Biotechnology Letters 21: 187–192, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

187

Purification and characterisation of PQQ-dependent glucose

dehydrogenase from Erwinia sp. 34-1

Liucija Marcinkevičienė, Irina Bachmatova, Rasa Semėnaitė, Rolandas Rudomanskis,

Gediminas Bražėnas, Rita Meškiene & Rolandas Meškys∗
Sector of Biosynthesis, Laboratory of Bioanalysis, Institute of Biochemistry, Mokslininku 12, Vilnius 2600,
Lithuania
∗ Author for correspondence (E-mail: meskys@bchi.lt)

Received 4 December 1998; Accepted 18 January 1999

Key words: PQQ, Erwinia, glucose dehydrogenase

Abstract
A pyrroloquinoline quinone-dependent glucose dehydrogenase from an isolate of Erwinia sp. has been purified to
homogeneity and characterised. SDS-PAGE showed a single band of 88.4 kDa. The enzyme activity was optimal
at 47 ◦ C and pH 7.5–8.5. The Michaelis constants for D-glucose and PMS were 3.2 mM and 132 µM, respectively
(50 mM glycine–NaOH, at pH 8.0).

Introduction stability are very desirable to design the more ben-

In Gram-negative bacteria, several membrane or
periplasmic dehydrogenases used for the primary
oxidation step of non-phosphorylated carbohydrates
are pyrroloquinoline quinone (PQQ)-dependent en-
zymes (Bouvet et al. 1989). The most studied glu-
cose dehydrogenases (GDH, EC 1.1.99.17) have been
isolated from Acinetobacter calcoaceticus (Geiger
& Gorisch 1986, Dokter et al. 1986, Olsthoorn
& Duine 1996, Olsthoorn et al. 1998, Dewanti
& Duine 1998), Gluconobacter oxydans (Ameyama
et al. 1981), Pseudomonas fluorescens (Matsushita
& Ameyama 1982) and Escherichia coli (Ameyama
et al. 1986). Some members of the Enterobacteri-
aceae, such as E. coli, Salmonella typhimurium or Ser-
ratia marcescens are seemingly unable to synthesize
PQQ, but are able to synthesize the glucose dehydro-
genase apo-enzyme (Neijssel 1987). PQQ containing
GDHs are very promising for biosensors development
since these enzymes do not need any additional cofac-
tor and are usually insensitive to oxygen (Mullen et al.
1985, Turner et al. 1987, Yokoyama et al. 1989, Ye
et al. 1993, Gorton 1995, Kurtinaitiene et al. 1995,
Smolander et al. 1992, 1995, 1995a, Schmidt 1997).
New GDHs exhibiting different substrate specificity or
eficial sensors for glucose determination. Although
some PQQ biosynthesis-encoding genes have been
isolated from Erwinia herbicola (Liu et al. 1992) the
PQQ-dependent glucose dehydrogenase from Erwinia
has neither been purified to homogeneity nor charac-
terised. The present work deals with the purification
and characterisation of a new PQQ-dependent glucose
dehydrogenase from Erwinia sp. 34-1.
Materials and methods
Materials
Yeast extract was obtained from Oxoid. DEAE Toyo-
pearl 650 M was from the Toyo-Soda. Carboxymethyl-
sepharose CL-6B, high viscosity carboxymethyl cel-
lulose, D-mannitol, phenazine methosulphate (PMS)
and 2,6-dichlorophenol indophenol (DCPIP) were
from Sigma. PQQ was purchased from Fluka.
Media and cultivation
GYC agar (g/l): glucose – 100, yeast extract – 2,
CaCO3 – 30, agar – 20. Erwinia sp. 34-1 was cul-
tivated aerobically at 30 ◦ C in 750 ml shaking flasks
188
containing 200 ml of A1 medium of following com-
position (g/l): yeast extract – 5, D-mannitol – 10,
(NH4 )2 HPO4 – 1.0, MgSO4 × 7H2 O – 2.0, pH 5.5.
Isolation of GDH producing microorganisms
Samples of soils and spoiled fruits were enriched aer-
obically at 30 ◦ C in the A1 medium. Once grown
in enrichment cultures, the aliquots were diluted and
spread on GYC agar plates. Positive colonies form-
ing a clear halo due to massive production of acids
from glucose were selected and purified by streaking
repeatedly on the same medium several times.
Assay of glucose dehydrogenase
Activities were determined at 30 ◦ C by measuring the
rate of discoloration of DCPIP at 600 nm in a mix-
ture containing 50 mM potassium phosphate buffer,
pH 7.3, and 20 mM substrate. Throughout the ex-
periments a molar absorption coefficient for DCPIP
was calculated from absorbance measurements at each
tested pH. One activity unit (U) corresponds with
the amount of enzyme converting 1 µmol of glucose
or DCPIP per minute under the specified assay con-
ditions. Protein concentration was estimated by the
Lowry method.
Purification of glucose dehydrogenase
The Erwinia sp. 34-1 was grown in the A1 synthetic
medium aerobically at 30 ◦ C for 20 h, harvested by
centrifugation (4, 500 × g, 30 min), washed with 0.9%
NaCl and suspended in Tris-HCl buffer, pH 8.0. Cells
were disrupted by ultrasonic treatment, the insoluble
fraction was collected by centrifugation (7, 000 × g,
30 min) and suspended in 50 mm potassium phosphate
buffer, pH 7.0, containing 1.3% Triton X-100 and
0.5 m NaCl. The suspension was stirred for 2 h at room
temperature and an insoluble material was removed
by centrifugation (7, 000 × g, 30 min). PEG 6000
was added to the clear GDH containing supernatant, to
15.5%, and the mixture was stirred at 4 ◦ C overnight.
Pellets were collected by centrifugation (7, 000 × g,
30 min) and GDH was extracted with 50 mM potas-
sium phosphate buffer, pH 7.0, containing 50 mM
NaCl and 1 mM MgSO4 . To the clear supernatant ob-
tained after centrifugation as above Triton X-100 was
added to a final concentration 0.5%. Then CaCl2 was
added (final concentration 0.5%) and clear solution
was obtained after removing all insoluble material by
centrifugation (4, 500 × g, 30 min). Cold ethanol was
added to the supernatant up to 50% (v/v), and the pre-
cipitate formed after incubation for 1 h was collected,
suspended in 75 mM potassium phosphate buffer,
pH 7.0, containing 0.1% Triton X-100 and 1 mM
MgSO4 . Glycerol was added to a final concentration
5% after discharging of all insoluble substances. The
enzyme solution was subjected to a DEAE-Toyopearl
650 column (1.5 × 12 cm) equilibrated with 75 mM
potassium phosphate buffer, pH 7.0, containing 0.1%
Triton X-100, 1 mM MgSO4 and 5% glycerol. The
enzyme was eluted with the same buffer. Fractions
with GDH activity were combined and concentrated
in a dialysis sack using a high viscosity CM-Cellulose
as an absorber. The concentrated enzyme was dialysed
against 5 mM potassium phosphate buffer, pH 7.0,
containing 0.05% of Triton X-100. The obtained so-
lution of active GDH was applied on hydroxyapatite
column equilibrated with 5 mM potassium phosphate
buffer, pH 7.0, containing 0.05% of Triton X-100 and
5% of glycerol. GDH was eluted by a linear gradient
of NaCl (0–0.5 M) in the same buffer. Active fractions
were pooled and concentrated in a dialysis sack us-
ing a high viscosity CM-Cellulose as an absorber. The
obtained solution of the enzyme was dialysed against
5 mM potassium phosphate buffer, pH 7.0, containing
0.05% of Triton X-100 and 5% of glycerol overnight
and stored at −20 ◦ C.
Electrophoresis
Polyacrylamide gel electrophoresis was performed ac-
cording to the method of Laemmli (1970). Subunit
composition studies were conducted by using 5%
stacking and 12.5% resolving gels in the presence
of SDS (0.1%). The standards were phosphorylase B
(97,400), bovine serum albumin (66,200), ovalbumin
(45,000), glyceraldehyde-3-phosphate dehydrogenase
from rabbit muscle (36,000), and soybean trypsin
(20,100).
Substrate specificity
Activity of purified enzyme towards sugars (20 mM)
was determined spectrophotometrically as described
above.
Kinetic parameters
Initial reaction rates were determined with various
concentrations of glucose and PMS at 30 ◦ C in the
glycine–NaOH buffer, at pH 8.0. The measured val-
ues and concentrations were plotted according to the