resolved, and the catalytic mechanism of the reductive half-cycle has been
elucidated [5, 6].
Very recently, a soluble aldose sugar dehydrogenase (ASDH) was purified from
Escherichia coli, and three-dimensional structure was determined . The cofactor (PQQ)
is bound in a large cleft of protein surface and is uniquely accessible to the water in
comparison to other PQQ enzymes. The ASDH has a low (18%) identity with the A.
calcoaceticus-soluble glucose dehydrogenase, but many amino acid residues involved in
binding of the PQQ cofactor are conserved in both enzymes. ASDH has a low affinity for
glucose and broad substrate specificity for aldose carbohydrates. The catalytic and
structural properties of ASDH propose that this enzyme belongs to a new subgroup of
PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus-soluble
dehydrogenase subgroup .
Practical application of carbohydrate dehydrogenases covers bioanalysis and carbohy-
drates modifications . During biocatalytical conversion, carbohydrates are oxidized by
oxidized enzyme following the reduced enzyme oxidation by electron acceptors. The high
reduced enzyme oxidation rate is an important step for effective enzyme turnover. PQQ-
dependent carbohydrate oxidases (COs) typically react slowly with dioxygen, and different
artificial electron acceptors are used [4, 9–11].
The aim of the present work was dual: (a) to determine the reactivity of PQQ-dependent
carbohydrate dehydrogenases with various electron acceptors and (b) to determine
specificity of mono-, di-, and oligosaccharide oxidation using artificial electron acceptors.
As PQQ dehydrogenases, glucose dehydrogenase from A. calcoaceticus (GDH) and aldose
sugar dehydrogenase from E. coli (ASDH) were used.
Materials and Methods
Soluble glucose dehydrogenase was purified from A. calcoaceticus LMD 79.41 as described
. The final product was the enzyme solution with specific activity ~1,800 U/mg of protein.
The recombinant soluble aldose sugar dehydrogenase encoded by yliI gene was purified from
E. coli. The pET M-11yliI construct  was transformed into E. coli BL21 (DE3) cells. Single
colonies were used to inoculate 5 ml of brain heart infusion (BHI) medium containing
kanamycin at 37 °C for 16 h. This culture was used to inoculate 500 ml of BHI medium
containing kanamycin. Cultures were grown until late log phase (A600 nmapproximately 0.8)
and then cooled to 25 °C prior to the induction of protein expression with 0.5 mM
isopropyl-β-D-thiogalactopyranoside. The cells were maintained at 25 °C for 5 h and
then harvested by centrifugation (3,000×g, 20 min, 4 °C). Purification of ASDH and
reconstitution with PQQ was carried out as described . The final product was ASDH
solution with specific activity 1.8 U/mg of protein. Molar concentration of enzymes was
determined by Lowry method  using molecular mass of GDH 100 kDa  and of
ASDH 39 kDa . The recombinant Coprinus cinereus peroxidase (rCiP) and Aspergillus
Appl Biochem Biotechnol (2011) 163:404–414405
niger catalase were received from Novo Nordisk A/S (Denmark) and used without further
purification. Concentration of rCiP and catalase was determined spectrophotometrically at
the wavelength of 405 nm using the molar extinction coefficient of 1.08×105M−1cm−1
Glucose, maltose, lactose, cellobiose, maltotriose, and maltotetraose were from “Sigma”;
cellotriose and cellotetraose were from “Merck”. PQQ, galactose, kanamycin, and
isopropyl-β-D-thiogalactopyranoside were from “Fluka”. Brain heart infusion medium
was purchased from “Oxoid”. Solutions of the carbohydrates were made in triple distilled
water and allowed to mutarotate 1 day before use. All the concentrations of carbohydrates
and calculated kinetic parameters are expressed in the terms of total amount of α and β
anomers. The solution of hydrogen peroxide was prepared from 30% hydrogen peroxide
(Carl Roth GmbH + Co), and the concentration was determined spectrophotometrically by
using extinction coefficient of 39.4 M−1cm−1at 240 nm . Buffer reagents and other
chemicals were of analytical grade.
3-(10H-Phenoxazin-10-yl)propionic acid (PPA), 3-(10H-phenoxazin-10-yl)-propane-1-
sulfonic acid (PPSA), and 10-methyl-10H-phenoxazine (MPX) were synthesized as
described in . 1-(N,N-Dimethylamine)-4-(4-morpholine)benzene (AMB) was synthe-
sized as described in . 2,6-Dichlorophenolindophenol sodium salt dihydrate (DCPIP)
was from Sigma. Radical cation of 5,10-dimethyl-5,10-dihydro-phenazine (DMDHP+•) was
synthesized by oxidizing DMDHP with hydrogen peroxide in ethanol containing hydro-
chlorous acid. The radical cations of the phenoxazine derivatives and AMB were prepared
by adding 20 μl rCiP (1 μM) and 4×25 μl of hydrogen peroxide (20 mM) to 2 ml
(0.5 mM) of the compound in the buffer solution. The reaction was terminated after 5 min
by adding 20 μl of catalase. Final concentration of catalase was 100 nM. The solutions of
the radical cations of PPA, PPSA, MPX, and AMB were prepared freshly before
measurements and were kept in ice during the measurements.
The reduction of radical cations and DCPIP was monitored spectrophotometrically. The
kinetic curves were recorded at the wavelength of maximum of absorbance of radical
cation. The concentrations of the electron acceptors were calculated using extinction
coefficients of 16 mM−1cm−1at 530 nm for PPA+•, PPSA+•, and MPX+•[15, 17, 18],
7.4 mM−1cm−1at 462 nm for DMDHP+•, 9.8 mM−1cm−1at 604 nm for AMB+•,
and 19.6 mM−1cm−1at 600 nm for DCPIP.
The kinetics of the dioxygen consumption was measured by using a homemade
computer-assisted membrane oxygen electrode and 1.1 ml cell thermostated at 25 °C. The
experiments were conducted in 5 mM Tris-HCl buffer solution, pH 8.0, containing 1 mM
CaCl2and 0.2 M glucose. The concentration of dioxygen in air-saturated buffer solution at
25 °C was assumed to be 0.25 mM .
The initial reaction rate of radical cations reduction was calculated as a slope of absorbance
change during 10–30 s. The exponential curve of DCPIP absorbance decrease was
approximated by first-order reaction kinetics, and initial reaction rate was calculated as
C0×k, where C0is initial concentration of the substrate and k is first-order reaction constant.
The dependence of dioxygen reduction rate on dioxygen concentration was obtained by
digital differentiation of the kinetic curves of dioxygen consumption at fixed dioxygen
concentration. In calculations, dioxygen and DCPIP were assumed as two electron
acceptors. The radical cations are single electron acceptors, and for this reason, the rate
of their reduction was divided by 2 to calculate the rate of substrate oxidation.
406Appl Biochem Biotechnol (2011) 163:404–414
The analysis of the dependence of the initial rate on acceptor (A) or substrate (S)
concentrations was performed by applying the ping-pong scheme [3, 4, 19]:
! Eredþ PEoxþ S $ Eox? S
Eredþ Aox! Eoxþ Ared
where Eox, Ered, Eox– S, S, P, Aox, and Areddenote the oxidized and reduced forms of
enzyme, the enzyme–substrate complex, the substrate, the product, and the oxidized and
reduced forms of electron acceptor, respectively. Following this scheme, the dependence of
the initial reaction rate on substrates’ and enzyme concentrations was expressed:
where kcat=k2, kred¼ k1? k2= k?1þ k2
oxidative constants, respectively, and [A]0and [S]0are initial acceptor and carbohydrate
concentrations, respectively. [E]tis a total enzyme concentration.
At large excess of substrate (carbohydrate), the dependence of the reaction initial rate on
substrate, enzyme, and electron acceptor concentrations could be simplified:
½ ?t=V0¼ 1=kcat
ð Þ þ 1= kred ? S ½ ?0
Þ, and kox=k3are apparent catalytic, reductive, and
???þ 1= kox? A
V0¼ kcat ? kox ? E
½ ?t? A
½ ?0= kox ? A
½ ?0þ kcat
At a low electron acceptor concentration, when kcat> kox×[A]0, the initial rate is linearly
proportional to the electron acceptor concentration:
V0¼ kox? E
½ ?t? A
At a high (saturated) concentration of electron acceptor, when kox×[A]0>kred×[S]0, the
Eq. 3 could be simplified:
V0¼ kcat? kred? E
½ ?t? S ½ ?0= kred? S ½ ?0þ kcat
At a low substrate concentration, when kcat>kred×[S]0, the initial rate is linearly
proportional to the carbohydrate concentration:
V0¼ kred? E
½ ?t? S ½ ?0
The dependence of initial rate on substrate (carbohydrate) or electron acceptor (A)
concentration was fitted by Michaelis–Menten (Eqs. 4 and 6) or linear anamorphosis
(Eqs. 5 and 7), and calculated parameters were used to evaluate koxand kred:
kox¼ slope= E
kred¼ slope= E
kox¼ Vmax= KMðAÞ? E
kred¼ Vmax= KMðSÞ? E
Appl Biochem Biotechnol (2011) 163:404–414407
where KM(A)= kcat/koxand KM(S)= kcat/kredare apparent Michaelis–Menten constants for
electron acceptor and carbohydrate, respectively.
Results and Discussion
Kinetics of Dehydrogenases with Artificial Electron Acceptors
It was demonstrated that dioxygen reacts slowly with PQQ-dependent dehydrogenases [9–
11]. GDH at concentration of 5.5 μM and in presence of 0.1 M of glucose did not show
significant dioxygen consumption. The estimated oxidation constant was less that
5 M−1s−1. ASDH, however, catalyzes glucose oxidation by dioxygen at higher rate
(Fig. 1). The initial reaction rate is directly proportional to enzyme concentration (Fig. 1,
insert). The dependence of reaction rate on dioxygen concentration at various ASDH
concentrations was also linear (Fig. 2). The calculated koxvalue was equal to (6.6±0.6)×
102M−1s−1. This value is significantly less in comparison to flavin oxidases; it is 4.5×
105M−1s−1for glucose oxidase from A. niger  and 1.7×105M−1s−1for carbohydrate
oxidase Microdochium nivale . Low oxidation constant with dioxygen does not allow
practical use of dioxygen as oxidizer even for ASDH catalyzed process.
In trying to find effective electron acceptors, radical cations of N-substituted
phenoxazines, N,N-substituted phenazine, and N,N-substituted p-phenylendiamine were
used. These radical cations are stable in water solution . The electrochemical
conversion of the radical cations is fast, and for this reason, electrochemical method of
18001500 1200900600 3000
Initial rate (µM s-1)
Fig. 1 Dioxygen concentration change at various ASDH amounts in presence of 0.2 M glucose at 25 °C.
Inserted graph shows the dependence of the initial rate of dioxygen consumption on ASDH concentration.
The curve drawn though the points is a linear approximation
408 Appl Biochem Biotechnol (2011) 163:404–414
the acceptors regeneration can be applied . High reactivity of some radical cations with
PQQ-dependent dehydrogenases was demonstrated in previous preliminary investigation
The reduction of radical cations proceeded at GDH concentration as low as 1 nM
(Fig. 3). Oxidative constant calculated using Eq. 9 was (2.2±0.4)×107M−1s−1for radical
cation of PPA (Table 1). The constants for other acceptors are even larger. They approached
diffusion-limited values for enzyme catalysis . The reactivity does not correlate with
redox potential of acceptors since E′0is 0.634, 0.339, and 0.390 V for PPA, DMDHP, and
AMB, respectively [15, 16, 25]. This in addition indicates possible diffusion limitation of
radical cations reactivity.
The reactivity of radical cations was compared with DCPIP, which was used in original
investigations . The calculated constant of GDH with DCPIP is lower than that of radical
cations (Table 1).
Reduced ASDH reacts with radical cations at 20–250 nM concentration. The calculated
constants varied between 1.0×106and 2.3×107M−1s−1(Table 1). The constant values do
not correlate with redox potential of acceptor.
The kinetics of DCPIP reduction was performed at various glucose concentrations. The
analysis of initial rate dependence on DCPIP concentration at various glucose concen-
trations was performed applying the ping-pong scheme of enzyme action (Eqs. 1 and 2) and
the expression of the initial rate dependence on enzyme, substrate, and electron acceptor
concentration (Eq. 3). According to the Eq. 3, the dependence of reverse rate on reverse
DCPIP concentration is linear, and the change of glucose concentration generates parallel
lines. Experimentally obtained dependences of reverse rate on reverse DCPIP concentration
were near linear and parallel at different glucose concentrations that confirm the ping-pong
scheme of ASDH action. The reactivity of DCPIP was lower than the reactivity of radical
cations, as in the case of GDH.
Dioxygen consumption rate (µM s-1)
2.25 µM ASDH
4.5 µM ASDH
6.75 µM ASDH
Fig. 2 The dependence of reaction rate on dioxygen concentration at various ASDH concentrations. The
curves drawn though the points are linear approximations. Conditions are the same as in Fig. 1
Appl Biochem Biotechnol (2011) 163:404–414409
Initial rate (µM s-1)
Fig. 3 Kinetic curves of PPA+•concentration change at 0.4 nM GDH and 20 mM glucose. The insert graph
shows the dependence of initial reaction rate on PPA+•concentration; the curve drawn through the points is
an approximation of the data by linear equation
Table 1 Oxidation constants of GDH and ASDH reaction with radical cations and DCPIP at 25 °C
Electron acceptorEnzymeBuffer solutionpHCglucose, Mkox, M−1s−1
410Appl Biochem Biotechnol (2011) 163:404–414
Specificity of Dehydrogenases
To evaluate specificity of dehydrogenases, radical cation of PPAwas chosen. The oxidation
of carbohydrates was performed at constant electron acceptor concentration at pH 7.2. The
calculated kredwas used as a parameter related to specificity.
The kinetic curves of the radical cation concentration decrease were almost linear up to
larger conversion (Figs. 3 and 4). The linear dependence of initial rate on substrate
(carbohydrate) concentration indicates that limiting reaction is substrate oxidation (Fig. 3).
In this case, kredwas calculated using Eq. 11. The saturating character of the dependence of
the initial rate on substrate concentration indicated unsatisfactory concentration of acceptor
at high substrate concentration. However, the slope calculated as Vmax/(KM(S)) can be used
for kredevaluation (Eq. 10).
In the case of GDH, the calculated bimolecular constants of carbohydrates oxidation span a
range 4.6×104–6.9×105M−1s−1(Table 2). The kredshows that GDH shows quite broad
specificity and reacts effectively with monosaccharides and disaccharides, such as maltose or
lactose. The reactivity of substrates depends on the length of the carbohydrate chain. Mono-
and disaccharides are more reactive in comparison to oligosaccharides. The determined
specificity is typical for the soluble PQQ-dependent carbohydrate dehydrogenases  (Fig. 5).
The reactivity ASDH toward carbohydrates is about 4 orders of magnitude less in
comparison to GDH (Table 2). A tendency of reactivity increase was indicated for
oligosaccharides. The larger ASDH reactivity toward oligosaccharides can be related to the
structure of the open active center .
Initial rate (µM s-1)
Fig. 4 GDH catalyzed reduction of PPA+•in presence of maltotetraose. The concentrations of maltotetraose
are shown on the curves. The concentration of GDH was 1.0 nM. The insert graph shows the dependence of
the initial reaction rate on maltotetraose concentration
Appl Biochem Biotechnol (2011) 163:404–414411
The low ASDH reactivity can be associated with non-optimal pH. Southall et al. 
demonstrated that the activity of ASDH toward the substrates increases at pH 8.75. For this
reason, reductive constant of ASDH for three substrates—glucose, galactose, and lactose—
was measured at pH 8.0. The obtained values of kredat pH 8.0 are higher than at pH 7.2, but
the relative specificity among these three substrates is the same at pH 7.2 and 8.0.
Table 2 The reactivity of GDH, ASDH, and CO at 25 °C
aData from 
Initial rate (µM s-1)
Fig. 5 GDH catalyzed reduction of PPA+•in presence of maltotriose. The concentrations of maltotriose are
shown on the curves. The concentration of GDH was 0.2 nM. The insert graph shows the dependence of the
initial reaction rate on maltotriose concentration
412Appl Biochem Biotechnol (2011) 163:404–414
In this study, we have characterized the reactivity of two PQQ-dependent carbohydrate
dehydrogenases toward various substrates and electron acceptors. Both enzymes show the
wide substrate specificity acting on mono-, di-, and oligosaccharides. Moreover, various
electron mediator systems might be applied to regenerate these dehydrogenases. One of
possible PQQ carbohydrate dehydrogenase applications is lactobionic acid (LA) synthesis
from lactose. LA is an excellent biodegradable chelator and can be used for preparation of
detergents. The use of carbohydrate oxidase from M. nivale for lactose oxidation was
suggested very recently . The reactivity of GDH toward lactose is similar in comparison
to CO (Table 2). However, the specificity of both enzymes is different. GDH catalyzes the
oxidation of di- and oligosaccharides with α-glycoside and β-glycoside bonds at a similar
rate. In contrast, CO shows much higher reactivity toward carbohydrates containing β-
glycoside bonds. Given a suitable electron acceptor, GDH can be used for LA production
from lactose. The constant of reactivity of ASDH with lactose is very low, and the use of
this enzyme for lactobionic acid might need large amount of protein. However, the wide
substrate specificity of ASDH means possibilities to use the enzyme in combination with an
appropriate hydrolase for modification of polysaccharides, in addition to development of
Acknowledgments The research was supported by Lithuanian State Science and Studies Foundation, grant
No N-09/2007. The authors thank Dr. S.M. Southall for the pET M-11yliI construct and Dr. R. Vidziunaite
and M. Dagys for assistance in measurements.
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