A chromogenic assay for limit dextrinase and pullulanase activity

Article abstract of DOI:10.1016/j.ab.2013.12.004

A new chromogenic substrate to assay the starch debranching enzymes limit dextrinase and pullulanase is described. The 2-chloro-4-nitrophenyl glycoside of a commercially available branched heptasaccharide (Glc-maltotriosyl- maltotriose) was found to be a suitable specific substrate for starch debranching enzymes and allows convenient assays of enzymatic activities in a format suited for high-throughput analysis. The kinetic parameters of these enzymes toward the synthesized substrate are determined, and the selectivity of the substrate in a complex cereal-based extract is established.

Full text of DOI:10.1016/j.ab.2013.12.004

Analytical Biochemistry 449 (2014) 45–51
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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
A chromogenic assay for limit dextrinase and pullulanase activity
1
⇑
⇑
Marie Bøjstrup , Caspar Elo Christensen, Michael Skovbo Windahl, Anette Henriksen , Ole Hindsgaul
Carlsberg Laboratory, Gamle Carlsberg vej 10, 1799 Copenhagen V, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 October 2013
Received in revised form 27 November 2013
Accepted 2 December 2013
Available online 10 December 2013
A new chromogenic substrate to assay the starch debranching enzymes limit dextrinase and pullulanase
is described. The 2-chloro-4-nitrophenyl glycoside of a commercially available branched heptasaccharide
(Glc-maltotriosyl-maltotriose) was found to be a suitable specific substrate for starch debranching
enzymes and allows convenient assays of enzymatic activities in a format suited for high-throughput
analysis. The kinetic parameters of these enzymes toward the synthesized substrate are determined,
and the selectivity of the substrate in a complex cereal-based extract is established.
Ó 2013 Elsevier Inc. All rights reserved.
Keywords:
Coupled assay
Limit dextrinase
Pullulanase
Chromogenic substrate
Natural starch consists of a mixture of amylose (20–30%) and
amylopectin (70–80%). Amylose is a linear polysaccharide of
presence of pullulanase or LD has been shown to be of major impor-
tance in order to achieve a complete and efficient conversion of the
branched polysaccharides into small fermentable sugars during the
saccharification process involved in these processes [7,8].
Some commercial kits to determine LD activity are available,
with the most commonly used being the Limit Dextrizyme kit
(Megazyme, Bray, Ireland) that is based on a dye-labeled cross-
linked pullulan [9]. This kit is not widely used, however, because
it is considered to give results with a large standard deviation
and is not suited for high-throughput screening using microtiter
plates. A defined selective chromogenic substrate suitable for
high-throughput screening and real-time analysis has not been
described to date.
Here we present the development of a sensitive chromogenic
assay for LD suited for high-throughput screening. We describe
the synthesis of the selective chromogenic substrate 1 and validate
the method by showing linearity of the assay for both recombi-
nantly derived LD and bacterial-derived pullulanase as well as for
native LD in crude barley malt extract.
a
(1 ? 4)-linked glucose units, whereas amylopectin is a branched
polysaccharide of (1 ? 4)-linked glucose units with approxi-
mately 5% branching formed by (1 ? 6)-glucosidic bonds [1].
During starch hydrolysis, the hydrolytic enzymes b-amylase,
-amylases, pullulanase/limit dextrinase, and -glucosidases act
a
a
a
a
together in the degradation of these polysaccharides [2,3]. Pullula-
nase (EC 3.2.1.41) and limit dextrinase (LD, EC 3.2.1.142)2 specifi-
cally hydrolyze the
a(1 ? 6)-glycosidic bonds in branched
oligosaccharide dextrins but usually have only low activity toward
polysaccharides [3,4]. Pullulanase is a bacterial enzyme, whereas
LD is present in a variety of plants. The substrate specificities of
these enzymes are similar; however, pullulanase has a significant
action on glycogen, whereas LD has no activity [5]. Barley is the only
plant where LD is of commercial interest because LD is the only
starch debranching enzyme in barley malt used for beer production
[6]. The enzymatic conversion of starch into glucose, maltose, and
fructose for use in the production of glucose or maltose syrups for
food sweeteners or during mashing in the brewing process repre-
sents an important growth area in industrial enzyme use [2]. The
Materials and methods
⇑
Corresponding authors. Fax: +45 33 27 47 08.
E-mail addresses: marie.bojstrup@carlsberglab.dk (M. Bøjstrup), ole.hindsgaul@
Synthesis of the chromogenic substrate 1
carlsberglab.dk (O. Hindsgaul).
1
The scheme for the synthesis of the chromogenic substrate 1 is
summarized in Scheme 1. First, the commercially available oligo-
saccharide Glc-maltotriosyl-maltotriose 2 was peracetylated in
high yield using acetic anhydride and pyridine catalyzed by
4-dimethylaminopyridine (DMAP). Subsequent formation of the
bromosugar 4 was achieved using 33% HBr in acetic acid in
dichloromethane (DCM) at 0 °C. The phase-transfer-catalyzed
Current address: Department of Protein Structure and Biophysics, Novo Nordisk
A/S, 2760 Måløv, Denmark.
2
Abbreviations used: LD, limit dextrinase; DMAP, 4-dimethylaminopyridine; DCM,
dichloromethane; ClNP, 2-chloro-4-nitrophenol; TBABr, tetrabutylammonium bro-
mide; UV, ultraviolet; MALDI–ToF–MS, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry; RT, room temperature; UPLC, ultra-performance
liquid chromatography; LDI, limit dextrinase inhibitor; DTT, dithiothreitol; Glc3–ClNP,
maltotrioside-glycoside 3.
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2013.12.004

46
Assay for limit dextrinase and pullulanase / M. Bøjstrup et al. / Anal. Biochem. 449 (2014) 45–51
Scheme 1. Synthesis of the chromogenic substrate 1. (a) Ac2O, pyridine, DMAP, RT, 100%; (b) 33% HBr in AcOH, Ac2O, DCM, 0 °C, 2 h, 100%. (c) 2-Chloro-4-nitrophenol, TBABr,
aqueous NaOH (0.75 M), DCM, RT, 20%. (d) MeOH/Et3N/H2O (2:2:1), RT, 24 h, 37%.
glycosylation of 2-chloro-4-nitrophenol (ClNP) was performed in
the presence of tetrabutylammonium bromide (TBABr) as phase-
transfer catalyst using 3 equivalents of ClNP in a two-phase system
(0.75 M aqueous sodium hydroxide/dichloromethane) [10]. Finally,
O-deacetylation of the peracetylated glycoside 5 provided us with
the target compound 1 in an unoptimized overall yield of 7.4%. The
experimental details are described below.
All commercially available solvents and reagents were used
without further purification. Organic solvents were removed under
diminished pressure at less than 40 °C (bath temperature). Thin
layer chromatography (TLC) was carried out on silica plates (Merck
60 F254 aluminum plates) with detection by ultraviolet (UV) light
(short wavelength) or 10% sulfuric acid in ethanol. Column chro-
matography was performed on silica gel (Merck, 230–400, 60 Å).
Matrix-assisted laser desorption/ionization time-of-flight mass
and the organic phase was washed with saturated NaHCO3 and
brine, dried (Na2SO4), filtered, and concentrated. The residue was
purified by flash chromatography (EtOAc/hexane, 2:1 and then
4:1). Fractions containing the desired compound were concen-
trated to give 5 as a colorless oil (11 mg, 20%).
The peracetylated glycoside 5 from above was dissolved in
MeOH/Et3N/H2O (2:2:1, 5 ml). The mixture was left stirring over-
night. The mixture was evaporated and passed through mixed
bed resin (MB-1) to remove salts. The fractions containing com-
pound were concentrated to give compound 1 as a white powder
(2.4 mg, 37%). The overall yield was 7.4%.
1H NMR (800 MHz, D2O): d 8.41 (d, 1H, J = 2.7 Hz), 8.21 (dd, 1H,
J = 2.7, 9.2 Hz), 7.38 (d, 1H, J = 9.2 Hz), 5.44 (d, 1H, J = 3.8 Hz), 5.35–
5.37 (m, 3H), 5.34 (d, 1H, J = 7.8 Hz), 4.93 (m, 2H), 3.44–4.01 (m,
41H), 3.41 (dd, 1H, J = 9.7, 9.7 Hz); 13C NMR (201 MHz, D2O): d
157.0, 142.2, 125.9, 124.0, 123.0, 115.3, 99.8, 99.7, 99.4, 99.4,
99.2, 97.8, 97.6, 77.3, 77.0, 76.9, 76.0, 75.5, 74.7, 73.1, 73.0, 73.0,
72.8, 72.8, 72.7, 72.1, 71.5, 71.4, 71.4, 71.3, 71.2, 71.2, 71.2, 71.1,
71.0, 70.9, 70.9, 70.0, 69.2, 69.2, 69.1, 66.1, 65.6, 60.4, 60.2, 60.2,
60.1, 60.1.
spectrometry (MALDI–ToF–MS) was performed on
a Bruker
Daltonics Microflex instrument operating in reflectron mode. A
340-nm laser was used, and mass spectra were typically accumu-
lated from 100 laser shots. Nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker Daltonics Avance 800-MHz
spectrometer equipped with a 5-mm TCI cryoprobe operating at
298 K. D2O was obtained from Cambridge Isotope Laboratory.
Spectra are internally referenced to the solvent residual proton.
Spectra were processed using Bruker Topspin 2.0. Glc-maltotrio-
syl-maltotriose was purchased from Megazyme. All other chemi-
cals were purchased from Sigma–Aldrich.
Standard malt extraction
Malt flour (0.5 g) was shaken with 4 ml of extraction buffer
(100 mM NaOAc and 5 mM CaCl2, pH 5.3) for 30 min. The slurry
was centrifuged for 10 min, and the supernatant was used
immediately.
Glc-maltotriosyl-maltotriose 2 (30.0 mg, 25 lmol) was sus-
pended in dry pyridine (3 ml). Acetic anhydride (2 ml) and a cata-
lytic amount of DMAP were added, and the mixture was left at
room temperature (RT) overnight. Additional acetic anhydride
(0.5 ml) was added, and the mixture was stirred at RT overnight.
Recombinantly derived barley LD
The barley LD gene (HvLD) was expressed in Escherichia coli or
codon optimized for Pichia pastoris expression by GenScript for
MALDI–ToF–MS (with
a-cyano-4-hydroxycinnamic acid [4-HCCA]
as matrix) showed completion of the reaction. The solvents were
evaporated in vacuo. The residue was dissolved in DCM, washed
with aqueous HCl (0.2 M), saturated NaHCO3, and brine, dried (Na2-
SO4), filtered, concentrated in vacuo, and coevaporated twice with
toluene. This gave the peracetylated product in quantitative yield.
cloning into the pPinka-HC vector. The pPinka-HC-HvLD vector
was linearized with Afl II and transformed into PichiaPink strain
1 from Invitrogen. Screening for colonies was done as described
in the Invitrogen user manual [11]. The expression level in Pichia-
Pink was at the same level as described by others [12].
The peracetylated sugar (53 mg, 25 lmol) was dissolved in
DCM (3 ml). Acetic anhydride (0.15 ml) was added, and the mix-
ture was cooled to 0 °C. Two drops of HBr (33% in AcOH) were
added, and the mixture was stirred at 0 °C for 10 min. HBr (33%
in AcOH, 3 ml) was added, and the mixture was stirred at 0 °C for
2 h. The mixture was concentrated in vacuo, coevaporated with
toluene, and used in the next step without further purification.
Enzymatic assay
The assay development and optimization was done in 50-ll
total volume in each well (Nunc 96-well plate, half area). Two
replicates were made. As background was used the concentration
of substrate with no debranching enzyme added. This blank was
subtracted from the measured values. All experiments were per-
formed using a 100-mM HOAc/NaOAc buffer containing 5 mM
The residual bromosugar 4 (53 mg, 25
DCM (2 ml) and aqueous NaOH (0.75 M, 1 ml). ClNP (14 mg,
80 mol) and TBABr (8 mg, 24.8 mol) were added, and the mix-
ture was stirred vigorously at RT overnight. DCM (5 ml) was added,
lmol) was dissolved in
l
l
CaCl2 (pH 5.3) at 45 °C.
750 U/ml), b-glucosidase (Agrobacterium sp., 380 U/ml), and
a-Glucosidase (Bacillus stereaothermophilus,

Assay for limit dextrinase and pullulanase / M. Bøjstrup et al. / Anal. Biochem. 449 (2014) 45–51
47
pullulanase (M1 from Kleibsella planticola, 699 U/ml) were pur-
chased from Megazyme. The mixtures were equilibrated for
5 min at 45 °C before the addition of the substrate. Kinetic UV
Recombinant LD: prepared as described, with the amount
varied from 1.3 to 8 g/ml
Pullulanase: amount varied from 0.1 to 10
l
l
g/ml
measurements were recorded at 45 °C on
340PC384 Absorbance Microplate Reader from Molecular Devices
a
SpectraMax
Crude barley malt extract: prepared as described, with the
amount varied from 10 to 50% (v/v).
at 405 nm with measurements every 20 s.
Enzyme kinetics
Data analysis
The experiments were performed as described in the ‘‘Enzy-
matic assay’’ section above, with the amount of
a-glucosidase
After an initial lag phase, the rate of hydrolysis was constant
and the maximum initial velocity was calculated from the slope
of the graph. A standard curve for ClNP was used to determine
the relationship between measured optical density (OD) and con-
centration of the chromophore. A nonlinear regression analysis
(GraphPad Prism version 4.02 for Windows, GraphPad Software,
San Diego, CA, USA) was used to determine Michaelis–Menten
parameters (Km and Vmax values).
being 30 U/ml in each well and the amount of b-glucosidase being
15 U/ml in each well. The amount of the starch debranching en-
zymes was fixed as indicated for the specific enzymes. Substrate
1 was added after equilibration in a concentration that varied from
50 to 2000 lM, and the absorbance was measured as described:
Recombinant LD: prepared as described, with 2.6
well
lg/ml in each
Pullulanase: 1.9 lg/ml in each well
UPLC analysis of products from hydrolysis
Crude barley malt extract: prepared as described, with 40% (v/v)
in each well.
The analysis of the products from enzymatic hydrolysis of 1
were analyzed on a Waters Acquity System. This was equipped
with a TUV detector (dual wavelength UV detector), an FLR detec-
tor (fluorescence detector, 320 nm, kem = 420 nm), an SQ detector
(single quadrupole electrospray ionization–mass spectrometry
[ESI–MS] detector), Binary Solvent Manager, Sample Manager,
and Column Oven Manager. Solvents used were HPLC (high-perfor-
mance liquid chromatography) grade from LabScan, and H2O was
Results and discussion
We designed the selective chromogenic substrate 1 (Scheme 2)
based on the commercially available heptasaccharide Glc-maltotri-
osyl-maltotriose 2 (Megazyme) (Scheme 1). The heptasaccharide 2
was predicted to be a substrate for LD because it contains
a
(1 ? 6)-glucosidic bonds. It is known that LD is unable to hydro-
lyze a single (1 ? 6)-linked glucose and requires at least one
(1 ? 4)-glycosidic bond on each side of the (1?6)-glucosidic
linkage [13]. Accordingly, LD should only cleave the internal
(1 ? 6) bond, resulting in initial formation of a maltotriosyl-gly-
MilliQ grade. Buffers were filtered through a 2-
use. The column used was an Acquity UPLC (ultra-performance
liquid chromatography) BEH Glycan column (1.7
m, 2.1 Â
150 mm) equipped with a VanGuard BEH Glycan precolumn
(1.7
lm filter prior to
a
a
a
l
a
l
m, 2.1 Â 5 mm). The eluent was 10 mM ammonium formate
coside 3 (Scheme 2). If the assay is performed on purified LD,
where no other endogenous enzymes are present, the initially
formed maltotriosyl-glycoside 3 would be stable and exogenous
enzymes would need to be added to give a coupled assay. Both
(pH 4.5)/acetonitrile (78–55:22–45, v/v), and the flow rate was
0.2 ml/min.
a
-glucosidase and b-glucosidase would be required to ensure com-
Determination of needed amount of a- and b-glucosidases
plete and fast hydrolysis of 3 to release the chromogenic reporter
group. Such coupled assays, using various chromogenic reporter
groups, have been described for other glycosidases [14,15]. The
exogenous enzymes must be selected to leave the substrate 1 in-
tact and act only on the product 3 released from the initial cleavage
by LD.
The experiment was performed as described in the ‘‘Enzymatic
assay’’ section above, either with the amount of -glucosidase
a
fixed at 30 U/ml in each well and the amount of b-glucosidase
varied as indicated below or with the amount of b-glucosidase
fixed at 15 U/ml in each well and the amount of a-glucosidase var-
Numerous chromogenic and fluorogenic compounds have been
employed for the detection of the hydrolytic activity of glycosi-
dases [16]. Nitrophenol-glycosides and coumarin-glycosides are
used in the most common commercial kits. The hydrolysis of
fluorogenic substrates can be detected with high sensitivity but
requires equipment that is not available in many laboratories
and especially not in industrial labs where often a UV–visible
spectrometer is the only standard equipment for analysis. The
activity of LD is normally measured at pH 5.5, meaning that the
most commonly used UV–visible chromophore, p-nitrophenol, will
not be colored at this pH but will require the addition of base to
generate a visible nitrophenolate. ClNP has a more favorable pKa
of 5.13, meaning that this chromophore will show acceptable
absorbance even at pH 5.5 [9,14]. Continuous absorbance measure-
ments should be possible, therefore, with no need for development
of the color by adding base. If end-point measurements are desired,
quenching with base would only further increase the sensitivity of
the assay [14].
ied as indicated below. Substrate 1, corresponding to 0.5 mM, was
added to the mixture after equilibration, and the absorbance was
measured as described:
a-Glucosidase: amount varied from 2 to 30 U/ml
b-Glucosidase: amount varied from 2 to 25 U/ml
Recombinant LD: prepared as described, with 5
well being used for the optimization
lg/ml in each
Pullulanase: 1.9 lg/ml in each well used for the optimization
Crude barley malt extract: prepared as described, with 40% (v/v)
in each well being used for the optimization.
Linearity of assay
The experiments were performed as described in the ‘‘Enzy-
matic assay’’ section above, with the amount of
a-glucosidase
being 30 U/ml in each well and the amount of b-glucosidase being
15 U/ml in each well. The amount of the starch debranching
enzymes was varied as indicated for the specific enzymes. Sub-
strate 1, corresponding to 0.5 mM, was added to the mixture after
equilibration, and the absorbance was measured as described:
To be able to correlate the formation of ClNP to LD activity,
a- and b-glucosidases must be added in amounts sufficient that
the rate of release of ClNP will depend only on the LD activity
and is not limited by the activity of endogenous or exogenous

48
Assay for limit dextrinase and pullulanase / M. Bøjstrup et al. / Anal. Biochem. 449 (2014) 45–51
Scheme 2. Concept of the assay, illustrated using 2-chloro-4-nitrophenol (ClNP) as chromophore. Initial cleavage by LD releases a maltotriosyl-glycoside 3 susceptible to
hydrolysis by endogenous and exogenous enzymes. The released chromophore (or fluorophore) can be quantified spectrophotometrically and directly correlated to the
activity of LD in the sample.
Fig.1. Investigation of linear range. (A) Correlation between the amount of added recombinant LD and the initial velocity of the hydrolysis of 1 ([S] = 0.5 mM). (B) Correlation
between the amount of added pullulanase from Kleibsella planticola and the initial velocity of the hydrolysis of 1 ([S] = 0.5 mM).
enzymes present. The required concentrations of
dases were determined by varying the concentrations of these
enzymes in the presence of recombinant LD (5 g/ml) or pullula-
nase (1.9 g/ml). In a typical solution phase assay, the conditions
used were 0.5 mM substrate 1, 30 U/ml -glucosidase, 15 U/ml b-
glucosidase in 0.1 mM NaOAc (pH 5.3), and 5 mM CaCl2 at 45 °C.
The linearity of the assay was evaluated using increasing
amounts of recombinant LD (Fig. 1A) as well as pullulanase from
K. planticola (Fig. 1B). A linear correlation (R2 > 0.99) was seen be-
tween the amount of added LD and the initial velocity (nmol/min)
a
- and b-glucosi-
The Michaelis–Menten constants Km and Vmax for a recombi-
nantly derived pullulanase and barley LD were determined by
using varying concentrations of the chromogenic substrate 1 rang-
l
l
ing from 50 to 2000
under standard assay conditions as mentioned earlier, and initial
rate (nmol/min) versus concentration of substrate ( M) was
lM (Fig. 2). Enzyme activities were measured
a
l
plotted. A nonlinear regression analysis to the standard Michae-
lis–Menten equation (GraphPad Prism version 4.02 for Windows)
was used to determine the Michaelis–Menten parameters Km and
Vmax
146.0 10 l
.
The Km was 154.3 19
M for pullulanase.
lM for recombinant LD and
of the hydrolysis of 1 up to a concentration of approximately 3
lg/
ml LD and approximately 3 g/ml pullulanase. Above this concen-
tration, the rate of hydrolysis of 1 is dependent on other factors
such as the concentration of the exogenous enzymes.
l
To evaluate the assay in a more complex matrix, we investi-
gated a crude extract from barley malt that contains several poten-
tially interfering glucosidases in addition to LD. Fortunately, we

Assay for limit dextrinase and pullulanase / M. Bøjstrup et al. / Anal. Biochem. 449 (2014) 45–51
49
Fig.2. (A) Determination of Km and Vmax for LD in recombinant barley LD using the chromogenic substrate 1. (B) Determination of Km and Vmax for pullulanase using the
chromogenic substrate 1. Error bars indicate the standard deviations for duplicate measurements.
can benefit from the fact that the limit dextrinase inhibitor (LDI),
which is endogenous in barley seeds, is known to act specifically
on LD [17]. LDI is a 14-kDa hydrophobic protein containing four
disulfide bonds and has been recombinantly expressed in high
yield [18]. The activity of LD detected in crude extracts from barley
malt is normally low due to the presence of endogenous LDI. It has
been shown that the LDI can be inactivated by using reducing
agents such as dithiothreitol (DTT). The reducing conditions
destroy the disulfide bonds of LDI, resulting in an increase in the
observed activity of LD [9]. Therefore, we envisaged that the
specificity of the substrate 1 could be confirmed by complete
inhibition of LD with the specific inhibitor LDI.
Chromogenic substrate 1 was added to barley malt extract with
and without added recombinant LDI present, and analysis of the
reaction products was monitored by hydrophilic interaction
ultra-performance liquid chromatography (HILIC–UPLC) using UV
detection (300 nm), as illustrated in Fig. 3. When substrate 1 was
added to barley malt extract (Fig. 3B), only formation of the malto-
trioside-glycoside 3 (Glc3–ClNP) from the expected hydrolysis by
LD was seen together with shorter ClNP-glycosides and ClNP. To
our great satisfaction, the chromogenic substrate 1 was not hydro-
lyzed by
a-glucosidases present in the malt extract given that no
formation of branched maltohexosyl-, maltoheptosyl-, or maltotet-
raosyl-glycosides is seen in the chromatogram (Fig. 3B). This con-
Fig.3. UPLC chromatograms of samples (Waters, BEH Glycan column, 1.7
lm) with UV detection at 300 nm. All samples were treated in NaOAc buffer (pH 5.3) at 60 °C for 1 h:
(A) substrate 1; (B) substrate 1 and 40% (v/v) barley malt extract; (C) substrate 1, 40% (v/v) barley malt extract, and exogenous barley LDI.

50
Assay for limit dextrinase and pullulanase / M. Bøjstrup et al. / Anal. Biochem. 449 (2014) 45–51
(Fig. 3C). This indicates that indeed the hydrolysis products seen
in Fig. 3B originated from the hydrolysis of 1 by LD to give the
intermediate maltotriosyl-glycoside (Glc3–ClNP) followed by fur-
ther hydrolysis of Glc3–ClNP with b-amylase,
a-glucosidases, and
b-glucosidases present in the barley malt extract.
The data in Fig. 4 confirm that the endogenous LD activity (trace
B) increases on the addition of 25 mM DTT (trace A), supporting the
inactivation of LDI by this agent. The addition of exogenous recom-
binant LDI in excess to the extract totally inhibits the activity of LD
given that no change in absorbance is seen (Fig. 4, trace C). As is
clearly evident from Fig. 4, a lag phase appears prior to reaching
the steady state velocity. This is well known for coupled enzyme
assays [19] and might be shortened by selecting more effective
exogenous enzymes.
As was done for the pure recombinant enzymes, we verified
that the initial velocity of the hydrolysis correlated with the
amount of barley malt extract added to 1. A linear correlation
(R2 > 0.96) between the amount of extract added (%, v/v) and the
corresponding initial rate (nmol/min) was found (Fig. 5). This
means that substances present in the barley malt extract do not in-
hibit the exogenous enzymes to any significant extent.
Fig.4. UV absorption at 405 nm measured by time, with all experiments at pH 5.3
and 45 °C: (A) substrate 1 treated with 40% (v/v) barley malt extract and 25 mM
DTT; (B) substrate 1 treated with 40% (v/v) barley malt extract; (C) substrate 1
treated with 40% (v/v) barley malt extract and excess LDI.
The Michaelis–Menten constants Km and Vmax for LD in a crude
extract from barley malt were determined by using varying
concentrations of the chromogenic substrate 1 ranging from 50
to 1000
the recombinant enzymes (Fig. 6). The Km determined was
134.9 17 M for LD in crude barley malt extract, which is in very
good agreement with the Km determined for recombinantly ex-
pressed barley LD (154.3 19 M).
lM with 40% (v/v) crude malt extract as described for
l
l
In summary, we have designed and synthesized a selective
chromogenic substrate suitable for use in a high-throughput assay
for LD and pullulanase and kinetic characterization of these en-
zymes. The initial velocity of the hydrolysis of this substrate can
be linearly correlated with the amount of enzyme added. This
correlation is seen for both recombinantly expressed LD and LD
in barley malt extract. The specificity of the assay toward starch
debranching enzymes from other cereals should be determined
in order to expand the utility of the assay.
Fig.5. Investigation of linear range for LD in barley malt extract. Correlation
between the amount of added barley malt extract (%, v/v) and the initial velocity of
the hydrolysis of 1 ([S] = 0.5 mM) is shown.
Acknowledgments
Birte Svensson and Marie Sofie Møller (Technical University of
Denmark, DTU) are acknowledged for their generous donation of
recombinantly expressed barley LD inhibitor. Monica Palcic and
Morten Munch Nielsen (Carlsberg Laboratory) are acknowledged
for their generous supply of recombinantly expressed barley LD.
Finn Lok and Ole Olsen (Carlsberg Research Centre) are acknowl-
edged for their advice and help regarding LD extraction and
activity measurements.
firmed that indeed the a(1 ? 6)-linked glucose at the nonreducing
end acted as a protecting group preventing hydrolysis by exo-act-
ing enzymes in barley malt. An additional experiment was per-
formed in which LDI was added in excess to inhibit the activity
of LD. The products from hydrolysis were analyzed by UPLC
(Fig. 3C). Only trace hydrolysis of the substrate was detected
Fig.6. Determination of Km and Vmax for LD barley malt extract (40%, v/v) using the chromogenic substrate 1. Error bars indicate the standard deviations for duplicate
measurements.

Assay for limit dextrinase and pullulanase / M. Bøjstrup et al. / Anal. Biochem. 449 (2014) 45–51
[11] PichiaPink Expression System, version A, 2009, Invitrogen.
51
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