Colourimetric and fluorometric substrates for measurement of pullulanase activity

Article abstract of DOI:10.1016/j.carres.2014.04.014

Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing these substrates have been developed. These mixtures comprise thermostable α- and β-glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-maltotrioside (BzCNPG₃G₃, 1) as a colourimetric substrate or 4,6-O-benzylidene-4-methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside (BzMUG₃G₃, 2) as a fluorometric substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, β-amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the non-reducing end d-glucosyl residue. The substrates are not hydrolysed by any α-amylases studied, (including those from Aspergillus niger and porcine pancreas) and are resistant to hydrolysis by Pseudomonas sp. isoamylase. On hydrolysis by pullulanase, the 2-chloro-4-nitrophenyl-β-maltotrioside (3) or 4-methylumbelliferyl-β- maltotrioside (4) liberated is immediately hydrolysed to d-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The reaction is terminated by the addition of a weak alkaline solution leading to the formation of phenolate ions in solution whose concentration can be determined using either spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation.

Full text of DOI:10.1016/j.carres.2014.04.014

Accepted Manuscript
Colourimetric and fluorometric substrates for measurement of pullulanase ac-
tivity
Barry V. McCleary, David Mangan, Vincent McKie, Claudio Cornaggia,
Edward Rooney
PII:
S0008-6215(14)00175-X
http://dx.doi.org/10.1016/j.carres.2014.04.014
CAR 6731
DOI:
Reference:
Carbohydrate Research
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Revised Date:
Accepted Date:
14 February 2014
16 April 2014
18 April 2014
Please cite this article as: McCleary, B.V., Mangan, D., McKie, V., Cornaggia, C., Rooney, E., Colourimetric and
fluorometric substrates for measurement of pullulanase activity, Carbohydrate Research (2014), doi: http://
dx.doi.org/10.1016/j.carres.2014.04.014
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Colourimetric and fluorometric substrates for measurement of pullulanase activity
Barry V. McCleary*, David Mangan*, Vincent McKie, Claudio Cornaggia and Edward Rooney
Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County
Wicklow, Ireland.
Corresponding authors. Tel.: +353 12861220.
E-mail addresses:
david@megazyme.com (David Mangan)
barry@megazyme.com (Barry McCleary)
Abstract
Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been
prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing
these substrates have been developed. These mixtures comprise thermostable α- and β-
glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-
maltotrioside (BzCNPG3G3, 1; Figure 1) as a colourimetric substrate or 4,6-O-benzylidene-4-
methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside (BzMUG3G3, 2) as a fluorometric
substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, β-
amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the
non-reducing end D-glucosyl residue. The substrates are not hydrolysed by any α-amylases
studied, (including those from A. niger and porcine pancreas) and are resistant to hydrolysis by
Pseudomonas sp. isoamylase. On hydrolysis by pullulanase (Figure 1), the 2-chloro-4-
nitrophenyl-β-maltotrioside (3) or 4-methylumbelliferyl-β-maltotrioside (4) liberated is
immediately hydrolysed to D-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The
reaction is terminated by the addition of a weak alkaline solution leading to the formation of
phenolate ions in solution whose concentration can be determined using either
1

spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific,
accurate, robust and readily adapted to automation.
Keywords: pullulanase, limit dextrinase, assay procedure, 4,6-O-benzylidene-2-chloro-4-
nitrophenyl-β-maltotriosyl (1-6)-α-D-maltotrioside, BzCNPG3G3, 4,6-O-benzylidene-
methylumbelliferyl-β-maltotriosyl (1-6)-α-D-maltotrioside, BzMUG3G3.
1. Introduction
Pullulanase (α-dextrin glucanohydrolase, pullulan 6-glucanohydrolase, limit dextrinase,
amylopectin 6-glucanohydrolase)1,2 (EC 3.2.1.4) cleaves the α-1,6-linkages in pullulan,
amylopectin and α- and β-limit dextrins of starch. Hydrolysis of α-1,6-linkages in 1,4:1,6-α-
gluco-oligosaccharides by pullulanase requires the presence of at least one α-1,4-linked glucosyl
residue on each side of the α-1,6-linkage. Thus the smallest substrate for pullulanase is the
tetrasaccharide 62-α-D-maltosyl maltose (7, Figure 2).3 Hydrolysis of glycogen is restricted by
the highly branched, short chain length and dense nature of the molecule.2,3 Several groups of
enzymes catalysing endo-hydrolysis of pullulan have been described including those that
hydrolyse the α-1,6-glucosidic bonds in pullulan and branched α-1,4;1,6 glucans (e.g.
amylopectin)1,4 (Type I) and those referred to as amylopullulanases5 (Type II) which can
hydrolyse both α-1,4- and α-1,6-glucosidic linkages. Other endo-acting enzymes active on
pullulan include pullulan hydrolase type I (neopullulanase) 6 and type II (isopullulanase);7 which
only cleave (α-1,4) linkages in pullulan releasing panose (8) or isopanose (9), respectively
(Figure 2). A fifth type of endo-hydrolase active on pullulan is referred to as pullulan hydrolase
type III. This enzyme has the ability to hydrolyse both (α-1,6) and (α-1,4) glucosidic linkages in
pullulan leading to the formation of panose (8) as well as maltose, maltotriose and glucose. 8
Pullulanase finds widespread application in the starch processing industry. In
combination with amyloglucosidase (AMG) it gives more efficient conversion of starch to
glucose, reducing the formation of isomaltose (10) and giving up to 1% greater yield of
2

glucose.9,10 High maltose syrups are produced from starch using combinations of pullulanase and
β-amylase.9,11
The industrial application of pullulanase has led to significant interest in this enzyme and
considerable screening efforts to locate new and interesting forms with different optimal
conditions of pH and temperature for activity.
Pullulanase activity in industrial enzyme preparations can conveniently be assayed with
reduced pullulan using an appropriate reducing sugar method such as that of Nelson12 and
Somogyi.13 In crude enzyme preparations, the activity can be more specifically assayed using
soluble dyed substrates (Red Pullulan) or dyed and cross-linked pullulan either in powder
(AZCL-Pullulan) or tablet (Limit-Dextrazyme) form.14 While the latter two substrates are very
useful and widely used, they do not readily lend themselves to automated analysis procedures.
The aim of this work was to develop simple assay procedures for pullulanase using the
well-defined oligosaccharide, 63-α-D-maltotriosyl maltotriose (11, Figure 2) based on similar
procedures previously developed and published for the measurement of α-amylase15,16 and endo-
cellulase.17 The two substrates developed were 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-63-
α-D-maltotriosyl maltotrioside (BzCNPG3G3; 1) and 4,6-O-benzylidene-4-methylumbelliferyl-β-
63-α-D-maltotriosyl maltotrioside (BzMUG3G3; 2). In this paper, the synthesis of these
oligosaccharide substrates and their use in enzyme-linked assays for pullulanase will be
described. During the course of this work the synthesis and use of the 2-chloro-4-nitrophenyl 63-
α-D-glucosyl 63-α-D-maltotriosyl maltotrioside (12) lacking a 4,6-O-benzylidene group for the
assay of pullulanase and limit dextrinase was described.18
2. Results and Discussion
63-α-D-Maltotriosyl maltotriose (11) was obtained in a highly pure form from Megazyme
International. 11 is derived from pullulan which is composed mainly of repeating maltotriosyl
units (~ 93%),19 but also contains approximately 7% maltotetraosyl units. It is important that
there is no contamination present in the 63-α-D-maltotriosyl maltotriose used for the synthesis of
these substrates as the most common contaminant (heptasaccharide containing a maltotetraosyl
3

unit) would be susceptible to hydrolysis by fungal α-amylases and thus would make the substrate
less specific for the assay of pullulananse activity in enzyme mixtures.
The 2-chloro-4-nitrophenyl- and 4-methylumbelliferyl- derivatives of 63-α-D-maltotriosyl
maltotriose (11) were prepared from oligosaccharide 11 following the procedure reported by
Planas et al.20 The 4,6-O-benzylidene protecting group was then introduced to prepare substrates
1 and 2 respectively as previously described for cello-oligosaccharides.17 The principle of the
assay procedures for pullulanase using 1 and 2 is shown in Figure 1. On hydrolysis of the
oligosaccharide substrates 1 or 2 at the 1,6-α-linkage, the released 2-chloro-4-nitrophenyl-β-
maltotrioside or 4-methylumbelliferyl-β-maltotrioside respectively are immediately hydrolysed
to glucose and either 2-chloro-4-nitrophenol or 4-methylumbelliferone by the concerted action of
the α-glucosidase and β-glucosidase enzymes in the reagent mixture. Reaction is terminated and
phenolate ions are developed by addition of dilute alkali.
Unlike the substrate described by Bøjstrup et al.18 these 4,6-O-benzylidine end-blocked
substrates are absolutely resistant to all known exo-acting α-glucosidases and α-glucanases, such
as AMG and β-amylase. Incubation of 1 under standard assay conditions with 200 Units of
Bacillus stearothermophilis α-glucosidase, Rhizopus sp. AMG or barley β-amylase for up to 4 h
gave no hydrolysis. In contrast, substrate 12 as used in the pullulanase assay procedure described
by Bøjstrup et al.18 is rapidly hydrolysed by AMG, and is even hydrolysed (albeit slowly) by the
B. stearothermophilis α-glucosidase which is used in the reagent mixture. At a concentration of
1 mM and a pH of 5.5, the terminal non-reducing end 1,6-α-linked D-glucosyl residue in 63-α-D-
glucosyl 63-α-D-maltotriosyl-maltotriose (13) is hydrolysed by Bacillus stearothermophilis α-
glucosidase at 2% of the rate of hydrolysis of the non-reducing end 1,4-α-linked D-glucosyl
residue in maltoheptaose. This observation introduces an instability issue into the substrate
mixture used by Bøjstrup et al.,18 meaning that the ancillary enzyme α-glucosidase must be
added to the reagent mixture immediately at the time of performing the assay. Since β-
glucosidase can only hydrolyse the substrate after removal of α-linked D-glucosyl residues,
substrate 12 is stable in the presence of this enzyme. A lag phase was reported in the release of 2-
chloro-4-nitrophenol from 12 by Bojstrup et al.18 This was attributed to inadequate levels of the
ancillary enzymes in the reagent mixture. With an unblocked substrate such as 12 the level of α-
4

glucosidase added to the reagent mixture must be balanced by the rate of hydrolysis of 12 by this
enzyme.
In enzyme-linked assays, the optimal assay conditions for the enzyme of interest
(pullulanase) must be matched by the properties of the ancillary enzymes, with particular
reference to the pH activity profiles of these enzymes. The pH activity profiles for B.
stearothermophilis α-glucosidase and T. maritima β-glucosidase are shown in Figure 3 and those
for pullulanase from K. planticola and B. licheniformis are shown in Figures 4a and 4b. K.
planticola pullulanase has optimal pH for activity on reduced pullulan at 5.0-5.5, while B.
licheniformis pullulanase shows optimal activity (on reduced pullulan) at pH 4.5-5.0. T. maritima
β-glucosidase shows over 65% of maximal activity over the pH range 4.5-7.0, but B.
stearothermophilis α-glucosidase has just 20% of maximal activity at pH 4.5 and just 42% at pH
5.0. Clearly, this mixture of α-glucosidase and β-glucosidase cannot be used in assays at pH
values below 5.0, and even at pH 5.0, high levels of α-glucosidase are required to ensure instant
hydrolysis of the 2-chloro-4-nitrophenyl β-maltotrioside released by pullulanase hydrolysis of 1
or 2. Since many of the pullulanases described in literature have a pH optimum at 5.0, we
decided to perform the assays at pH 5.0.
The concentration of β-glucosidase required to optimise the sensitivity of hydrolysis of 1
by pullulanase was determined by using K. planticola pullulanase and assay solutions containing
substrate 1 (2 mM), 24 U/mL of B. stearothermophilis α-glucosidase and 0.7-6.5 U/mL of T.
maritima β-glucosidase (Figure 5). The concentration of α-glucosidase required to optimise the
sensitivity of hydrolysis of 1 by pullulanase was determined using assay solutions containing 1 (2
mM), 6.5 U/mL of T. maritima β-glucosidase and 2.4-24 U/mL of B. stearothermophilis α-
glucosidase (Figure 6). An aliquot of substrate solutions (0.1 mL) was incubated with
pullulanase (13.6 mU on this substrate) at 40 oC and the reaction terminated after 0-9 min by the
addition of 1.5 mL of 2% Tris solution (pH 10.0). β-Glucosidase at a concentration of 0.39
U/assay (3.9 U/mL) was sufficient to give maximal colour-release on hydrolysis of the substrate
(Figure 5), but a level of 0.65 U/assay was routinely used to allow for possible loss of enzyme
activity on storage of the substrate over longer times. For α-glucosidase, a higher concentration
of 2.4 U/assay (24 U/mL) was required to give maximal colour-release on hydrolysis of substrate
1 (Figure 6) because pH 5.0 is a sub-optimal pH for this particular enzyme. The activities of the
5

α-glucosidase and β-glucosidase stated here are those measured at the optimal pH for these
enzymes (pH 6.5 for both).
Under the assay conditions employed, the effect of substrate concentration was
determined by incubating 0.1 mL of substrate mixture containing 1 (0.2 to 4.0 mM), β-
glucosidase (at 0.65 U/assay) and α-glucosidase (at 2.4 U/assay) with 0.1 mL of buffered
solution containing either K. planticola (7.1 mU/assay on this substrate; 40 mU/assay on reduced
pullulan) or B. licheniformis pullulanase (14.5 mU/assay on this substrate; 74 mU/assay on
reduced pullulan). The reaction was terminated after fixed time intervals by the addition of 1.5
mL of 2 % Tris solution (pH 9.0) (Figure 7a and 7b). The maximum rate of hydrolysis for K.
planticola pullulananse was obtained at a concentration of substrate 1 of approximately 0.5 mM
in the assay mixture. Above this value there was slight substrate inhibition.. For B. licheniformis
pullulanase maximal rate was achieved at a concentration of substrate 1 of 1.2 mM, but there was
no apparent inhibition at concentrations up to 2.0 mM. In the final assay mixture, a concentration
of 2.0 mM substrate 1 was chosen to ensure a linear reaction range for the assay up to 1.0
absorbance units. The Michaelis-Menten curves for action of these two pullulanases on 1 are
shown in Figure 8a and 8b. K. planticola pullulanase had a lower Km value (0.032 mM) than B.
licheniformis pullulanase (0.124 mM). The value obtained for K. planticola pullulanase (0.032
mM) is much lower than the value of 0.146 mM found by Bøjstrup et al.18 using substrate 12.
Standard curves relating absorbance increase at 400 nm to hydrolysis of 1 by K.
planticola and B. licheniformis pullulanases standardised on borohydride-reduced pullulan
(Nelson/Somogyi assay) are shown in Figure 9. It is evident that the slopes of the curves are
dissimilar, indicating some difference in the ability of the two enzymes in hydrolysing the
modified hexasaccharide relative to the pullulan polysaccharide. The reason for this difference is
not clear, but may relate to interference of the chromogen or end-blocking group in the ability of
the enzyme to accommodate substrate 1 in the active site. Equally, it could be due to differences
in transglycosylation activities between the two enzymes. Indeed, a related enzyme, barley malt
limit-dextrinase, has been shown to have strong transglycosylation properties.21
In some applications, highly sensitive substrates and assay procedures are required. It is
well known that in assays such as those described here, fluorometric substrates give much higher
sensitivity than colourimetric ones based on nitrophenyl- or chloronitrophenyl- chromogen
groups. We have thus synthesised and evaluated compound 2 as a substrate for pullulanase. The
6

levels of α-glucosidase and β-glucosidase to ensure maximum sensitivity of the assay were, as
expected, the same as those required in the assay employing compound 1.
Under the assay conditions employed, the effect of substrate concentration was
determined by incubating 0.1 mL of substrate mixture containing substrate 2 (0.1 to 1.0 mM), β-
glucosidase (0.65 U/assay, i.e. per 0.1 mL) and α-glucosidase (2.4 U/assay, i.e. per 0.1 mL) with
0.1 mL of buffered solution containing K. planticola (1.4 mU/assay on this substrate, 6.6
mU/assay on reduced pullulan) or B. licheniformis pullulanase (1.8 mU/assay on this substrate,
9.1 mU/assay on reduced pullulan) and the reaction was terminated after fixed time intervals by
the addition of 3.0 mL of 2% Tris solution (pH 10.0) (Figure 10a and 10b). Michaelis-Menten
plots (Figure 11a and 11b) revealed Km values of 0.057 mM and 0.063 mM for K. planticola and
B. licheniformis pullulanases respectively. At substrate concentrations above 0.2 mM in the
assay mixture, K. planticola pullulanase showed some substrate inhibition. Fluorescence values
were determined with a portable fluorimeter, Quantifluor® ST, from Promega which gave very
stable and reproducible fluorescence readings in final sample volumes as low as 2.0 mL in the
fluorimeter tube. A standard curve for 4-methylumbelliferone in 2% Tris solution (pH 10.0) is
shown in Figure 12. The curve is linear up to a meter value of 1000 and allows direct conversion
of fluorescence values to micromoles of 4-methylumbelliferone. Standard curves relating
fluorescence increase to the level of either K. planticola or B. licheniformis pullulanases are
shown in Figure 13.
The stability of the reagent mixture containing 1, thermostable β−glucosidase (6.5 U/mL)
and thermostable α−glucosidase (24 U/mL) at -20oC, 4oC and 20oC is shown in Figure S1
(Supplementary Information). Similar stability studies were performed with reagent mixtures
containing substrate 2. On storage of the reagent mixtures for up to 66 days, 0.1 mL aliquots
were removed and the blank absorbance was determined as well as the absorbance on incubation
of the substrate with a set amount of K. planticola pullulanase (13.8 mU on substrate 1) at 40oC
for 10 min. With reagent mixtures stored at 20oC there was a significant increase in the blank
absorbance value, albeit only after 65 days. The analogous studies on substrate 2 using K.
planticola pullulanase (7.4 mU on reduced pullulan) showed a similar stability of the substrate
(results not shown). The increase in reaction absorbance value parallels the increase in the blank
value, such that on subtracting the blank value from the reaction value, the determined
absorbance value was very similar to those obtained on storage at -20oC and 4oC. This
7

demonstrates that the α-glucosidase and β-glucosidase in the reagent mixture are stable even on
storage at 4oC for 50 days. For much longer storage times, a storage temperature of -20oC is
recommended. These studies would indicate that the prepared substrate should be stable for > 2
years at -20oC.
For screening purposes in microplate format, the volumes of reagent mixture containing 2
and of the solution being assayed for pullulanase activity is reduced to 20 µL and the sensitivity
is increased by reducing the volume of Tris solution used to terminate the reaction and to adjust
the final pH, to 50 µL.
In this paper, the synthesis of two new substrates for the specific assay of pullulanase,
namely BzCNPG3G3 (1) and BzMUG3G3 (2), has been described and assay formats have been
suggested. Traditionally, pullulanase has been assayed with pullulan and more recently with
borohydride-reduced pullulan using a reducing sugar method such as the Nelson/Somogyi
procedure.12,13 Dyed pullulan-based substrates were introduced to simplify the assay of
pullulanase (and barley malt limit-dextrinase) in crude microbial fermentation broths and in grain
and malted grain extracts where high levels of sugars preclude the use of reducing sugar
methods. The most commonly used dyed substrates are Red Pullulan14 (a soluble, dyed pullulan)
and AZCL-Pullulan (a dyed and crosslinked, insoluble pullulan substrate). These dyed substrates
have found widespread application since their introduction approximately 20 years ago. AZCL-
Pullulan is most conveniently used in a tablet form (Limit Dextrizyme tablets).14 For comparison
purposes, the two pullulanases used in this study have been standardised on Red Pullulan and
Limit Dextrizyme tablets and the results are shown in Figures 14 and 15. The new substrates
described here (BzCNPG3G3, 1 and BzMUG3G3, 2) are completely specific for the assay of
pullulanase in the presence of all other hydrolytic enzymes studied. They are simple to use,
extremely stable and are readily incorporated into automated formats. The major limitation in the
introduction of these are the difficulties associated with the production of pure oligosaccharide
11 and the low overall yields for the synthesis of the colourimetric and fluorometric derivatives 1
and 2.
3. Experimental
8

3.1 Materials
Thermostable β-glucosidase (Thermotoga maritima; Accession number Q08638, Megazyme Cat.
No. E-BGOSTM), thermostable α-Glucosidase (Bacillus stearothermophilus; Accession number
P94451, Megazyme Cat. No. E-TSAGS), Pullulanase M1 (Klebsiella planticola; Megazyme Cat.
No. E-PULKP) and Pullulanase M2 (Bacillus licheniformis; Megazyme Cat. No. E-PULBL),
Barley β-amylase (Megazyme Cat. No. E-BARBL100), Amyloglucosidase (Rhizopus sp.;
Megazyme Catalogue No. E-AMGPU), 63-α-D-maltotriosyl maltotriose (Megazyme Cat. No. O-
MTMT) and Glucose Test Kit (Megazyme Cat. No. K-GLUC) were obtained from Megazyme
International Ireland. All other chemicals used in organic synthesis were purchased from Sigma
Aldrich, Lennox Laboratory Supplies, Fischer Scientific Ireland or Apollo Scientific and were
analytical reagent grade. A Bruker Avance 400 was employed for 1H (400.13 MHz) and 13C
(100.61 MHz) NMR spectra. Resonances δ, are in ppm units downfield from an internal
reference in C2D6SO (δH = 2.50). Mass spectrometry analysis was performed with a Q-Tof
Premier Waters Maldi-quadrupole time-of-flight (Q-Tof) mass spectrometer equipped with Z-
spray electrospray ionization (ESI) and matrix assisted laser desorption ionisation (MALDI)
sources. Silica gel Florisil (200 mesh; Aldrich) was used for column chromatography. Analytical
thin-layer chromatography was performed using Merck 60 F254 silica gel (pre-coated sheets, 0.2
mm thick, 20 cm x 20 cm) and visualised by UV irradiation or 5% H2SO4/EtOH staining.
3.2 Colourimetric substrate synthesis
3.2.1 2-Chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-maltotrioside and 4-
methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside synthesis
The 2-chloro-4-nitrophenyl and 4-methylumbelliferyl derivatives of 11 were synthesised
according to the general methodology employed by Planas et al.20 These compounds were not
purified following global deacetylation but were instead carried forward into the benzylidene
acetal formation reaction as crude isolates (~75% w/w purity).
3.2.2 4,6-O-Benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl-α-(1,6)-maltotrioside
(BzCNPG3G3, 1)
9

To a solution of 2-chloro-4-nitrophenyl-β-maltotriosyl-α-(1,6)-maltotrioside (1 g, 0.87
mmol) and p-toluenesulfonic acid monohydrate (43 mg, 0.22 mmol) in anhydrous
dimethylformamide (10 mL) containing activated 4Å molecular sieves (200 mg), was added
benzaldehyde dimethylacetal (1.05 mL, 6.98 mmol) over 1 hour via syringe. The reaction was
stirred for 14 hours under an argon atmosphere at 50ºC. After this time, triethylamine (43 µL,
0.31 mmol) was added and the reaction cooled to room temperature. The crude reaction mixture
was absorbed onto silica gel and purified by flash chromatography to afford BzCNPG3G3 (1) as a
pale yellow solid (215 mg, 0.17 mmol) in 6% overall yield from maltotriosyl-maltotriose.
mp (H2O) 196-199ºC (dec.); 1H NMR (400MHz, C2D6SO) δ 3.03-3.16 (m, 1H), 3.21-3.82 (m,
33H), 3.87-3.99 (m, 1H), 4.08-4.19 (m, 1H), 4.52 (t, J = 5.52 Hz, 1H), 4.64 (t, J = 5.27 Hz, 1H),
4.67-4.73 (m, 2H), 4.81 (t, J = 5.52 Hz, 1H), 5.00 (d, J = 3.77 Hz, 1H (anom)), 5.02-5.13 (m,
5H), 5.14 (d, J = 3.77 Hz, 1H (anom)), 5.32 (d, J = 7.78 Hz, 1H (anom)), 5.35 (d, J = 5.02 Hz,
1H), 5.43 (d, J = 3.02 Hz, 1H), 5.47 (d, J = 2.51 Hz, 1H), 5.51 (d, J = 3.26 Hz, 1H), 5.59 (s, 1H),
5.60-5.75 (m, 6H), 7.35-7.43 (m, 3H), 7.43-7.51 (m, 3H), 8.23 (dd, J = 9.29, 2.76 Hz, 1H), 8.35
(d, J = 2.76 Hz, 1H); 13C NMR (100MHz, C2D6SO) δ 60.5, 60.8, 60.8, 61.3, 63.8, 67.5, 68.5,
70.5, 70.6, 71.2, 72.1, 72.2, 72.2, 72.3, 72.6, 72.9, 72.9, 73.1, 73.5, 73.5, 73.7, 73.8, 74.0, 76.0,
76.5, 79.4, 79.8, 80.6, 81.4, 81.4, 99.2, 100.1, 100.9, 101.2, 101.3, 101.7, 101.9, 116.1, 122.7,
124.7, 126.1, 126.9, 126.9, 128.5, 128.5, 129.4, 138.2, 142.1, 158.1; HRMS MALDI+ [M+Na]+
Calc. 1256.3260, Found 1256.3219.
3.2.3 4,6-O-Benzylidene-4-methylumbelliferyl-β-maltotriosyl-α-(1,6)-maltotrioside
(BzMUG3G3, 2)
To a solution of 4-methylumbelliferyl-β-maltotriosyl-α-(1,6)-maltotrioside (1 g, 0.87
mmol) and p-toluenesulfonic acid monohydrate (43 mg, 0.22 mmol) in anhydrous
dimethylformamide (10 mL) containing activated 4Å molecular sieves (200 mg), was added
benzaldehyde dimethylacetal (1.05 mL, 6.98 mmol) over 1 hour via syringe. The reaction was
stirred for 14 hours under an argon atmosphere at 50ºC. After this time, triethylamine (43 µL,
0.31 mmol) was added and the reaction cooled to room temperature. The crude reaction mixture
was absorbed onto silica gel and purified by flash chromatography to afford BzMUG3G3 (2) as a
pale yellow solid (167 mg, 0.14 mmol) in 5% overall yield from maltotriosyl-maltotriose.
10

mp (H2O) 230-235 ºC (dec.); 1H NMR (400MHz, C2D6SO) δ 2.39 (d, J = 1.25 Hz, 3H), 3.01-
3.11 (m, 1H), 3.17-3.77 (m, 34H), 3.84-3.94 (m, 1H), 4.06-4.16 (m, 1H), 4.49 (t, J = 6.02 Hz,
1H), 4.63, (t, J = 5.02 Hz, 1H), 4.66 (d, J = 3.51 Hz, 1H (anom)), 4.68 (t, J = 5.27 Hz, 1H), 4.98
(d, J = 3.76 Hz, 1H (anom)), 5.02 (d, J = 3.77 Hz, 1H (anom)), 5.03-5.20 (m, 6H)), 5.30-5.38
(m, 1H), 5.41-5.79 (m, 10H), 6.24 (d, J = 1.00 Hz, 1H), 6.99-7.06 (m, 2H), 7.32-7.40 (m, 3H),
7.40-7.47 (m, 2H), 7.66-7.73 (m, 1H); 13C NMR (100MHz, C2D6SO) δ 18.7, 60.7, 60.79, 60.82,
61.4, 63.8, 67.5, 68.5, 70.49, 70.54, 71.2, 72.09, 72.12, 72.2, 72.3, 72.6, 72.8, 72.9, 73.2, 73.5,
73.6, 73.7, 73.8, 74.0, 75.8, 76.5, 79.7, 79.8, 80.5, 81.4, 81.4, 99.1, 100.1, 100.8, 101.1, 101.3,
101.6, 101.8, 103.6, 112.2, 113.7, 114.6, 126.8, 126.8, 126.9, 128.5, 128.5, 129.3, 138.3, 153.9,
154.9, 160.5, 160.6; HRMS ESI+ [M+Na]+ Calc. 1259.3854, Found 1259.3854.
3.3
Dissolution of colourimetric and fluorometric oligosaccharide substrates
BzCNPG3G3 (1) and BzMUG3G3 (2)
Compound 1 was dissolved at a concentration of 12.4 mM in dimethylsulfoxide and
stored at -20oC between use. In this form 1 is stable for > 4 years. For use as a substrate, 1.0 mL
of this solution was added to 2.0 mL of distilled water and mixed thoroughly. Thermostable β-
glucosidase (50 µL, 3.2 M (NH4)2SO4, 400 U/mL) and α-glucosidase (50 µL, 3.2 M (NH4)2SO4,
1500 U/mL) was then added, mixed thoroughly and stored at -20ºC between use (final
concentration of substrate 1 (4 mM). In this form, the substrate is stable for > 2 years.
Compound 2 was dissolved at a concentration of 3.1 mM in dimethylsulfoxide and
stored at -20oC between use. In this form 2 is stable for > 4 years. For use as a substrate, 1.0 mL
of this solution was added to 2.0 mL of distilled water and mixed thoroughly. Thermostable β-
glucosidase (50 µL, 3.2 M (NH4)2SO4, 400 U/mL) and α-glucosidase (50 µL, 3.2 M (NH4)2SO4,
1500 U/mL) was then added, mixed thoroughly and stored at -20ºC between use (final
concentration of substrate 2 (1.0 mM). In this form, the substrate is stable for > 2 years.
3.4 Standard assay of pullulanase using BzCNPG3G3 (substrate 1) (4 mM) in the presence
of β-glucosidase (6.5 U/mL) and α-glucosidase (24 U/mL)
11

A pre-equilibrated aliquot of pullulanase (0-40 mU on reduced pullulan) in 200 mM
sodium acetate buffer, pH 5.0 containing 0.5 mg/mL of BSA (0.1 mL) was added to a pre-
equilibrated aliquot of substrate 1 solution (0.1 mL, 4 mM) containing thermostable β-
glucosidase (0.65 U) and thermostable α-glucosidase (2.4 U) and the mixture was incubated at
40oC for exactly 10 min. The reaction was terminated by adding 1.5 mL of 2% Tris (pH 10.0).
The tube contents were well mixed and the absorbance measured at 400 nm against a reagent
blank. One Unit of activity is the amount of enzyme required to release one micromole of 2-
chloro-4-nitrophenolate per minute at 40oC and pH 5.0.
3.5
Standard assay of pullulanase using BzMUG3G3 (substrate 2) (1 mM) in the
presence of β-glucosidase (6.5 U/mL) and α-glucosidase (24 U/mL)
A pre-equilibrated aliquot of pullulanase (0-6 mU on borohydride-reduced pullulan) in
200 mM sodium acetate buffer, pH 5.0 containing 0.5 mg/mL of BSA (0.1 mL) was added to a
pre-equilibrated aliquot of substrate 2 solution (0.1 mL, 1 mM) containing thermostable β-
glucosidase (0.65 U) and thermostable α-glucosidase (2.4 U) and the mixture was incubated at
40oC for exactly 10 min. The reaction was terminated by adding 3.0 mL of 2% Tris (pH 10.0).
The tube contents were well mixed and the fluorescence measured with a portable fluorimeter,
Quantifluor ST, from Promega, U.K.
3.6
Standard assay of pullulanase using borohydride reduced pullulan (10 mg/mL) by
reducing sugar determination.
A pre-equilibrated aliquot of pullulanase (0.2 mL; 0-100 mU on borohydride-reduced
pullulan) in 100 mM sodium acetate buffer, pH 5.0 containing 0.5 mg/mL of BSA (0.1 mL) was
added to a pre-equilibrated aliquot of borohydride-reduced pullulan (Lot 100902, 0.5 mL, 10
mg/mL) in 100 mM sodium acetate buffer (pH 5.0). The resulting mixture was agitated on a
vortex stirrer and incubated at 40oC. Incubations were terminated by the addition of Nelson–
Somogyi solution C12,13 (0.5 mL) after 0, 3, 6, 9 and 12 min. The colour was developed
according to the Nelson–Somogyi procedure.12,13 The contents of tubes were stirred vigorously
12

and the absorbance was measured at 520 nm against a substrate/reagent blank and a glucose
standard solution (50 µg) was included. One Unit of activity is the amount of enzyme required to
release one micromole of glucose reducing sugar equivalents per minute at 40oC and pH 5.0.
3.7
Determination of the pH activity profiles of α-glucosidase, β-glucosidase and
pullulanases.
To determine the effect of pH on the activity of α-glucosidase and β-glucosidase on 4-
nitrophenyl α-glucoside or 4-nitrophenyl β-glucoside respectively, the substrates were prepared
at a concentration of 10 mM in 100 mM buffer solutions over a range of pH values; citrate-
phosphate (pH 4.0-7.0), phosphate (pH 7.0-9.0), acetate (pH 4.5-5.0) or maleate (pH 5.5-6.5). A
pre-equilibrated aliquot of the enzyme (0.1 mL, 140 mU) in 100 mM KCl containing 0.5 mg/mL
BSA was added to a pre-equilibrated aliquot of substrate solution (0.1 mL, 10 mM) at the various
pH values and the mixtures were incubated at 40oC for exactly 10 min. The reactions were
terminated by adding 3.0 mL of 2% Tris (pH 10.0). To determine the effect of pH on the activity
of pullulanase on borohydride-reduced pullulan, a pre-equilibrated aliquot of pullulanase (0.2
mL; 60 mU on borohydride-reduced pullulan) in 100 mM KCl containing 0.5 mg/mL of BSA
was added to a pre-equilibrated aliquot of borohydride-reduced pullulan (Lot 100902, 0.5 mL,10
mg/mL) in 100 mM sodium acetate buffer (pH 4.5 or 5.0) or sodium maleate buffer (pH 5.0, 5.5,
6.0 or 6.5). The resulting mixture was agitated on a vortex stirrer and incubated at 40oC.
Incubations were terminated by the addition of Nelson–Somogyi solution C12,13 (0.5 mL) after 0,
3, 6, 9 and 12 min. The colour was developed according to the Nelson–Somogyi procedure.12,13
The contents of tubes were stirred vigorously and the absorbance was measured at 520 nm
against a substrate/reagent blank and a glucose standard solution (50 µg) was included. One Unit
of activity is the amount of enzyme required to release one micromole of glucose reducing sugar
equivalents per minute at 40oC and pH 5.0.
3.8
Assay of pullulanase on BzCNPG3G3 (1) and BzMUG3G3 (2).
To determine the effect of substrate concentration (Figure 8) on the rate of hydrolysis of 1 by
13

Klebsiella planticola and Bacillus licheniformis pullulanase, 0.1 mL of 1 (0.2–4.0 mM) in the
presence of thermostable β-glucosidase (0.65 U) and thermostable α-glucosidase (2.4 U), was
incubated with either 0.1 mL of K. planticola pullulanase (7.1 mU on this substrate; 40 mU on
reduced pullulan) or B. licheniformis pullulanase (14.5 mU on this substrate; 74 mU on reduced
pullulan) in 100 mM sodium acetate buffer (pH 5.0) at 40oC. The reaction was terminated after
fixed time intervals by adding 1.5 mL of 2% Tris solution (pH 9.0) and absorbance measured at
400 nm. To determine the effect of substrate concentration (Figure 10) on the rate of hydrolysis
of 2 by K. planticola and B. licheniformis pullulanase, 0.1 mL of 2 (0.05–1 mM) in the presence
of thermostable β-glucosidase (0.65 U) and thermostable α-glucosidase (2.4 U), was incubated
with 0.1 mL of K. planticola pullulanase (1.39 mU on this substrate, 6.6 mU on reduced
pullulan) or B. licheniformis pullulanase (1.8 mU on this substrate, 9.1 mU on reduced pullulan)
in 100 mM sodium acetate buffer (pH 5.0) at 40oC. Reaction was terminated after fixed time
intervals by adding 3.0 mL of 2% Tris solution (pH 9.0) and the fluorescence measured with a
portable fluorimeter, Quantifluor® ST, from Promega, U.K. The effect of β-glucosidase
concentration on the rate of increase in absorbance at 400 nm was determined by preparing
solutions containing 1 (4 mM), α-glucosidase (24 U/mL) and β-glucosidase (0.7-6.5 U/mL) and
was determined as follows (Figure 6): aliquots (0.1 mL) of the solutions were incubated with K.
planticola pullulanase (13.6 mU on this substrate) at 40oC and reaction terminated after 0, 3, 6
and 9 min by adding 1.5 mL of 2% Tris solution (pH 9.0). The effect of α-glucosidase
concentration on the rate of increase in absorbance at 400 nm was determined by preparing
solutions containing 1 (4 mM), β-glucosidase (6.5 U/mL) and α-glucosidase (2.4 - 24 U/mL) and
was determined as follows (Figure 7): aliquots (0.1 mL) of the solutions were incubated with K.
planticola pullulanase (13.6 mU on this substrate) at 40oC and reaction terminated after 0, 3, 6
and 9 min by adding 1.5 mL of 2% Tris solution (pH 9.0). The effect of α-glucosidase and β-
glucosidase concentrations on the rate of increase in fluorescence on hydrolysis of 2 was
determined by preparing solutions containing 2 (1 mM) , β-glucosidase and α-glucosidase
mixtures as described for studies on substrate 1. The fluorescence was measured with a
Quantifluor® ST portable fluorimeter (Promega, U.K).
Standard curves for the action of K. planticola and B. licheniformis pullulanases on 1
(Figure 10) were determined by incubating 1 (4 mM, containing β-glucosidase at 6.5 U/mL and
14

α-glucosidase at 24 U/mL) with 0.1 mL of K. planticola and B. licheniformis pullulanases (0-40
mU/assay on borohydride-reduced pullulan in pH 5.0, 100 mM sodium acetate buffer containing
0.5 mg/mL BSA) at 40oC for 10 min. The reaction was terminated by the addition of 1.5 mL of
2% Tris solution (pH 10.0). The tube contents were well mixed and the absorbance measured at
400 nm against a reagent blank. Standard curves for the action of K. planticola and B.
licheniformis pullulanases on 2 (Figure 14) were determined by incubating 2 (1 mM, containing
β-glucosidase at 6.5 U/mL and α-glucosidase at 24 U/mL) with 0.1 mL of K. planticola and B.
licheniformis pullulanases (0-6 mU/assay on borohydride-reduced pullulan in pH 5.0, 100 mM
sodium acetate buffer containing 0.5 mg/mL BSA) at 40oC for 10 min. The reaction was
terminated by the addition of 3.0 mL of 2% Tris solution (pH 10.0). The tube contents were well
mixed and the fluorescence was measured with a Quantifluor® ST portable fluorimeter
(Promega, U.K).
Long term stability of compound 1 in the presence of β-glucosidase at 6.5 U/mL and α-
glucosidase at 24 U/mL) was determined by storing the substrate mixture at -20oC, 4oC and 20oC
for up to 66 days. After the various time intervals, stability was determined by running a standard
assay by mixing 0.1 mL of the substrate mixture with 0.1 mL of K. planticola pullulanase (6.8
mU on substrate 1 mixture) for 10 min at 40 oC and stopping the reaction with 1.5 mL of 2% Tris
solution (pH 10.0) (Figure S1 - Supplementary Information). Changes in blank absorbance
values were determined by adding 1.5 mL of 2 % Tris solution (pH 10.0) to 0.1 mL of the
substrate solution and 0.1 mL of the buffer solution, mixing well and measuring the absorbance
at 400 nm. Long term stability of compound 2 (in the presence of β-glucosidase at 6.5 U/mL and
α-glucosidase at 24 U/mL) was determined by storing the substrate mixture at -20oC, 4oC and
20oC for up to 66 days. After the various time intervals, stability was determined by running a
standard assay by mixing 0.1 mL of the substrate mixture with 0.1 mL of K. planticola
pullulanase (28 mU on reduced pullulan) for 10 min at 40 oC, stopping the reaction with 3.0 mL
of 2% Tris solution (pH 10.0) and measuring the fluorescence with a Quantifluor® ST portable
fluorimeter. Changes in blank absorbance values were determined by adding 3.0 mL of 2% Tris
solution (pH 10.0) to 0.1 mL of the substrate solution and 0.1 mL of the buffer solution and
mixing well. It appears clearly from the results shown in Figure S1 (Supplementary Information)
that mixtures containing 1 are very stable at 4oC and -20oC, but less stable at 20oC. On storage at
15

20oC, the absorbance of the blank solution of the substrate mixture containing 2 in the assay
(containing no enzyme) increased from 0.025 to 0.104 over 66 days (i.e., four-fold), but the
assay absorbance increased by the same amount such that the determined enzyme activity
remained unchanged. This increase in absorbance value represents a degradation of
approximately 3% of the substrate in the assay mixture. At -20oC and 4oC, the absorbance of the
blank solution in the assay increased by just 0.000 and 0.015 respectively, over the same period.
In the assay, the substrate behaved exactly as in the freshly prepared reagent solution. Similar
stability was observed with the substrate mixture containing compound 2.
3.9
Hydrolysis of 61-α-D-glucosyl 63-α-D-maltotriosyl maltotriose (13) and
maltoheptaose by α-glucosidase and amyloglucosidase
Pre-equilibrated aliquots of B. stearothermophilus α-glucosidase (0.1 mL; 1.0 U) or A.
niger amyloglucosidase (0.1 mL, 1.0 U) in 100 mM sodium maleate buffer, pH 5.0 containing
0.5 mg/mL of BSA was added to a pre-equilibrated aliquots of 61-α-D-glucosyl 63-α-D-
maltotriosyl maltotriose (13) or maltoheptaose in 100 mM sodium maleate buffer, pH 5.5 and the
mixture was incubated at 40oC. The reaction was terminated after 0, 5, 10, 20, 40 and 90 min by
incubating the tubes in a boiling water bath for 2 min. Free glucose was measured using glucose
oxidase/peroxidase reagent from a Glucose (GOPOD) test kit (Megazyme Cat. No. K-GLUC).
The relative rates of hydrolysis of these substrates by α-glucosidase was calculated from these
values. Under these conditions, AMG gave complete hydrolysis of both substrates within 20 min.
3.10 Hydrolysis of BzCNPG3G3 (1) and BzMUG3G3 (2) by α-glucosidase, β-amylase and
Rhizopus sp. amyloglucosidase
Hydrolysis of substrates 1 and 2 by exo-acting enzymes was determined using substrate
solutions containing 1 (4 mM), 24 U/mL of B. stearothermophilis α-glucosidase and 6.5 U/mL
of T. maritima β-glucosidase. Aliquots of substrate solution were incubated with B.
stearothermophilus α-glucosidase (0.1 mL; 20 U), A. niger amyloglucosidase (0.1 mL, 20 U) or
barley β-amylase (0.1 mL, 20 U) in 100 mM sodium maleate buffer, pH 5.0 containing 0.5
16

mg/mL of BSA. The tubes were incubated at 40oC and the reaction was terminated after 0-4 h by
the addition of either 1.5 mL (substrate 1) or 3.0 mL (substrate 2) of 2% Tris solution (pH 10.0).
3.11 Standard assay of pullulanase using Red Pullulan
A pre-equilibrated aliquot of pullulanase (1.0 mL; 0-400 mU on reduced pullulan) in 200
mM sodium acetate buffer, pH 5.0 containing 0.5 mg/mL of BSA was added to a pre-
equilibrated aliquot of Red Pullulan (Lot 100906, 0.5 mL, 20 mg/mL) in 500 mM KCl. The
resulting mixture was agitated on a vortex stirrer and incubated at 40oC. The incubations were
terminated after 10 min by the addition of 95/5 v/v ethanol/H2O (2.5 mL) which precipitated non-
hydrolysed, high-molecular weight Red Pullulan. The tube contents were stirred vigorously on a
vortex mixer and the tubes were allowed to equilibrate at room temperature for 10 min. The
tubes were again stirred and then centrifuged at 1,000 g for 10 min. The supernatant solutions
were then poured directly from the tubes into spectrophotometer cuvettes and the absorbance of
the blank and of the reaction solutions were measured at 510 nm against distilled water (Figure
14). Activity was determined by reference to a standard curve. A reagent blank was prepared by
replacing the enzyme preparation with 100 mM sodium acetate buffer (pH 5.0, containing 0.5
mg/mL BSA).
3.12 Standard assay of pullulanase using Limit Dextrizyme tablets
A Limit Dextrizyme tablet (Lot 121101) was added to a pre-equilibrated aliquot of
pullulanase (0.5 mL; 0-25 mU on reduced pullulan) in 100 mM sodium acetate buffer (pH 5.0
containing 0.5 mg/mL BSA). The tablet was allowed to swell without agitation. The suspension
was incubated at 40oC for 10 min and the reaction was terminated by the addition of 10 mL of
2% Tris solution (pH 10.0) with vigorous agitation on a vortex mixer and the tubes were left at
room temperature. After approximately 10 min the tube contents were agitated again on a vortex
mixer and the contents were filtered through Whatman No. 1 (9 cm) filter circles. The filtrates
were poured into spectrophotometer cuvettes and the absorbance of the blank and of the reaction
solutions were measured at 590 nm against distilled water (Figure 15). Activity was determined
17

by reference to a standard curve. A reagent blank was prepared by replacing the enzyme
preparation with 100 mM sodium acetate buffer (pH 5.0 containing 0.5 mg/mL BSA).
Acknowledgements:
The authors thank Ms. Ruth Ivory and Mr. Niall McCormack for invaluable technical support.
References
1. Bender, H.; Wallenfels, K. Meth. Enzymol. 1966, 8, 555–559.
2. Lee, E. Y. C.; Whelan, W. J. The Enzymes (3rd ed.). New York: Academic Press. 1972,
vol. 5, 191–234.
3. Banks, W.; Greenwood, C. T.; Starch and its components. Aberdeen University Press,
1975, 233-234.
4. Kim, C. H.; Nashiru, O.; Ko, J. H. FEMS Microbiol. Lett. 1996, 138, 147-152.
5. Roy, A.; Messaoud, E. B.; Bejar, S. Enzyme Microbiol. Technol. 2003, 33, 720-724.
6. Imanaka, T.; Kuriki, T. J. Bacteriol. 1989, 171, 369–374.
7. Sakano, Y.; Masuda, N.; Kobayashi, T. Agric. Biol. Chem. 1971, 35, 971–973.
8. Niehaus, F.; Peters, A.; Groudieva, T.; Antranikian, G. FEMS Microbiol. Lett. 2000,
190, 223-229.
9. Norman, B. E. Starch 1982, 34, 340-346.
10. Crabb, W. D.; Mitchinson, C. Trends Biotechnol. 1997, 15, 349-352.
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11. Shaw, J. F.; Sheu, J. R. Biosci. Biotechnol. Biochem. 1992, 56, 1071-1073.
12. Nelson, N. J. Biol. Chem. 1944, 153, 375-380.
13. Somogyi, M. J. Biol. Chem. 1952, 195, 19-23.
14. McCleary, B. V. Carbohydr. Res. 1992, 227, 257-268.
15. Blair, H. E. U.S. Patent 4 794 078, 1988.
16. McCleary, B. V.; Sheehan, H. J. Cereal Sci., 1987, 6, 237-251.
17. McCleary, B. V.; Mangan, D.; Daly, R.; Fort, S.; Ivory, R.; McCormack, N.
Carbohydr. Res. 2014, 385, 9-17.
18. Bøjstrup, M.; Christensen, C. O.; Windahl, M. W.; Henriksen, A.; Hindsgaul, O. Anal.
Biochem. 2014, 449, 45-51.
19. Catley, B. J. Carbohydr. Res. 1978, 61, 419-424.
20. Planas, A.; Abel, M.; Millet, Ó.; Palasí, J.; Pallarés, C.; Viladot, J.-L. Carbohydr. Res.,
1998, 310, 53-64.
21. McDougall, G. J.; Ross, H. A.; Swanston, J. S.; Davies, H. V. Planta, 2004, 218, 542-
551.
Figures
Figure 1. Overview of pullulanase assay procedure using BzCNPG3G3 (1) and BzMUG3G3 (2).
19

Figure 2. Pullulan-derived structures of interest.
Figure 3. pH activity profiles for α-glucosidase and β-glucosidase.
Figure 4. pH activity curves for (a) K. planticola and (b) B. licheniformis pullulanases on
borohydride-reduced pullulan (Lot 100901) at 40oC.
Figure 5. Effect of β-glucosidase concentration on reaction kinetics over 9 min in the presence of
K. planticola pullulanase (13.6 mU/assay) and BzCNPG3G3 (1) (2 mM) in pH 5.0 100 mM
sodium acetate at 40oC.
Figure 6. Effect of α-glucosidase concentration on reaction kinetics over 9 min in the presence of
K. planticola pullulanase (13.6 mU /assay) and BzCNPG3G3 (1) (2 mM) in pH 5.0 100 mM
sodium acetate at 40oC.
Figure 7. Effect of BzCNPG3G3 (1) concentration on reaction kinetics over 6 min in the
presence of (a) K. planticola pullulanase (7.1 mU/assay on this substrate, 40 mU/assay on
reduced pullulan) and (b) B. licheniformis pullulanase (14.5 mU/assay on this substrate, 74
mU/assay on reduced pullulan) plus β-glucosidase (0.65 mU/assay) and α-glucosidase (2.4
mU/assay) in pH 5.0 100 mM sodium acetate at 40oC.
Figure 8. Determination of the Michaelis-Menten Km constant for (a) K. planticola and (b) B.
licheniformis pullulanases on BzCNPG3G3 (1). Initial rates of reaction were determined as
described in Figure 7 and in the Experimental Section.
Figure 9. Standard curves relating absorbance increase at 400 nm observed with BzCNPG3G3 (1)
to level of K. planticola and B. licheniformis pullulanase activity on borohydride reduced
pullulan at 40oC in pH 5.0 100 mM sodium acetate.
20

Figure 10. Effect of BzMUG3G3 (2) concentration on reaction kinetics over 6 min in the presence
of (a) K. planticola pullulanase (1.39 mU/assay on this substrate, 6.6 mU/assay on reduced
pullulan) and (b) B. licheniformis pullulanase (1.8 mU/assay on this substrate, 9.1 mU/assay on
reduced pullulan) in the presence of β-glucosidase (0.65 mU/assay) and α-glucosidase (2.4
mU/assay) in pH 5.0 100 mM sodium acetate at 40oC.
Figure 11. Determination of the Michaelis-Menten Km constant for (a) K. planticola and (b) B.
licheniformis pullulanases on BzMUG3G3 (2). Initial rates of reaction were determined as
described in Figure 10 and in Experimental Section.
Figure 12. Standard curve relating fluorescence (arbitrary units) against concentration
(micromolar) of 4-methylumbelliferone in 2% Tris (pH 10.0).
Figure 13. Standard curves relating increase in fluorescence observed with BzMUG3G3 (2) to
level of K. planticola and B. licheniformis pullulanase activity on borohydride-reduced pullulan
(Lot 100901) at 40oC in pH 5.0 100 mM sodium acetate.
Figure 14. Standard curve relating the activity of pullulanase (mU/assay, i.e. /1.0 mL) to increase
of absorbance at 510 nm on hydrolysis of Red-Pullulan (Lot 100906). Incubations were
performed under standard conditions at pH 5.0 for 10 min at 40oC. Pullulanase activity
(mU/assay) was determined on borohydride-reduced pullulan (Lot 100901) using the Nelson-
Somogyi reducing-sugar assay.
Figure 15. Standard curve relating the activity of pullulanase (mU/assay, i.e. /0.5 mL) to increase
of absorbance at 590 nm on hydrolysis of Azurine-CL-Pullulan (in Limit-Dextrizyme tablets, Lot
121101). Incubations were performed under standard conditions at pH 5.0 for 10 min at 40oC.
Pullulanase (U/mL) was determined on borohydride reduced pullulan (Lot 100901) using the
Nelson-Somogyi reducing-sugar assay.
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