Activity of three β-1,4-galactanases on small chromogenic substrates

Article abstract of DOI:10.1016/j.carres.2011.05.017

β-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal β-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one ba

Full text of DOI:10.1016/j.carres.2011.05.017

Carbohydrate Research 346 (2011) 2028–2033
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Activity of three b-1,4-galactanases on small chromogenic substrates
Søs Torpenholt a, Jérôme Le Nours a, , Ulla Christensen a, Michael Jahn b, Stephen Withers b,
Peter Rahbek Østergaard c, Torben V. Borchert c, Jens-Christian Poulsen a, Leila Lo Leggio a,
⇑
a Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
b Centre for High-throughput Biology (CHiBi) and Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1
c Novozymes A/S, Smørmosevej 25, DK-2880 Bagsværd, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
b-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalac-
tan side chains of the complex pectin network in plant cell walls. Two fungal b-1,4-galactanases from
Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been
Received 1 February 2011
Received in revised form 28 April 2011
Accepted 15 May 2011
kinetically characterized using the chromogenic substrate analog 4-nitrophenyl b-1,4-D-thiogalactobio-
Available online 26 May 2011
side synthesized by the thioglycoligase approach. Values of kcat/Km for this substrate with A. aculeatus
b-1,4-galactanase at pH 4.4 and for M. giganteus b-1,4-galactanase at pH 5.5 are 333 Mꢀ1 sꢀ1 and
62 Mꢀ1 sꢀ1, respectively. By contrast the B. licheniformis b-1,4-galactanase did not hydrolyze 4-nitro-
Keywords:
b-1,4-Galactanase
Enzyme kinetics
Chromogenic substrates
Transglycosylation
Chemoenzymatic synthesis
Thioglycoligase
phenyl b-1,4-D-thiogalactobioside. The different kinetic behavior observed between the two fungal and
the bacterial b-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the struc-
ture of B. licheniformis b-1,4-galactanase. This loop contains substrate binding subsites ꢀ3 and ꢀ4, which
presumably cause B. licheniformis b-1,4-galactanase to bind 4-nitrophenyl -1,4-b-
D
-thiogalactobioside
-thiogalactobioside, the two fun-
gal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-b- -galactopyranoside, but
non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-b-
D
D
kinetic parameters could not be determined because of transglycosylation at substrate concentrations
above 4 mM.
Ó 2011 Elsevier Ltd. All rights reserved.
A plant cell is surrounded by an essential cell wall that has di-
verse functions in the plant’s life. At a physical level it protects
the plant against pathogens and herbivores and provides rigid sup-
port. At a cellular level it takes part in the cell to cell communica-
tion, intracellular signaling and transport processes. The cell wall
consists of a primary wall and in many cells also of a secondary
wall. The primary cell wall consists, among other components, of
the polysaccharide pectin, which is especially abundant in
non-woody plants.1 Pectins are composed of regions of homogalac-
turonan and regions of rhamnogalacturonans I and II. Rhamnoga-
sidic bond in the side chains of b-1,4-galactan and arabinogalactan
type I are hydrolyzed by endo-b-1,4-galactanase. Some, but not all,
endo-b-1,4-galactanases can additionally hydrolyze small galacto-
oligosaccharides (e.g., trisaccharides) formed during the initial
polysaccharide degradation,3 as well as the polysaccharide itself.
b-1,4-Galactanases are glycoside hydrolases that belong to fam-
ily GH 53 in clan GH A,4,5 the largest of the glycoside hydrolase
clans, according to the CAZY classification.6–8 All the families in
glycoside hydrolase clan GH A share a (b/a)
barrel fold4,5 and
8
the same retaining catalytic mechanism, a double displacement
mechanism involving two carboxylic acid residues, one functioning
as a catalytic nucleophile and one as a general acid/base.
Enzyme kinetic investigations have been carried out for endo-b-
1,4-galactanases from several species9–12 mostly on heterogenous
and ill-defined polysaccharide substrates. Here, the activities of
Aspergillus aculeatus (AAGAL; GenBank ID: AAA32692.1), Meripileus
giganteus (MGGAL; GenBank: AAQ77827.1) and Bacillus lichenifor-
mis (BLGAL; GenBank ID: AAO31370.1) endo-b-1,4-galactanases to-
wards the well defined thiodisaccharide derivative 4-nitrophenyl
lacturonan I is a polysaccharide consisting of
b-1,4-galactan and arabinogalactan type I attached to C4 of the
Rha residues in a backbone made up of repeating units of 1,2-
-rhamnopyranosyl-1,4-
-galacturonic acid.2 The b-1,4 O-glyco-
a-1,5-arabinan,
a-
L
a-D
Abbreviations: AAGAL, b-1,4-galactanase from Aspergillus aculeatus; BLGAL, b-
1,4-galactanase from Bacillus licheniformis; MGGAL, b-1,4-galactanase from Merip-
ileus giganteus; GH, glycoside hydrolase; 4-GTB, 4-nitrophenyl-1,4-b- -thiogalacto-
bioside; 2-NPG, 2-nitrophenyl-1,4-b- -galactopyranoside; 2-NPGB, 2-nitrophenyl-
-galactobioside; DNP, 2,4-dinitrophenol; PNP, 4-nitrophenol.
D
D
1,4-b-
D
1,4-b-
D
-thiogalactobioside (4-GTB) as well as the commercially
-galactopyranoside (2-NPG) were
⇑
Corresponding author. Tel.: +45 35320295.
available 2-nitrophenyl b-
D
E-mail addresses: leila@chem.ku.dk, leila@kemi.ku.dk (L. Lo Leggio).
Present address: Monash University, Department of Biochemistry and Molecular
Biology, School of Biomedical Sciences, Clayton, Vic 3800, Australia.
investigated. Small, well defined substrates, as opposed to heterog-
enous polymers, allow monitoring of hydrolysis of specific
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.05.017

S. Torpenholt et al. / Carbohydrate Research 346 (2011) 2028–2033
2029
glycosidic linkages, and thus a more direct interpretation of activ-
1
0.8
0.6
0.4
0.2
0
ity measurements in terms of molecular mechanisms. The use of a
thio-linked disaccharide glycoside ensures that internal degrada-
tion of the disaccharide does not occur, simplifying analysis. The
results are discussed in the light of the known biochemical charac-
teristics and 3D-structures of AAGAL13,14 (PDB IDs: 1FHL, 1FOB)
and BLGAL3,15 (PDB IDs: 1R8L, 1UR0, 1UR4, 2CCR, 2GFT, 2J74).
2. Results and discussion
2.1. Production of thiogalactobioside
The thiodisaccharide 4-nitrophenyl -1,4-b-D-thiogalactobioside
0
20
40
60
was produced by the thioglycoligase approach (Scheme 1), which
requires a catalytic acid/base mutant of a retaining glycoside
hydrolase, an activated sugar donor and a thiosugar acceptor. We
used Agrobacterium sp. b-glucosidase E170G,16,17 2,4-dinitrophenyl
Time [sec]
Figure 1. Progress curves for cleavage of 2-NPG by b-1,4-galactanases. Measure-
ments with AAGAL are shown with solid a line, MGGAL with dashed line and BLGAL
with a dotted line. The figure was generated using the software package Grafit.18
b-D-galactopyranoside and 4-nitrophenyl 4-deoxy-4-thio-b-D-
galactopyranoside, respectively.
produce galactose and galactobiose when incubated with 2-NPG.
The free galactose arises from hydrolysis, occurring in competition
with transglycosylation, of the initially formed galactosyl enzyme
intermediate. This experiment demonstrates that transglycosyla-
tion occurs and explains the accelerating progress curves. No such
behavior is seen with BLGAL either in the spectrophotometric or
TLC assay (Figs. 1 and 2). Most probably this is because BLGAL does
not bind 2-NPG productively, but rather at distal-subsites. This is
consistent with earlier investigations,3 which revealed that the
smallest oligosaccharide BLGAL can hydrolyze is b-1,4-galac-
totetraose.
2.2. Activity against polymeric substrates
The specific activities of the enzymes were determined using
the dyed substrate azo-galactan by a colorimetric assay involving
measurement of the release of dye by monitoring at 540 nm. The
specific activities determined for the whole assay duration
(20 min) were 0.47 0.02 AU/
dium citrate pH 5.5, 0.12 0.01 AU/
citrate pH 4.4 and 0.041 0.001 AU/
l
g enzyme for MGGAL in 4 mM so-
g for AAGAL in 4 mM sodium
g for BLGAL in 25 mM MES
l
l
pH 6.5 and 0.5 mM CaCl2. All measurements were carried out at
room temperature.
2.4. Enzymatic hydrolysis of 4-nitrophenyl -1,4-b-D-
thiogalactobioside (4-GTB)
2.3. Action of b-galactanases on 2-nitrophenyl b-D-
galactopyranoside
The two fungal fungal b-1,4-galactanases AAGAL and MGGAL
Initial spectrophotometric monitoring of the reaction of the two
fungal b-1,4-galactanases AAGAL and MGGAL showed very low
had considerably higher specific activity on 4 mM 4-GTB than on
2-NPG, 1.8
for MGGAL. The initial velocities observed for cleavage of a range of
concentrations of the substrate 4-GTB by AAGAL (33 M) and
MGGAL (11 M) are plotted in Figure 3. Normal saturable Michae-
l l
mol minꢀ1 mgꢀ1 for AAGAL and 0.44 mol minꢀ1 mgꢀ1
hydrolytic activity on 4 mM 2-nitrophenyl b-
(2-NPG) over long reaction times (20 min). AAGAL had under these
conditions specific activity of 0.06
mol minꢀ1 mgꢀ1 while
MGGAL a specific activity of 0.03
mol minꢀ1 mgꢀ1. However,
incubation with higher concentrations of 2-NPG resulted in non-
linear progress curves exemplified in Figure (12 mM and
D-galactopyranoside
l
a
l
l
l
lis–Menten kinetics are observed for AAGAL, while a linear depen-
dency is seen for MGGAL, in the measurable concentration range of
4-GTB. The maximum measurable substrate concentration was
12.5 mM, due to the high background signal. Fitting of data for AA-
GAL to the Michaelis–Menten equation yielded Km and kcat values
of 18.6 5.5 mM and 6.2 1.3 sꢀ1, respectively, using the software
package Grafit.18 The kcat/Km was calculated as 333 Mꢀ1 sꢀ1. The
linear dependence for MGGAL in the v versus [S] plot indicates a
very high Km value (ꢁ12.5 mM) and does not allow determination
of individual values of kcat and Km. However a reliable value for
1
16 mM 2-NPG with AAGAL and MGGAL, respectively). Such behav-
ior is most likely due to the occurrence of an initial transglycosyla-
tion to form a disaccharide, 2-nitrophenyl 1,4-b-D-galactobioside
(2-NPGB), which serves as a much better substrate for the enzyme
and is rapidly cleaved to galactobiose without significant accumu-
lation (Scheme 2). Consistent with this, TLC analysis (Fig. 2)
showed that the two fungal galactanases AAGAL and MGGAL
mutated acid/base
OH
OH
H
H
SH
OH
H
O
OH
OH
OH
DNP
OH
HO
S
OH
O2N
OH
O
O
O
O
HO
HO
O
HO
O-PNP
HO
O-PNP
OH
OH
NO2
-
OH
O
O
OH
O
O
-
O
O
catalytic nucleophile
Scheme 1. Thioglycoligase reaction mechanism.

2030
S. Torpenholt et al. / Carbohydrate Research 346 (2011) 2028–2033
OH
OH
O2
N
OH
OH
O
HO
O
O
OH
OH
HO
O
O2
N
OH
O
OH
OH
HO
O
O2
N
O2
N
O
OH
HO
HO
O
OH
O2
N
HO
OH
OH
O
OH
HO
O
OH
O
HO
OH
OH
Scheme 2. Galactanase-catalyzed formation of galactobiose by transglycosylation.
6
4
2
0
0
2
4
6
8
10
12
14
4-GTB [mM]
Figure 3. Michaelis–Menten curve for hydrolysis of 4-GTB by b-1,4-galactanases.
Measurements with AAGAL are marked with circles and MGGAL with squares. The
figure was generated using the software package GraFit.18
of 43% as calculated by ClustalW19 (Fig. 4), and can be expected
to have very similar active sites. Eight key residues involved di-
rectly or indirectly in catalysis (Glu136, Glu246, Trp297, Arg45,
Ser213, His81, Asn135, and Gly40), and reported to be functionally
conserved21 or to have functional counterparts22 for Clan GH-A, are
all found in both AAGAL and MGGAL. The Trp115 and Asp117 res-
idues implicated in substrate binding at the ꢀ2 subsite for BLGAL
(GenBank ID: AAO31370.1, PDB ID: 1UR0)3 are conserved in both
AAGAL and MGGAL, while Lys120 of BLGAL is only conserved in
MGGAL and cannot make similar interactions in AAGAL. The puta-
tive BLGAL residues3,15 interacting with the substrate at subsite
ꢀ1, Asn164, Tyr234 and Ala116, are conserved in both AAGAL
and MGGAL. This similarity is consistent with the fact that their
Figure 2. Thin layer chromatographic analysis of the products of reaction of b-1,4-
galactanases with 2-NPG. Galactose, galactobiose, 2-NPG and 2-nitrophenol are
shown as standards. UV-active compounds are marked with a circle. 2-Nitrophenol
is in the broad band at the top of the plate.
kcat/Km of 62 Mꢀ1 sꢀ1 is obtained from the slope and the Km value
must be at least 60 mM according to the following reasoning: the
linear dependency for MGGAL in the v versus [S] plot should only
be true for Km ꢁ [S]. Therefore a conservative estimate for a mini-
mum value of Km can be 5 ꢂ max [S] equal to 60 mM. Since Km is at
least 60 mM, then kcat must be at least 3.7 sꢀ1. No hydrolysis of
4-GTB by BLGAL was observed, even at enzyme concentrations as
high as 5.5 mg/mL.
kcat/Km values are only fivefold different—corresponding to less
than 1 kcal/mol difference in transition state affinities for the
two enzymes. Such a difference is comparable to those observed
for disruptions of single hydrogen bonds in other enzyme carbohy-
drate complexes,23 and can be rationalized by small structural
differences.
2.5. Structural interpretation of results
The bacterial b-1,4-galactanase BLGAL, unlike the two fungal b-
1,4-galactanases MGGAL and AAGAL, does not cleave 4-GTB and 2-
NPG while showing activity on dye-modified galactan. Using the
The two fungal enzymes, AAGAL (GenBank ID: AAA32692.1)
and MGGAL (GenBank ID: AAQ77827.1), have a sequence identity

S. Torpenholt et al. / Carbohydrate Research 346 (2011) 2028–2033
2031
Figure 4. Sequence alignment. Eight functionally conserved key galactanase residues in Clan GH-A (Glu136, Glu246, Trp297, Arg45, Ser213, His81, Asn135 and Gly40, AAGAL
numbering)21 are coloured grey. The residues lining the subsites ꢀ1 (Asn164, Tyr234, Ala116) and -2 (Trp115, Asp117, Lys120) based upon the BLGAL structure (BLGAL
numbering) are marked with a dotted square and a solid square, respectively. Loop 8 is colored black in the BLGAL sequence. Secondary structure elements are annotated as
in.3 This figure was made with ClustalW19 and Stride.20
known crystal structures for AAGAL and BLGAL3,14 (PDB IDs: 1FOB,
1UR0), we conclude that this is due to the presence of an especially
long loop 8 in the structure of BLGAL, which is much shorter in the
structure of AAGAL and in the sequence of MGGAL. This long loop
contains two important residues Trp347 and Trp363 that, together
with the conserved Trp115, constitute aromatic platforms at the
substrate binding subsites ꢀ2, ꢀ3, and ꢀ4 (Fig. 5). The loop enables
BLGAL to bind 4-GTB non-productively by the use of subsites ꢀ3
and ꢀ4 or ꢀ2 and ꢀ3, most likely subsites ꢀ2 and ꢀ3, where galac-
tobiose has been shown bound in the crystal structure.3 The lack of
cleavage of 2-NPG could be due to non-productive binding, or
because a single subsite is insufficient for binding.
yielding Km and kcat values of 2.3 mM and 32 sꢀ1, respectively. C.
japonicus b-1,4-galactanase, like BLGAL, contains a tryptophan res-
idue (Trp 347) in subsite ꢀ3 but lacks Trp363 in subsite ꢀ4. A large
part of the loop containing subsite ꢀ3 and ꢀ4 is missing in C. japo-
nicus b-1,4-galactanase (Fig. 4), so subsite ꢀ3 might well be absent
or may be very different, thereby explaining why C. japonicus b-
1,4-galactanase is unlikely to have the same kinetic properties as
BLGAL.
3. Conclusion
Kinetic measurements were performed on two well defined aryl
glycoside substrates 2-NPG and 4-GTB, yielding values of Km and
kcat and kcat/Km for 4-GTB of 18.6 5.5 mM, 6.2 1.3 sꢀ1 and
333 Mꢀ1 sꢀ1 for AAGAL, while a kcat/Km value for MGGAL of
62 Mꢀ1 sꢀ1 was found. BLGAL did not hydrolyze 4-GTB. Kinetic
studies with 2-NPG at concentrations above 4 mM yielded non-lin-
ear progress curves, suggesting very effective transglycosylation
Kinetic studies have been performed on b-1,4-galactanases
from other species than those investigated here. Most of this work
was however done on heterogenous polymeric substrates and can
therefore not be used for comparison. Kinetic studies have been
carried out with the bacterial Pseudomonas fluorescens sub. cellulosa
(now known as Cellvibrio japonicus) (GenBank ID: CCA62990.1) b-
1,4-galactanase using 2,4-dinitrophenyl b-1,4-galactobioside,12

2032
S. Torpenholt et al. / Carbohydrate Research 346 (2011) 2028–2033
lated and worked up by standard procedures (Ac2O/pyridine) and
characterized by 1H NMR.
1H NMR (400 MHz, CDCl3): d 8.20 (m, 2H, aryl), 7.06 (m, 2H,
0
0
aryl), 5.46 (dd, 1H, J2,1 7.5 Hz, J2,3 9.6 Hz, H-2). 5.42 (dd, 1H, J4 ,3
0
0
0
3.4 Hz, J4 ,5 , 1 Hz, H -4), 5.14 (dd, 1H, J3,2 9.6 Hz, J3,4 4.4 Hz, H-3),
5.16 (dd, 1H, J2 ,3 = J2 ,1 10.0 Hz, H0-2), 5.10 (d, 1H, J1,2 7.5 Hz, H-
0
0
0
0
0
0
0
0
0
0
0
1), 5.00 (dd, 1H, J3 ,4 3.4 Hz, J3 ,2 10 Hz, H-3 ), 4.51 (d, 1H, J1 ,2
10 Hz, H0-1), 4.46 (dd, 1H, J6a,6b 12.2 Hz, J6a,5 7.7 Hz, H-6a), 4.36
(dd, 1H, J6b,6a 12.2 Hz, J6b,5 3.5 Hz, H-6b), 4.19–4.06 (m, 3H, H-5,
2 ꢂ H-6), 3.84 (m, 1H, H0-5), 3.66 (dd, 1H, J4,3 4.4 Hz, J4,5 2.0 Hz,
H-4), 2.20–1.95 (m, 21H, –COCH3).
4.2. Enzymes
4.2.1. Cloning, expression, and purification of AAGAL, BLGAL,
and MGGAL
Cloning and expression of AAGAL (Gen bank ID: AAA32692.1),
BLGAL (Gen bank ID: AAO31370.1) and MGGAL (Gen bank ID:
AAQ77827.1) are described in detail in US patent 5474922,
6,331,426 B1 and 6,329,185 B1. The molecular weights of AAGAL,
BLGAL and MGGAL as calculated from the gene sequences were
36758.7 Da, 43591.4 Da and 34610 Da, respectively. AAGAL and
BLGAL were purified as previously described3,27 to a final concen-
tration of 5.8 mg/mL and 4.1 mg/mL, respectively. The gene encod-
ing MGGAL was expressed in Aspergillus oryzae essentially as
described for Humicola insolens galactanase.14 The culture broth
was centrifuged (10,000ꢂg, 20 min) and the supernatant filtered
through a Nalgene 0.2
ual Aspergillus host cells. The filtrate was concentrated approx. 15
times by ultrafiltration on Filtron omega cassette (cut-
lm filtration unit in order to remove resid-
a
Figure 5. Comparison of the loop region 7/8 of BLGAL (gray) and AAGAL (red) in the
published crystal structures.3,14 The ligand galactotriose (bound in the BLGAL
structure) is shown in yellow. Trp347 and Trp363 in subsite ꢀ3 and ꢀ4 are shown
in blue. This figure was generated with Pymol24 using coordinates with PDB codes
1UR0 and 1FOB for BLGAL and AAGAL, respectively.
off = 3000 Da). Ammonium sulfate was added to give a 1.2 M final
concentration and the filtrate was applied to a Phenyl Toyopearl
650S column (from TosoHaas) equilibrated in 20 mM succinic
acid/NaOH, 1.2 M ammonium sulfate, pH 6.0 (buffer A). After
washing with equilibration buffer the column was eluted with a
linear ammonium sulfate gradient (1.2–0 M) over four column vol-
umes. The eluted fractions showing galactanase activity were ad-
justed to 1.2 M ammonium sulfate and applied to a SOURCE
Phenyl column (from GE Healthcare) equilibrated in buffer A. After
washing with equilibration buffer the column was step-eluted
with 20 mM succinic acid/NaOH, pH 6.0. The peak containing the
galactanase activity was collected and applied to a Superdex 75
column (GE Healthcare) equilibrated in 10 mM dimethylglutaric
acid, 100 mM H3BO3, 2 mM CaCl2, 100 mM NaCl, pH 6.0 and the
column was eluted with the same buffer. Fractions containing
galactanase activity and where only one band was seen on a Coo-
massie stained gel were pooled and concentrated to 2.0 mg/mL.
for AAGAL and MGGAL, while BLGAL showed no activity. Transgly-
cosylation was confirmed by thin layer chromatography. The lack
of BLGAL activity against 2-NPG and 4-GTB is probably a result of
an extra long loop 8 so far only observed in the structure of BLGAL.
4. Experimental
4.1. Chemicals
4.1.1. General and previously described chemicals
All chemicals were purchased from Sigma Aldrich A/S unless
otherwise stated. 2,4-Dinitrophenyl b-
4-nitrophenyl 4-deoxy-4-thio-b- -galactopyranoside were synthe-
sized as described previously.25,26
D-galactopyranoside and
D
4.3. Methods
4.1.2. Synthesis of 4-nitrophenyl-1,4-b-
4-Nitrophenyl 4-deoxy-4-thio-b-
-galactopyranoside25 (80 mg,
252 mmol, 20 mM), 2,4-dinitrophenyl b- -galactopyranoside
(130 mg, 390 mmol, 30 mM) and the mutant Abg E170G (ꢃ1 mg/
ml) were incubated for 1 h at room temperature in phosphate buf-
fer (80 mM, pH 7.5) as reported previously for similar systems.16,17
The reaction was monitored by TLC (7:2:1 EtOAc/MeOH/H2O),
which revealed a complete consumption of donor and the forma-
tion of a new UV-active compound. Additional 2,4-dinitrophenyl
D-thiogalactobioside
4.3.1. Kinetic analysis with 2-nitrophenyl b-
D-galactopyranoside
D
and 4-nitrophenyl-1,4-b- -thiogalactobioside
D
D
Rates were determined by measuring the absorbance continu-
ously (every second) at 410 nm after mixing enzyme, buffer and
substrate at room temperature for 60 s. For detecting hydrolytic
activity at low 2-NPG concentrations (4 mM) the absorbance was
monitored for up to 20 min. The initial velocities in mM/min were
calculated from the slope of the absorbance versus time plot using
the extinction coefficients determined for the reaction product
b-D-galactopyranoside was then added to a final concentration of
4-nitrophenol in 4 mM citrate pH 4.4 and pH 5.5 of e410nm
=
0.0544 mMꢀ1 and 410nm = 0.628 mMꢀ1, respectively, at room tem-
e
55 mM and the reaction was maintained at room temperature for
24 h. TLC indicated complete consumption of donor but no further
consumption of acceptor. The pH of the reaction mixture was ad-
justed to ꢃ2.5 using a concentrated solution of acetic acid, and
the dinitrophenol (DNP) was extracted with CH2Cl2. The thiogalac-
toside was initially purified by solid phase extraction, then acety-
perature (data not shown). The kinetic values were determined
from the v versus [S] plot.
The conditions for the 2-NPG assay were 0.40 mg/mL MGGAL in
4 mM sodium citrate pH 5.5 and 12 mM substrate and 1.2 mg/mL
AAGAL in 5 mM sodium citrate pH 4.4 and 16 mM substrate. For

S. Torpenholt et al. / Carbohydrate Research 346 (2011) 2028–2033
2033
the 4-GTB assay 0.40 mg/mL MGGAL in 4 mM sodium citrate pH
Acknowledgments
5.5 and 1.2 mg/mL AAGAL in 4 mM sodium citrate pH 4.4 and
16 mM substrate were used. For BLGAL 0.94 mg/mL BLGAL in
20 mM MES, 0.4 mM calcium chloride pH 6.5 and 16 mM substrate
was used for the 2-NGP assays and 25 mM MES, 0.5 mM calcium
chloride pH 6.5 for the 4-GTB assay. The buffers were chosen
according to previously reported pH optima for AAGAL27 and
MGGAL (US patent 6329185) or conditions previously used for oli-
gosaccharide degradation with BLGAL.3 4-GTB activity was also
measured with 5.5 mg/mL BLGAL. All the measurements were
done at 410 nm on a Perkin Elmer LAMBDA 800 UV/VIS spectro-
The authors acknowledge funding from the NABIIT program
(from the Danish Council for Strategic Research), the Danish Coun-
cil for Independent Research, the Danish National Research Foun-
dation, and the Natural Sciences and Engineering Research
Council of Canada (NSERC). Dorthe Boelskifte is thanked for techni-
cal assistance.
References
1. Willats, W. G.; McCartney, L.; Mackie, W.; Knox, J. P. Plant Mol. Biol. 2001, 47, 9–
27.
photometer at room temperature, using a 10
with a path length of 1 cm from Hellma Analytics.
The 10 l ultra-micro cuvette assay was tested and found to
give reliable rate measurements. For testing, kinetic parameters
obtained in a 1500 l semi micro cuvette were compared with
those obtained in a 10 l ultra-micro cuvette for 0.8 U/mL Aspergil-
ll ultra-micro cuvette
2. Caffall, H. K.; Mohnen, D. Carbohydr. Res. 2009, 344, 1879–1900.
3. Ryttersgaard, C.; Le Nours, J.; Lo Leggio, L.; Jorgensen, C. T.; Christensen, L. L.;
Bjornvad, M.; Larsen, S. J. Mol. Biol. 2004, 341, 107–117.
4. Jenkins, J.; Lo Leggio, L.; Harris, G.; Pickersgill, R. FEBS Lett. 1995, 362, 281–285.
5. Henrissat, B.; Callebaut, I.; Fabrega, S.; Lehn, P.; Mornon, J. P.; Davies, G. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 7090–7094.
l
l
l
6. Henrissat, B. Biochem. J. 1991, 280, 309–316.
lus niger b-1,4-galactosidase (Megazyme Ltd.) and 200–5000 lM 2-
NPG, at room temperature.
7. Henrissat, B.; Bairoch, A. Biochem. J. 1996, 316, 695–696.
8. Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat,
B. Nucleic Acids Res. 2009, 37, D233–D238.
9. Kimura, I.; Yoshioka, N.; Tajima, S. J. Ferment. Bioeng. 1998, 85, 48–52.
10. Van de Vis, J. W.; Searle-van Leeuwen, M. J. F.; Siliha, H. A.; Kormelink, F. J. M.;
Voragen, A. G. J. Carbohydr. Polym. 1991, 16, 167–187.
11. Hinz, S. W.; Pastink, M. I.; van den Broek, L. A.; Vincken, J. P.; Voragen, A. G.
Appl. Environ. Microbiol. 2005, 71, 5501–5510.
12. Braithwaite, K. L.; Barna, T.; Spurway, T. D.; Charnock, S. J.; Black, G. W.;
Hughes, N.; Lakey, J. H.; Virden, R.; Hazlewood, G. P.; Henrissat, B.; Gilbert, H. J.
Biochemistry 1997, 36, 15489–15500.
13. Ryttersgaard, C.; Lo Leggio, L.; Coutinho, P. M.; Henrissat, B.; Larsen, S.
Biochemistry 2002, 41, 15135–15143.
14. Le Nours, J.; Ryttersgaard, C.; Lo Leggio, L.; Østergaard, P. R.; Borchert, T. V.;
Christensen, L. L.; Larsen, S. Protein Sci. 2003, 12, 1195–1204.
15. Le Nours, J.; De Maria, L.; Welner, D.; Jørgensen, C. T.; Christensen, L. L.;
Borchert, T. V.; Larsen, S.; Lo Leggio, L. Proteins Struct. Funct. Bioinform. 2009, 75,
977–989.
4.3.2. Activity measurements against the polymeric substrate
azo-galactan
The activity of the enzymes with the polymeric dyed substrate
azo-galactan was estimated by an assay carried out according to
the manufacturer’s (Megazyme Ltd.) instructions described in
AGALP 11/99 except for the buffers in the reaction mixture which
were 4 mM sodium citrate pH 4.4 for AAGAL, 4 mM sodium citrate
pH 5.5 for MGGAL and 25 mM MES pH 6.5 and 0.5 mM CaCl2 for
BLGAL. The protein concentrations used in the assay were
0.48 lg/mL AAGAL, 0.20 lg/mL MGGAL, and 1.4 lg/mL BLGAL.
A590 was measured with a Perkin Elmer LAMBDA 800 UV/VIS
spectrometer.
16. Jahn, M.; Marles, J.; Warren, R. A. J.; Withers, S. G. Angew. Chem. 2003, 42, 352–
354.
17. Jahn, M.; Withers, S. G. Biocatal. Biotransform. 2003, 21, 159–166.
18. Leatherbarrow, R. J. GraFit Version 7; Erithacus Software Ltd: Horley, UK,
2009.
19. Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.;
McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.;
Gibson, T. J.; Higgins, D. G. Bioinformatics 2007, 23, 2947–2948.
20. Heinig, M.; Frishman, D. Nucleic Acids Res. 2004, 32, 500–502.
21. Sakon, J.; Adney, W. S.; Himmel, M. E.; Thomas, S. R.; Karplus, P. A. Biochemistry
1996, 35, 10648–10660.
4.3.3. Thin layer chromatography
Three separate enzyme reactions were monitored: MGGAL
(0.40 mg/mL MGGAL, 4 mM sodium citrate pH 5.5, 12 mM 2-
NPG), BLGAL (0.94 mg/mL, 16 mM 2-NPG, 15 mM MES, 0.3 mM
CaCl2, pH 6.5, 3) and AAGAL (1.2 mg/mL AAGAL, 16 mM 2-NPG,
5 mM sodium citrate pH 5.5). The reactions were stopped by freez-
ing in liquid nitrogen after 10 and 30 min of reaction time. Approx-
22. Durand, P.; Lehn, P.; Callebaut, I.; Fabrega, S.; Henrissat, B.; Mornon, J. P.
Glycobiology 1997, 7, 277–284.
imately 20 ll of each sample and standards (50 mM galactose,
10 mM galactobiose, 40 mM 2-NPG and 10 mM 2-nitrophenol)
were transferred to a silica gel plate (Kiesel Gel 60, Merck) at room
temperature by a capillary tube and eluted using ethyl acetate–
MeOH–water (7:2:1 by vol) until the liquid front was 1–2 cm from
the top of the plate. The UV active compounds were visualized by a
UV lamp (UVGL-25 from UVP) at 365 nm. The plate was visualized
by soaking briefly in 10 w/v% ammonium heptamolybdate tetrahy-
drate in 2 M sulfuric acid and air dried completely.28 The plate was
then heated for ꢃ5 min at 105 °C to visualize the carbohydrates as
blue spots.
23. Sierks, M. R.; Bock, C.; Refn, S.; Svensson, B. Biochemistry 1992, 31, 8972–
8977.
24. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, USA; 2002.
25. Chen, H.; Withers, S. G. Carbohydr. Res. 2010, 345, 2596–2604.
26. Namchuk, M.; McCarter, J.; Becalski, A.; Andrews, T.; Withers, S. G. JACS 2000,
122, 1270–1277.
27. Christgau, S.; Sandal, T.; Kofod, L. V.; Dalbøge, H. Curr. Genet. 1995, 27, 135–
141.
28. Zweig, G.; Sherma, J. Handbook Ser. Chromatogr. Sect. A 1992, 2, 162.