Fungal glucuronoyl esterases and substrate uronic acid recognition

Article abstract of DOI:10.1271/bbb.90486

Glucuronoyl esterases are enzymes involved in microbial plant cell-wall degradation. In this study we purified and characterized two recombinant Phanerochaete chrysosporium glucuronoyl esterases, PcGE1 and PcGE2. The catalytic activity of these and previo

Full text of DOI:10.1271/bbb.90486

Biosci. Biotechnol. Biochem., 73 (11), 2483–2487, 2009
Fungal Glucuronoyl Esterases and Substrate Uronic Acid Recognition
y
ˇ
Miroslava DURANOVA, Jan HIRSCH, Katarına KOLENOVA, and Peter BIELY
´
´
´
´
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences,
´ ´
Dubravska cesta 9, 845 38 Bratislava, Slovakia
Received July 7, 2009; Accepted August 19, 2009; Online Publication, November 7, 2009
[doi:10.1271/bbb.90486]
Glucuronoyl esterases are enzymes involved in micro-
bial plant cell-wall degradation. In this study we
purified and characterized two recombinant Phanero-
chaete chrysosporium glucuronoyl esterases, PcGE1 and
PcGE2. The catalytic activity of these and previously
described glucuronoyl esterases was investigated on new
synthetic substrates, methyl esters of uronic acids and
their glycosides, prepared by esterification with ethereal
diazomethane.
terized to date was from the thermophilic fungus
Sporotrichum thermophile.9) The P. chrysosporium
genome also contains two GE genes (pcge1 and pcge2),
which have been heterologously expressed.10) In this
study, the corresponding gene products were purified
and characterized, and their substrate specificity was
investigated on new synthetic substrates.
Materials and Methods
The data obtained indicate that the enzymes hydro-
lyzed efficiently not only esters of 4-O-methyl-D-glucur-
onic acid, but also methyl esters of D-glucuronic acid
carrying a 4-nitrophenyl aglycon. Moreover, the fact
that they did not recognize the 4-epimers of these
compounds, the D-galacturonic acid derivatives, sup-
ports the hypothesis that these carbohydrate esterases
attack ester linkages between 4-O-methyl-D-glucuronic
acid of glucuronoxylan and lignin alcohols.
Purification of P. chrysosporium glucuronoyl esterases. Recombi-
nant strain P. cinnabarinus UU-P001.19 was used in PcGE1 produc-
tion and S. commune UU-S002.2 in PcGE2 production by procedures
described previously.10) The supernatant of a 14-d-old culture was
concentrated on 10 kDa cut-off membrane, and the concentrate,
containing PcGE1 or PcGE2, was loaded onto a weak anion exchange
DEAE Sepharose FF column (2:5 ꢀ 30 cm) equilibrated by 50 mM
sodium-phosphate buffer, pH 6.0 (buffer A). Elution of proteins was
achieved by 400 ml of linear gradient of 0–1 M NaCl in buffer A. The
flow rate was set at 4.0 ml/min, and fractions of 8 ml were collected.
Fractions containing GE activity were pooled, desalted, and concen-
trated. After the addition of ammonium sulfate to the resulting 1 M
concentration, the sample was loaded onto a Phenyl Sepharose FF
column (1:5 ꢀ 30 cm) equilibrated by 1 M ammonium sulfate in
buffer A. The flow rate was set at 3.0 ml/min, and fractions of
6.0 ml were collected. Bound proteins were eluted by 240 ml of a
decreasing gradient of ammonium sulfate, and then by 240 ml of
buffer A. The fractions containing GE were pooled and concentrated
by ultrafiltration. In the last purification step, two different ion
exchange columns were used. The sample containing PcGE1 was
diluted in 50 mM sodium acetate, pH 4.0 (buffer B), and loaded onto a
weak cation CM HiTRAP FF 5 ml column, and the sample containing
PcGE2 was diluted in 50 mM buffer A, and loaded onto a strong anion
MonoQ 5/50 GL 1-ml column. Elution of both columns was controlled
by an AKTAbasic system (GE Life Science, Piscataway, NJ). Proteins
adsorbed on the CM HiTRAP column were eluted by 50 ml of NaCl
gradient in buffer B (0–0.5 M NaCl), and proteins adsorbed on MonoQ
5/50 GL by 10 ml of NaCl gradient in buffer A (0–0.4 M NaCl) at a
flow rate 2.0 ml/min. Fractions of 1.0 ml were collected. Fractions with
GE activity were pooled, desalted and concentrated. The purity of the
proteins was determined by SDS–PAGE.
Key words: glucuronoyl esterase; Phanerochaete chrys-
osporium; lignin-carbohydrate complex;
glucuronoxylan; 4-O-methyl-D-glucuronic
acid
Glucuronoyl esterases (3.1.1.-., GEs) might be en-
zymes hydrolyzing ester bonds occurring in plant cell-
walls between the 4-O-methyl-D-glucuronic acids of
hemicelluloses and the hydroxyl alcohols of lignin.1–3)
This hypothesis was suggested by the fact that GEs show
activity on (aryl)alkyl esters of 4-O-methyl-D-glucuronic
acid,4) substrates that mimic the ester bonds mentioned
above. GE activity was detected for first time in the crude
cellulolytic system of the fungus Schizophyllum com-
mune, and its purification and characterization3) resulted
in the establishment of a new carbohydrate esterase
family (CE15, http://www.cazy.org/fam/CE15.html).5)
On the basis of the internal amino acid sequence of
S. commune glucuronoyl esterase, numerous homolo-
gous sequences were found in microbial genomes.5) The
sequences included the Hypocrea jecorina Cip2 protein
of unknown function over-expressed during growth on
cellulose,6) and the carboxy-terminal domain of the
Ruminococcus flavefaciens multidomain protein.7) Now
both proteins are considered to be GEs belonging to
CE15. The catalytic domain of Cip2 was crystallized by
Wood et al.,8) and it awaits solution of its three-
dimensional structure. The last GE purified and charac-
Glucuronoyl esterase activity assays. Methyl 4-O-methyl-D-gluco-
pyranuronate (Compound I, Fig. 1)11) was used in TLC monitoring3) of
GE activity during the purification procedures. Specific enzyme
activities, kinetic parameters, temperature, and pH optima were
determined by HPLC on chromogenic substrate 4-nitrophenyl 2-O-
(methyl-4-O-methyl-ꢀ-D-glucopyranosyluronate)-ꢁ-D-xylopyranoside
(Compound VI, Fig. 1),12) as described previously.3) One unit of
glucuronoyl esterase activity was defined as the amount of enzyme
de-esterifying 1 mmol of the substrate in 1 min at 30 ꢁC.
y
To whom correspondence should be addressed. Fax: +421-2-5941-0222; E-mail: chempbsa@savba.sk
Abbreviations: CBD, cellulose binding domain; GEs, glucuronoyl esterases; PcGE1, Phanerochaete chrysosporium glucuronoyl esterase 1;
PcGE2, Phanerochaete chrysosporium glucuronoyl esterase 2

ˇ
´
M. DURANOVA et al.
2484
Fig. 1. Synthetic Substrates for Determination of GE Activity by TLC (compound I), HPLC (compound VI) and by the Hestrin Procedure
(compounds I–V).
Cellulose-binding studies. PcGE1 (2 mg) was added to 100 ml of
5% w/v suspension of Avicel PH101 in 50 mM sodium phosphate
buffer (pH 6.0), and mixture was incubated for 4 h at 4 ꢁC. Control was
run in parallel in the absence of cellulose. Samples were centrifuged at
10;000 ꢀ g for 10 min. The activity of GE in the supernatant and the
control enzyme solution was determined by HPLC.
144.1, 126.7 (ꢀ2), 117.7 (ꢀ2) (6 ꢀ Ar), 101.5 (C1), 77.0, 76.7, 74.4,
72.8 (C2, C3, C4, C5), 53.0 (OCH3).
Methyl D-galactopyranuronate (IV) (a mixture of ꢀ and ꢁ anomers)
1H-NMR (D2O, 400 MHz) ꢂ: 5.20 (1H, d, H1ꢀ), 4.64 (1H, m, H5ꢀ),
4.49 (1H, d, H1ꢁ), 4.34 (1H, m, H5ꢁ), 4.21 (1H, m, H4ꢀ), 4.15 (1H,
dd, H4ꢁ), 3.82 (1H, dd, H3ꢀ), 3.70 (4H, m, OCH3, H2ꢀ), 3.60 (1H, dd,
H3ꢁ), 3.38 (1H, m, H2ꢁ). 13C-NMR (D2O, 100.6 MHz) ꢂ: 171.4
(COOCH3 ꢀ), 170.5 (COOCH3 ꢁ), 96.2 (C1ꢁ), 92.3 (C1ꢀ), 74.1, 72.1,
71.1, 70.4, 70.0, 69.6, 68.4, 67.7 (C2ꢀ, C2ꢁ, C3ꢀ, C3ꢁ, C4ꢀ, C4ꢁ,
C5ꢀ, C5ꢁ), 52.8 (OCH3).
4-Nitrophenyl methyl-ꢁ-D-galacturonide (V) 1H-NMR (CD3OD,
400 MHz) ꢂ: 8.21 (2H, d, Ar), 7.25 (2H, d, Ar), 5.08 (1H, d, H1), 4.56
(1H, s, H5), 4.22 (1H, s, H4), 3.85 (1H, t, H2), 3.77 (3H, s, OCH3),
3.69 (1H, dd, H3). 13C-NMR (CD3OD, 100.6 MHz) ꢂ: 170.2
(COOCH3), 163.9, 144.1, 126.6 (ꢀ2), 117.8 (ꢀ2) (6 ꢀ Ar), 101.8
(C1), 75.8 (C5), 74.1 (C3), 71.4 (C2), 71.2 (C4), 52.8 (OCH3).
The ester character of compounds II–IV was also confirmed by
FTIR. Absorption peaks characteristic of esters were found at
1,732 cmꢂ1, 1,730 cmꢂ1, 1,735 cmꢂ1, and 1,713 cmꢂ1 for II, III, IV,
and V, respectively.
New substrates of glucuronoyl esterases and their de-esterification.
Methyl esters of D-glucuronic acid (compound II, Fig. 1), 4-nitro-
phenyl ꢁ-D-glucuronide (compound III, Fig. 1), D-galacturonic acid
(compound IV, Fig. 1), and 4-nitrophenyl ꢁ-D-galacturonide (com-
pound V, Fig. 1) were prepared by esterification (Sigma-Aldrich, St.
Louis, MO) with ethereal diazomethane.
Preparation of methyl esters of D-uronic acids. Dried uronic acid or
its glycoside (1 g) was dissolved in 15 ml of absolute methanol in a
two-necked flask equipped with a dropping funnel, a stirrer, and a
CaCl2 drying tube. The solution was cooled to ꢂ10 ꢁC, and throughout
esterification the temperature of the solution was kept below 0 ꢁC.
Then the freshly prepared solution of ethereal diazomethane was added
dropwise. When the color of the reaction mixture remained slightly
yellow permanently, esterification was complete. Usually 3 g of
N-nitrosomethyl urea is required for the preparation of diazomethane
for complete esterification of 1 g of D-saccharide acid. The ether and
methanol were removed under reduced pressure to obtain the uronic
acid methyl ester. Ethereal diazomethane was prepared by the
Action of GEs on methyl esters of uronic acids and their glycosides.
The ability of the GEs to hydrolyze compounds II-V and also
compound I (Fig. 1) was followed by determination of the remaining
esters following Hestrin.14) The low sensitivity of this method does not
allow determination of kinetic constants, and hence only specific
activities were determined. The reaction mixture, containing 20 mM
substrate and 3 mg of enzyme in 50 mM sodium phosphate buffer
(pH 6.0), was incubated at 30 ꢁC for 15 min (compounds I–III) or 20 h
(compounds IV–V). The remaining esters were converted to hydroxa-
mic acid by a reaction with 2 volumes of solution containing 2 M
procedure described by Muller et al.13)
¨
Characterization of the new esters by NMR and FTIR. 1H and 13C-
NMR spectra were recorded in D2O or CD3OD at 25 ꢁC on a Varian
400MR spectrometer operating at 400 MHz and 100.6 MHz respec-
tively. COSY and HSQC pulse programs were also used. IR spectra
were measured with an FTIR spectrometer Nicolet 6700, and the
Diamond ATR technique (Smart Orbit accessory, Thermo Fisher
Scientific Inc., Waltham, MA) was applied for measuring in a solid
state.
.
NH2OH HCl and 3.5 M NaOH (1:1, v/v). One volume of HCl:H2O
solution (1:2, v/v) was added to the mixture after 60 s, and
.
subsequently, after the addition of 1 volume of FeCl3 6H2O in 0.1 M
HCl, the hydroxamic acid was complexed with ferric ions, resulting in
a measurable chromophore (540 nm). The performance of the GEs
from P. chrysosporium were compared with the action of GEs from
S. commune3) and H. jecorina.5)
Methyl D-glucopyranuronate (II) (a mixture of ꢀ and ꢁ anomers)
1H-NMR (D2O, 400 MHz) ꢂ: 5.16 (1H, d, H1ꢀ), 4.58 (1H, d, H1ꢁ),
4.27 (1H, m, H5ꢀ), 3.96 (1H, d, H5ꢁ), 3.70 (3H, s, OCH3), 3.71–3.36
(5H, m, H2ꢀ, H3ꢀ, H3ꢁ, H4ꢀ, H4ꢁ), 3.20 (1H, m, H2ꢁ). 13C-NMR
(D2O, 100.6 MHz) ꢂ: 174.5 (COOCH3 ꢀ), 173.5 (COOCH3 ꢁ), 98.7
(C1ꢁ), 94.9 (C1ꢀ), 77.7, 77.1, 76.2, 74.8, 74.0, 73.8, 73.5, 73.2 (C2ꢀ,
C2ꢁ, C3ꢀ, C3ꢁ, C4ꢀ, C4ꢁ, C5ꢀ, C5ꢁ), 55.6 (OCH3).
4-Nitrophenyl methyl-ꢁ-D-glucuronide (III) 1H-NMR (CD3OD,
400 MHz) ꢂ: 8.21 (2H, d, Ar), 7.21 (2H, d, Ar), 5.18 (1H, d, H1),
4.13 (1H, d, H5), 3.77 (3H, s, OCH3), 3.63 (1H, m, H2), 3.53 (2H, m,
H3, H4). 13C-NMR (CD3OD, 100.6 MHz) ꢂ: 170.8 (COOCH3), 163.5,
Results
Purification of P. chrysosporium glucuronoyl esterases
The P. chrysosporium GEs produced by recombinant
strains were purified by a three-step procedure that
included ion-exchange chromatography and hydropho-

Glucuronoyl Esterases and Substrate Uronic Acid Recognition
2485
Table 1. Summary of Recombinant P. chrysosporium GE1 (A) and GE2 (B) Purification
A
Volume
(ml)
Total protein
(mg)
Total activity
(U)
Specific activity
(U/mg)
Yield
(%)
Purification
(fold)
Purification step
Culture fluid
Ultrafiltration
681
40
166
369
261
219
70
2.23
5.95
100
70.7
59.3
19
1
2.7
43.84
31.60
2.54
0.84
DEAE Sepharose FF
Phenyl Sepharose FF
HiTrap CM FF
6
6.93
27.56
38.81
3.1
0.80
0.17
12.4
17.4
33
8.9
B
Volume
(ml)
Total protein
(mg)
Total activity
(U)
Specific activity
(U/mg)
Yield
(%)
Purification
(fold)
Purification step
Culture fluid
Ultrafiltration
825
150
15
112.8
38
3.3
402
311
89
3.56
8.18
100
77.3
22.1
14.7
8
1
2.3
7.6
12.7
15
DEAE Sepharose FF
Phenyl Sepharose FF
MonoQ 5/50 GL
26.96
45.38
53.33
0.75
0.21
1.3
0.6
59
32
bic interaction chromatography. A summary of PcGE1
purification is presented in Table 1A, and of PcGE2 in
Table 1B. Concentrated filtrate of P. cinnabarinus
growth medium containing PcGE1 was loaded onto a
DEAE Sepharose column at pH 6.0 for the purpose of
pre-purification. GE activity was found in the protein
fractions unadsorbed by the resin. During subsequent
chromatography on a Phenyl Sepharose column, PcGE1
together with other proteins remained trapped on the
column. Most of the proteins were eluted with a
decreasing gradient of ammonium sulfate, but PcGE1
was not eluted until we washed the column with plain
buffer. Several other proteins still present in the sample
were removed by chromatography on a weak cation
exchange HiTRAP CM FF column. PcGE1 adsorbed to
the column at pH 4.0, but the other proteins did not.
Protein PcGE1 was eluted by 0.1 M NaCl, and showed
homogeneity on SDS–PAGE (Fig. 2A, lane 2). The
same purification procedure was applied to PcGE2. In
contrast to PcGE1, this protein was adsorbed to a DEAE
Sepharose at pH 6.0 and was eluted by 0.05 M NaCl.
Subsequent chromatography on a Phenyl Sepharose
column disclosed that the protein had hydrophobicity
similar to that of PcGE1. In contrast to most of the
proteins eluted during the decreasing ammonium sulfate
gradient, PcGE2 was released from the column during
elution with a buffer free of ammonium sulfate. The
protein was further purified by chromatography on a
strong anion exchange Mono Q resin. PcGE2 did not
adsorb to the resin at pH 6.0. The isolated protein
showed homogeneity on SDS–PAGE (Fig. 2B, lane 2).
A
B
Fig. 2. SDS–PAGE of Recombinant P. chrysosporium GEs.
A, Lane 1, molecular weight markers (Biorad, Hercules, CA);
lane 2, purified PcGE1 (2 mg). B, Lane 1, molecular mass standards
(Fermentas, Ontario); lane 2, purified PcGE2 (3 mg).
and 4.7 vs. calculated 4.8 for PcGE2. The presence of
CBD in the protein PcGE1 was experimentally con-
firmed. Only 20% of the enzyme activity remained in the
supernatant after 4 h of incubation of the enzyme with a
5% suspension of cellulose (Avicel PH101, Sigma-
Aldrich, St. Louis, MO).
The kinetic parameters of the P. chrysosporium GEs
were determined on 4-nitrophenyl 2-O-(methyl-4-O-
methyl-ꢀ-D-glucopyranosyluronate)-ꢁ-D-xylopyranoside
(compound VI, Fig. 1) (Table 2). PcGE1 showed 2
times higher catalytic affinity (Km) than PcGE2, but the
maximum velocity (Vmax) of substrate hydrolysis by
PcGE2 was 6 times higher that PcGE1. The catalytic
efficiency parameter (kcat=Km) was 2.5 times higher for
PcGE2 than for PcGE1. The results obtained were
compared with previously reported kinetic parameters
for H. jecorina, S. commune, and S. thermophile GEs
for the same substrate. P. chrysosporium GEs showed
lower catalytic affinity for the substrate, but a consid-
erably higher maximum velocity of hydrolysis than the
other GEs (Table 2).
Properties of P. chrysosporium glucuronoyl esterases
The molecular masses of the P. chrysosporium GEs
were determined by SDS–PAGE (Fig. 2). The molecular
mass appeared to be 47 kDa (calculated at 47.2 kDa) for
PcGE1, and 42 kDa (calculated at 42.3 kDa) for PcGE2.
These masses were similar to the calculated values,
although we expected higher masses considering the
presence of putative glycosylation sites (one for PcGE1
and seven for PcGE2).10) pH and temperature optima
were very similar for both enzymes: pH optimum 5.0–
6.0 and temperature optimum 45–55 ꢁC. The isoelectric
point for PcGE1 was found to be 6.5 vs. calculated 5.5
New substrates for glucuronoyl esterases
The de-esterification rates of methyl esters of uronic
acids and their glycosides (Fig. 1) by GEs from strains

ˇ
M. DURANOVA et al.
´
2486
Fig. 3. Specific Activities (U/mg) of GEs on Compounds I to V (see Fig. 1) Determined at a Substrate Concentration of 20 mM.
The error bars represent standard deviations of three independent measurements.
Table 2. Kinetic Parameters of P. chrysosporium GEs Determined
on 4-Nitrophenyl 2-O-(methyl-4-O-methyl-ꢀ-D-glucopyranosyluro-
nate)-ꢁ-D-xylopyranoside and a Comparison with Other Characterized
GEs
as recombinant proteins by two different hosts, P.
cinnabarinus and S. commune.10) Selection of the
regulatory element located in the front of the reading
frames made possible their production and purification
from glucose media. The enzyme containing CBD
showed binding affinity to crystalline cellulose and also
a higher molecular mass than the enzyme without CBD.
In spite of the fact that the PcGE1 and PcGE2 sequences
contain putative glycosylation sites, their estimated
molecular masses correspond to the calculated ones.
It seems that the CBD influences the pI values of
the enzymes, which reflects their different affinities
towards the ion-exchangers used in enzyme purification.
Consequently, identical purification steps might not
be applied to the two GEs. The differences in physical
properties between two P. chrysosporium GEs are
similar to those observed between the CBD-free S.
commune GE3) and the CBD-containing GEs from
H. jecorina5) and S. thermophile.9)
Since there is no experimental evidence that GEs
hydrolyze the ester linkages between uronic acids in
hemicellulose and lignin alcohols,15–20) the physiological
role of these carbohydrate esterases in microbial plant
cell-wall degradation remains unclarified. In this study,
we tested and compared the catalytic properties of the
two P. chrysosporium GEs and previously isolated GEs
on new synthetic substrates prepared by esterification of
commercially available uronic acids and their deriva-
tives using ethereal diazomethane. These compounds
mimic the ester linkages in lignin-carbohydrate com-
plexes. Examination of the GE activity on the methyl
esters of different uronic acids and their derivatives
indicates the important role of the 4-O-methyl group in
substrate recognition. The methyl ester of 4-O-methyl-D-
glucuronic acid was hydrolyzed by all four GEs at least
65 times faster than the methyl ester of D-glucuronic
acid. Previous study has also shown that S. commune
GE hydrolyzes esters of 4-O-methyl-D-glucuronic acid
with much higher efficiency than those of D-glucuronic
acid. Here we found that the change of the methyl ester
of free glucuronic acid into its 4-nitrophenyl ꢁ-glycoside
considerably increased the rate of hydrolysis by GEs.
The introduction of a hydrophobic aglycon increased
substrate affinity for the enzyme catalytic site. In
Km
Vmax
kcat
(sꢂ1
kcat=Km
(mM/s)
Enzyme
Ref.
(mM)
mmol/min/mg
)
PcGE1
PcGE2
ScGE
Cip2
0.83
1.82
0.31
0.50
1.3
14.2
88.4
4.4
11.2
62.3
3.2
13.5
34.2
10.3
9.0
—
—
3)
5)
9)
5.5
—
4.5
0.8
StGE
0.6
S. commune, H. jecorina, and P. chrysosporium were
followed by determination of residual esters by the
Hestrin procedure14) (Fig. 3). All of the enzymes showed
specific activity between 9.7–21.0 U/mg on methyl 4-O-
methyl-D-glucuronic acid (compound I), whereas the
activity on methyl ester of D-glucuronic acid (compound
II) was negligible, 65–242 times lower. These surprising
results were obtained with the methyl ester of 4-
nitrophenyl ꢁ-D-glucuronide (compound III), on which
all the GEs showed specific activities between 5.8–
17.8 U/mg despite the lack of the methyl group at
position 4. These values are comparable to the specific
activities obtained for compound I possessing the 4-O-
methyl group. Finally, the substrates in which D-
glucuronic acid was replaced by D-galacturonic acid
were also tested (compounds IV and V). As can be seen
in Fig. 3, the specific activities of the GEs on the
galacto-derivates were insignificant.
Discussion
To date, GEs from S. commune,3) H. jecorina,5) and
S. thermophile9) have been characterized. Two genes
(pcge1 and pcge2) coding for this recently discovered
carbohydrate esterase have been identified in the
genome of the white-rot basidiomycete P. chrysospo-
rium.5) While the gene pcge2 codes for an enzyme that
has only catalytic domain, pcge1 codes for an enzyme
that also has a family 1 CBD. Because the purification
of both P. chrysosporium GEs from sugar-beet pulp
containing growth media of the fungus proved to be
very difficult, the enzymes were successfully produced

Glucuronoyl Esterases and Substrate Uronic Acid Recognition
2487
ˇ
4) Spanikova S, Polakova M, Joniak D, Hirsch J, and Biely P,
´
Arch. Microbiol., 188, 185–189 (2007).
´
´
´
addition, this result serves as evidence that GEs accept
esterified D-glucuronic acid as their substrate also in the
form of its ꢁ-anomer. The de-esterification of 4-nitro-
phenyl methyl-ꢁ-D-glucuronide (compound III) by all
four tested enzymes points to the possibility of elabo-
rating a simple enzyme coupled assay of GEs using
ꢁ-glucuronidase as the auxiliary enzyme.
The above remarks do not apply to the methyl ester of
free D-galacturonic acid and its 4-nitrophenyl ꢁ-glyco-
side. These two compounds did not serve practically as
substrates for any of the tested GEs. Based on these
observations, we conclude that the gluco-configuration
on C-4 is decisive for enzyme activity. These results
underline the uniqueness of GE substrate specificity in
confrontation with pectin methyl esterases and the need
for a further search for GE native substrates.
ˇ
5) Li X-L, Spanikova S, de Vries RP, and Biely P, FEBS Lett., 581,
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´ˇ
´ˇ
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¨ ¨
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NMR, and Dr. V. Sasinkova (Institute of Chemistry,
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Slovak Academy of Sciences) for FTIR measurements.
This work was supported by a grant from the Slovak
Research and Development Agency under contract
no. APVV-51-003805.
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