Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.
A diversity of starch-acting enzymes are applied in the food
industry.1,2 Most of them hydrolyze starches and belong to
glycosyl hydrolase (GH) family 13.3 Also, starch-acting 4-α-
glucanotransferase enzymes are known (found in families
GH13, GH77), mostly catalyzing disproportionation reactions
(e.g., amylomaltase,4,5 cyclodextrin glucanotransferase6), thus
modifying starches but largely maintaining their highmolecular-
mass properties.7,8 In recent years, we have
characterized various family GH70 α-glucanotransferases
from subfamilies GtfB, GtfC, and GtfD.9 These enzymes are
4,6- and 4,3-α-glucanotransferases that catalyze the cleavage of
(α1 → 4)-linkages in amylose/amylopectin and maltodextrins
and introduce glucose units with (α1 → 6)-10 or (α1 → 3)-
linkages,11 respectively. The carbohydrates made by these
enzymes from starch are soluble and have clearly reduced
digestibility, making these products of interest for food
applications.12−14
The well-characterized GtfB enzyme of Limosilactobacillus
reuteri 121 is the first representative of such a 4,6-α-
glucanotransferase.10 Following cleavage of (α1 → 4)-linkages
in amylose (from the nonreducing end of donor molecules),
GtfB introduces linear (α1 → 6)-glucan segments to the
nonreducing end of acceptor molecules. This results in the
synthesis of isomaltose/maltose polysaccharide (IMMP), a
linear α-glucan polymer.15,16 Similar to the L. reuteri 121 GtfB
enzyme, the GtfC 4,6-α-glucanotransferase from Exiguobacterium
sibiricum was found to cleave the (α1 → 4)-linkages of
starch-like substrates and synthesize consecutive (α1 → 6)-
linkages. The Euastrum sibiricum GtfC activity, however, results
in the synthesis of oligosaccharides (designated as isomaltomalto/
oligosaccharides, IMMOs), instead of polymeric material.
Later on, 4,6-α-glucanotransferases producing reuteranlike
polymers from amylose have been found in GtfB and GtfD
subfamilies. Unlike IMMP and IMMO, reuteran consists of
(α1 → 4)-glucan segments interconnected by single (α1 → 6)
linkages in linear or branched orientations.13,17−19
GtfB enzymes thus far have only been identified in
mesophilic lactic acid bacteria,9,13 whereas GtfD enzymes
were found in taxonomically diverse plant-associated bacteria.
9,18,19 Interestingly, database searches showed that the
related GtfC 4,6-α-glucanotransferase enzymes occur in Grampositive
bacteria of the genera Exiguobacterium,17 Bacillus,17
and Geobacillus,9 adapted to growth at more extreme (low and
high) temperatures. Enzymes of thermophilic bacteria may be
more thermostable, allowing starch conversions at relatively
high temperatures. One field of application for such more
thermostable enzymes would be bread baking. The only 4,6-α-
glucanotransferase that has been tested in bread baking is the
Streptococcus thermophilus GtfB, slightly improving textural
properties and increasing the fraction of slowly digestible
starch of bread.20 This S. thermophilus GtfB, however, has the
highest activity at 40 °C and rapidly lost activity at higher
temperatures,21 e.g., before starch gelatinization occurred.
More thermostable starch-acting GtfC enzymes also may
display clear antistaling properties in bread products.
Bread has been an important food for thousands of years,
not only because of the energy and nutrients it delivers but also
because of its pleasant aroma, taste, and mouthfeel.
Unfortunately, the freshness of bread is lost rapidly, in a
process called staling, limiting the shelf-life of bread. Staling
starts directly after baking and involves both chemical and
physical changes that negatively affect the taste, aroma, and
mouthfeel of bread.22,23 Immediately after baking, the starch is
gelatinous, but upon cooling and over time, it starts to
retrograde/recrystallize, reducing the elasticity and increasing
the hardness of the bread crumb, which is a major indicator of
bread staling. In gluten-free breads, the staling is even
considerably faster,24 further increasing the demand for
antistaling agents, such as sugars, emulsifiers, and fats.25 Also,
starch-acting enzymes capable of trimming the exterior side
chains of the amylopectin, thereby strongly reducing the
process of starch retrogradation, are widely used as antistaling
agents.23 It is key that these enzymes only trim the exterior side
chains, without destroying the high-molecular-mass property of
the amylopectin. Therefore, exo-acting starch hydrolases such
as maltogenic α-amylase are the preferred antistaling
enzymes.26,27 On top of that, the enzyme should be active
around and above the starch gelatinization temperature (∼65
°C) but, at the same time, be inactivated by the temperature
reached at the end of the baking process (nearly 100 °C) to
avoid the presence of an active enzyme in the final bread
product.
Geobacillus sp. 12AMOR1 is a thermophilic bacterium
isolated from a 90 °C hot deep-sea sediment sample.28 It
encodes a putative GtfC enzyme.4 Characterization of this
GtfC enzyme as thermostable 4,6-α-glucanotransferase showed
that it possesses a novel maltose-transferring product
specificity, and its activity results in clear antistaling properties
in bread bakery products.
Protein Sequence Analysis and Cloning of the Geobacillus
GtfC Gene. The nucleotide and protein sequence of the Geobacillus
sp. 12AMOR1 GtfC encoding gene and enzyme (AKM18207.1) were
retrieved from the NCBI database. Its
putative signal peptide-encoding sequence was predicted using the
Signal P5 server.
Searches for conserved domains were done by using the Pfam server
and by comparing them to the conserved domains of the GtfC protein
of E. sibiricum 255-15 (ACB62096.1). For this, the GtfC protein
sequences were aligned by MUSCLE in MEGA 729 using default
parameters and analyzed with Jalview 2.10.3b1.30 The DNA fragment
encoding domains A, B, C, and IV (amino acids 33−738) was
amplified by PCR (forward 5′-CAGGGACCCGGTTATAGCTCCGGCCCGGAATTG-
3′ and reverse 5′- CGAGGAGAAGCCCGGTTACGCCTTATCCTTCGTCGGCA-
3′) from
Geobacillus sp. 12AMOR1 (DSM 17290) genomic DNA (DSMZ,
Braunschweig, Germany) with Phusion DNA polymerase (Thermo
Fisher, The Netherlands) and cloned into a modified pET15b vector
by ligation-independent cloning (LIC). The construct (pET15b-gtfC
12AMOR1) was verified by nucleotide sequencing (GATC, Cologne,
Germany) and transformed into Escherichia coli BL21 Star (DE3).
GtfC Protein Expression and Purification. Luria broth (LB)
medium supplemented with ampicillin (100 mg/L) was inoculated
with 1% (v/v) of an overnight culture of E. coli BL21 (DE3) with the
pET15b-gtfC 12AMOR1 and grown at 37 °C under shaking (200
rpm). A pET15b vector carrying the gtf B gene of L. reuteri 121
(pET15b-gtf B-ΔN-ΔV encoding GtfB-ΔN-ΔV, residues 762−1619-
His6)31 and an empty pET15b vector (negative control) were
expressed and purified using the same conditions as for the Geobacillus
gtfC construct. Expression was induced by the addition of 0.1 mM
IPTG, and incubation was continued for 20 h at 18 °C, 160 rpm.
Cells were harvested by centrifugation at 4 °C at 10 875g for 15 min
and washed once with 20 mM Tris−HCl, pH 8 plus 1 mM CaCl2.
From the Geobacillus GtfC culture, negative control, and GtfB culture,
respectively, 8, 7, and 10 grams of wet cell pellet per liter LB were
obtained. Pellets were stored at −20 °C or immediately used for
protein isolation. To this end, pellets were resuspended in washing
buffer (50 mM Tris−HCl, pH 8; 250 mM NaCl; 1 mM CaCl2; 0.25%
(v/v) Triton X; 5 mM β-ME) and broken by sonication (Soniprep,
9.5 mm probe, 12 μm amplitude, 6 cycles of 30 s on and 60 s off).
During sonication, the cell suspensions were kept on ice. Cell lysates
were centrifuged at 4 °C at 17 226g for 15 min, and the cell-free
extracts (CFEs) were stored at 4 °C. To determine whether the
proteins were expressed successfully, the CFEs were analyzed by SDSPAGE
(Supporting Information Figure S1). The Geobacillus GtfC and
GtfB-ΔN-ΔV constructs were purified by Ni2+-nitrilotriacetic acid
(NTA) affinity chromatography.19 Proteins were desalted using an
Amicon size-exclusion column (30 kDa MWCO) and desalting buffer
(20 mM Tris−HCl, pH 8; 1 mM CaCl2) (Supporting Information
Figure S1). Protein concentrations were determined by the Bradford
assay or by measuring the absorbance at 280 nm using a NanoDrop
2000 spectrophotometer (Isogen Life Science, De Meern, The
Netherlands).
Standard Reaction Buffer. All enzymatic reactions were
performed at 40 °C in 25 mM sodium citrate buffer, pH 6.0,
containing 1 mM CaCl2 unless mentioned otherwise.
Enzyme Activity Assays to Determine pH and Temperature
Optima. The Geobacillus GtfC activity was determined by measuring
the initial rate in the presence of 0.125% (w/v) amylose V (Avebe,
The Netherlands) using the amylose−iodine staining method.17,31
Assays were performed with 50 μg/mL enzyme in a reaction buffer at
40 °C. The decrease in absorbance (660 nm) of the α-glucan−iodine
complex resulting from transglycosylation and/or hydrolytic activity
was monitored for the first 6.5 min. One unit of activity was defined as
the amount of amylose V (mg) converted by 1 mg of enzyme per
minute. The pH profile and optimum pH of the Geobacillus GtfC
were determined in a reaction buffer with different pH values between
4.0 and 7.5. The temperature optimum of Geobacillus GtfC was
determined with 50 μg/mL enzyme and 0.125% amylose V at varying
temperatures between 40 and 80 °C. The thermostability was
investigated by measuring the residual enzyme activity after
incubation at 60 °C for different time periods. To this end, 50 μg/
mL enzyme was incubated in a reaction buffer in the absence of
amylose V for 0, 10, 30, and 60 min at 60 °C and pH 6.0 and then
immediately cooled to 4 °C. The residual enzyme activity was
measured at 40 °C with 0.125% amylose V. All enzymatic assays were
performed in quadruple.
Differential Scanning Fluorimetry Assay. The melting temperature
of Geobacillus GtfC was determined using the ThermoFluor
method.32 A real-time-PCR thermocycler was used to denature the
protein and measure the fluorescence. The stock solution of SYPRO
Orange (Thermo Fisher, The Netherlands) was diluted 100 times; 5
μL of this solution was added to 45 μL of 0.5 mg/mL enzyme in 20
mM Tris−HCl, pH 8, plus 1 mM CaCl2 (in duplicate). The
temperature was increased from 20 to 99 °C with 0.5 °C increments.
After incubation for 10 s, the fluorescence of SYPRO Orange was
measured.
Synthesis and Isolation of Geobacillus GtfC Products. The
purified Geobacillus GtfC (40 μg/mL) was incubated with 25 mM
maltoheptaose (G7), 0.5% amylose V, or 1% amylopectin (Eliane 100,
Avebe, The Netherlands) for 24 h (amylopectin 48 h). Reactions
were stopped by heating at 95 °C for 10 min. Incubations of 25 mM
G7 or 0.5% amylose V with GtfB-ΔN-ΔV were done as described for
Geobacillus GtfC, with the exception that reactions were performed in
25 mM sodium acetate buffer at pH 5.0. The amylose V incubations
were dialyzed in snakeskin tubing (MWCO for proteins of 3.5 kDa;
ThermoScientific) for 48 h against running tap water and for 24 h
against 20 L MilliQ water, after which the dialyzed material was
lyophilized. The Geobacillus GtfC products from amylopectin were
analyzed by TLC and NMR spectroscopy without prior dialysis.
Geobacillus GtfC Product Analysis with Hydrolytic Enzymes.
The purified α-glucan polymer produced by Geobacillus GtfC was
dissolved at a concentration of 3 mg/mL in 50 mM sodium acetate
buffer, pH 5.0, and incubated separately with an excess of α-amylase
(Aspergillus oryzae; Megazyme), dextranase (Chaetomium erraticum;
Sigma-Aldrich), and pullulanase M1 (Klebsiella planticola; Megazyme)
for 24 h at 37 °C. Amylose V, dextran (MW 70 kDa; Sigma-Aldrich),
and pullulan (Megazyme) were used as positive controls for the α-
amylase, dextranase, and pullulanase treatments, respectively, resulting
in complete degradation under these conditions. The degree of
hydrolysis was examined by TLC analysis.
Thin-Layer Chromatography. For thin-layer chromatography
(TLC) analysis, samples were spotted in 1 cm lines on TLC sheets
(Merck Kieselgel 60 F254, 20 × 20 cm). Plates were developed with nbutanol/
acetic acid/water = 2:1:1 (v/v/v). Bands were visualized by
orcinol/sulfuric acid staining and compared with a maltooligosaccharide
standard from DP1 to DP7.
High-pH Anion-Exchange Chromatography. High-performance/
pH anion-exchange chromatography (HPAEC) was performed
on an ICS-3000 workstation (Dionex, Amsterdam, The Netherlands),
equipped with an ICS-3000 ED pulsed amperometric detection
system (PAD). Samples were diluted 1:100 in MilliQ water and
filtered through a 0.2 μm cellulose filter prior to injection (25 μL
injection volume). The oligosaccharides were separated on a
CarboPac PA-1 column (Dionex; 250 × 2 mm) by using an isocratic
gradient of 100 mM NaOH for 5 min, followed by a linear gradient of
0−270 mM sodium acetate in 100 mM NaOH over 30 min with a
flow of 0.25 mL/min. The gradient was followed by a cleaning step
and a re-equilibration step to the starting conditions of the analysis.
Commercial oligosaccharide standards were used to identify the
peaks.
High-Performance Size-Exclusion Chromatography. The
molecular mass distribution of the purified Geobacillus GtfC polymer
was determined by high-performance size-exclusion chromatography
(HPSEC), as described previously.11 The HPSEC system (Agilent
Technologies 1260 Infinity) was equipped with a multiangle laser
light scattering detector (SLD 7000 PSS, Mainz, Germany), a
viscometer (ETA-2010 PSS, Mainz), and a differential refractive index
detector (G1362A 1260 RID Agilent Technologies). Separation was
performed by using three PFG-SEC columns with porosities of 100,
300, and 4000 Å, coupled with a PFG guard column. DMSO-LiBr
(0.05 M) was used as an eluent at a flow rate of 0.5 mL/min. The
system was calibrated and validated using a standard pullulan kit
(PSS, Mainz, Germany), with Mw ranging from 342 to 708 000 Da.
The specific RI increment (dn/dc) value for pullulan was determined
as 0.072 mL/g (PSS, Germany). The dn/dc value for the GtfC
polysaccharide in this system was taken to be the same as for pullulan.
The molecular mass (MW) was determined by the universal
calibration method. WinGPC Unity software (PSS, Mainz) was
used for data processing.
Methylation Analysis. Samples (in triplicate) were permethylated
using CH3I and solid NaOH in (CH3)2SO, as described
previously,33 and then hydrolyzed with 2 M trifluoroacetic acid (2 h,
120 °C) to give the mixture of partially methylated monosaccharides.
After evaporation to dryness, the mixture was dissolved in H2O and
was reduced with NaBD4 (2 h, room temperature). Subsequently, the
solution was neutralized with 4 M acetic acid and boric acid was
removed by repeated coevaporation with methanol. The obtained
partially methylated alditol samples were acetylated with 1:1 (v/v)
acetic anhydride−pyridine (30 min, 120 °C). After evaporation to
dryness, the mixtures of partially methylated alditol acetates
(PMAAs), dissolved in dichloromethane, were analyzed by GLC-EIMS
on an EC-1 column (30 m × 0.25 mm; Alltech), using a GCMSQP2010
Plus instrument (Shimadzu Kratos Inc., Manchester, U.K.)
and a temperature gradient (140−250 °C at 8 °C/min).34
NMR Spectroscopy. The freeze-dried polymer sample was
exchanged twice in 500 μL of D2O (99.9 atom % D, Cambridge
Isotope Laboratories, Inc., Andover, MA) with intermediate
lyophilization and finally dissolved in 650 μL of D2O, spiked with
0.005% of acetone as an internal standard. One-dimensional (1D) 1H,
two-dimensional (2D) TOCSY (200 ms), and 2D natural abundance
1H−13C HSQC spectra (1H frequency of 600.13 MHz, 13C frequency
of 150.91 MHz) were recorded on a Bruker 600 MHz spectrometer
(NMR Center, University of Groningen) at a probe temperature of
300 K. The NMR data were processed using the MestReNova 12
program (Mestrelab Research SL, Santiago de Compostella, Spain).
Chemical shifts (δ) were expressed in ppm by reference to internal
acetone (δ 2.225 for 1H and δ 31.08 for 13C).
Gluten-Free Bread Baking. The dry ingredients (Supporting
Information Table S1), except the yeast, were mixed into a
homogeneous mixture by hand. The mixture was added to a bowl
containing the oil, and the contents were mixed for 1 min at gear 1 of
a Hobart mixer with a flat beater (Hobart N50 planetary mixer,
Hobart Corporation, Troy, Ohio). The yeast was added, and mixing
was continued for 1 additional min. Subsequently, the water of 39 °C,
with or without enzyme, was added while mixing for 2 min. Then, the
batter was kneaded by hand and mixed at gear 1 for 2 min and then 6
min at gear 2. Batter pieces of 300 g were placed in a baking tin and
incubated at 30 °C with a relative moisture content of 90% for 30 min
for proofing. Baking profile: 30 min at 235 °C upper and 245 °C of
bottom temperature, with two steam injections in advance.
Wheat Bread Baking. The dry ingredients (Supporting
Information Table S2), except the yeast, were mixed into a
homogeneous mixture using a Diosna spiral mixer (Diosna SP24,
Diosna Dierks & Söhne GmbH, Osnabrück, Germany). The yeast w
hair was added, and mixing was continued until a homogeneous
mixture was obtained. Subsequently, the water of 40 °C, with or
without enzyme, was added and the dough was mixed for 4 min at low
speed. Oil was added and mixed for 1 additional min. Then, the
dough was mixed for 8 min at high speed yielding a solid dough. The
dough was divided into pieces of 220 g and fermented at 26−28 °C
for 60 min with a relative moisture content of 80−90%. The gas was
pressed out of the dough, shaped, and fermented again under the
same conditions for 50 min. Baking profile: an initial temperature of
220 °C, a baking temperature of 200 °C for 50 min, with two steam
injections in advance.
Texture Analysis. The hardness of the bread crumbs was
measured using the EZ-SX Texture Analyzer of Shimadzu (The
Netherlands). Cylindrical shaped pieces with a diameter of 4.5 cm
were taken from the center of 2.0 cm thick slices. They were
compressed at a speed of 1 mm/s, a total distance of 3 mm in case of
gluten-free bread and 4 mm in case of wheat bread, with a cylindrical
probe (P75; diameter 7.5 cm). The hardness of the crumb is defined
as the maximum peak force encountered (gram-force) during the
compression, average of four measurements.
Phylogenetic Analysis, Domains Present, and Order
of Conserved Motifs. Phylogenetic analysis showed that
Geobacillus sp. 12AMOR1 GtfC clusters with GtfC-like 4,6-α-
glucanotransferases. There is a 56% sequence identity to the
well-characterized GtfC protein of E. sibiricum 255-159,17 and a
60% sequence identity to the very recently characterized
Bacillus coagulans DSM 1 GtfC.35 The E. sibiricum GtfC
protein was the first example of a new subfamily within family
GH70, with a different domain order than the GtfB-type
enzymes and the glucansucrase enzymes (with both α-amylase
domains A, B, and C and domains IV and V). In fact, the GtfClike
proteins lack domain V and have the same domain
organization as the GH13 enzymes.17 Compared to GH13
enzymes, the (β/α)8 barrel of the GtfB-type and glucansucrase
enzymes is circularly permutated, causing an altered order of
the conserved regions (II−III−IV−I). Similar to the GtfC
protein of E. sibiricum, Geobacillus GtfC has two extra Ig2-like
domains with unknown function (Figure 1). The Geobacillus
GtfC protein (726 AA, 82 kDa) with complete domains A, B,
C, and IV, but lacking the Ig2-like domains, was produced,
yielding about 30 mg of purified GtfC protein per liter of
culture, which was used in all subsequent studies described
here.
Substrate Specificity and Biochemical Properties of
the Geobacillus GtfC. Maltoheptaose (G7) and amylose V
were individually incubated with Geobacillus GtfC, and the
products formed were analyzed by TLC and HPAEC-PAD.
Reactions with the truncated L. reuteri 121 GtfB enzyme
lacking the N-terminal and domain V (GtfB-ΔN-ΔV) were
performed in parallel. The analyses showed that Geobacillus
GtfC synthesized a range of shorter and longer oligosaccharides
from G7, reflecting its main disproportionation (transglycosidase)
activity (Figures 2A and 3A). However, its
product spectrum is very different from that of GtfB-ΔN-ΔV
(Figure 2A), which synthesizes linear products with consecutive
(α1 → 6)-linkages.15 GtfC did not form polymeric
material from G7, unlike GtfB-ΔN-ΔV. Furthermore, GtfB-
ΔN-ΔV released a significant amount of glucose from G7,
whereas Geobacillus GtfC mainly released maltose but not
glucose. When acting on amylose V, Geobacillus GtfC
accumulated considerably higher amounts of oligosaccharides
than GtfB-ΔN-ΔV, which mainly synthesized polymeric
material (Figure 2B).
HPAEC-PAD analysis showed that Geobacillus GtfC mainly
released maltose from amylose V and only a small amount of
G3 (Figure 3B). It has to be noted that peaks are present that
do not correspond to the G1−G7 peaks [standards containing
only (α1 → 4)-linkages], suggesting product oligosaccharides
with different linkages. TLC (Figure 2B) shows that besides
small oligosaccharides, Geobacillus GtfC also synthesized
polymeric material from amylose V, clearly differing from the
E. sibiricum GtfC only forming oligosaccharides. The polymeric
material produced by Geobacillus GtfC was further investigated
with HPSEC (see below).
Geobacillus GtfC displayed high activity within the pH range
4.5−7.0 at 40 °C, with a maximum activity of 4.1 U/mg at pH
6.0 (Figure 4A). At the optimal pH of 6.0, the activity of
Geobacillus GtfC increased with increasing temperature,
reaching 6.6 U/mg at 60 °C, and then gradually decreased
to 3 U/mg at 75 °C (Figure 4B). The activity of the
Geobacillus GtfC enzyme on amylose V is significantly higher
than that of GtfB-ΔN-ΔV (2.8 U/mg) and GtfC of E. sibiricum
(2.2 U/mg) on amylose V.17,36 Furthermore, Geobacillus GtfC
hardly lost any activity when incubated for 1 hour at 60 °C in
the absence of substrate (Figure 4C), showing that it is a
thermostable enzyme. GtfC was rapidly inactivated at 95 °C
(data not shown). The thermal unfolding of Geobacillus GtfC
was determined by differential scanning fluorimetry and
revealed a melting temperature of 68 °C, supporting its high
thermostability (Figure 4D).
Geobacillus GtfC Has a Unique Product Specificity,
Synthesizing a Linear (α1 → 6)/(α1 → 4)-Glucan. The
polymeric fraction resulting from the Geobacillus GtfC activity
on amylose was isolated and characterized in detail. As
revealed by HPSEC analysis, the Geobacillus GtfC synthesized
a low-molecular-mass polymer from amylose V with an average
MW of 2.5 kDa (Figure 5), equivalent to a degree of
polymerization (DP) of ∼15. This polysaccharide is much
smaller than the IMMP produced by the GtfB-ΔN-ΔV from
amylose (MW of ∼ 12.3 kDa) (Figure 5).
The GtfC polymer was treated with a high dose of hydrolytic
enzymes, α-amylase (Aspergillus oryzae), dextranase (Chaetomium
erraticum), and pullulanase M1 (Klebsiella planticola), to
further explore its structure (Figure 6). TLC analysis showed
that the Geobacillus GtfC polymer is almost completely
resistant to the endo-(α1 → 4)-hydrolase action of α-amylase,
whereas amylose V is completely hydrolyzed. The GtfC
polymer also appeared mostly resistant to the endo-(α1 → 6)-
hydrolase activity of dextranase (completely hydrolyzing
dextran), indicating the absence of consecutive (α1 → 6)-
linkages. The GtfC polymer (and pullulan) was efficiently
hydrolyzed by pullulanase M1, which cleaves alternating (α1
→ 6)/(α1 → 4)-linkages and (α1 → 4,6)-branching points.
Pullulanase M1 treatment yielded mostly maltose as the
hydrolysis product. These results suggest that the Geobacillus
GtfC polymer mostly contains consecutive maltose units linked
by single (α1 → 6)-bonds. In accordance with this structural
analysis, the GtfC enzyme thus cleaves off mainly maltose units
from the linear (α1 → 4)-chain of amylose V donor molecules
and reattaches these to α-glucan acceptor molecules (e.g.,
amylose V, maltose formed by the hydrolytic side reaction, or
earlier formed transglucosylation products) with an (α1 → 6)-
linkage. Whereas the 4-α-glucanotransferase enzymes of
families GH13 and 77 may be able to transfer maltose units
(disproportionation reactions), the maltose transfer by the
family GH70 Geobacillus GtfC 4,6-α-glucanotransferase clearly
represents a novel product specificity, creating an (α1 → _____6)-
linkage with transfer and reattachment of every maltose unit.
To structurally characterize the 2.5 kDa polymer produced
by the Geobacillus GtfC from amylose V in more detail, 1D/2D
1H−13C NMR spectroscopy was performed (Figure 7). The
assignments of protons and carbons were obtained by
TOCSY/HSQC experiments and comparison of chemical
shift values to literature values.11,15,37−39
Further significant signals are at δ 4.02 [H-3 of →4)Glc(α1
→ 6)], at δ 3.48 [H-4 of →6)Glc(α1 → 4)], and at δ 3.42 [H-
4 of nonreducing end Glc(α1 → 4)]. The 2D 1H−13C HSQC
spectrum shows proton/carbon signals of unsubstituted C-6 at
δC 61.4, stemming from nonreducing-terminal Glc(α1 → 4)
(δH 3.85/3.76) and mainly →4)Glc(α1 → 6) (δH 3.88/3.84).
Substituted C-6 is shown by downfield-shifted signals at δC
67.2, stemming from →6)Glc(α1 → 4) (δH 3.96/3.75). There
is no →6)Glc(α1 → 6) present due to the absence of C-6 (δC
66.7) and H-6 (δH 3.98/3.76) signals.37−39 Further confirmation
of assignments was obtained by HSQC signals at δH
3.27/δC 74.9 [H-2/C-2 of reducing end →4)Glcβ], at δH 3.42/
δC 70.1 [H-4/C-4 of nonreducing end Glc(α1 → 4)], and at
δH 3.48/δC 70.4 [H-4/C-4 of →6)Glc(α1 → 4)], at δH 3.60/
δC 72.5 [H-2/C-2 of →4)Glc(α1 → 6) and nonreducingterminal
Glc(α1 → 4)]. The complete assignment is
summarized in Table 1.
So far, these results indicate a significant amount of α-glucan
polymer containing alternating (α1 → 6)/(α1 → 4)-linked
glucose units. The presence of a minor amount of substrate
amylose V left over, containing [→4)Glc(α1 → 4)], cannot be
excluded due to the known typical overlap of its proton and
carbon signals with the assigned signals above.37−39 The
anomeric proton signals at δ 5.40−5.36 could include H-1 of a
branching unit →4,6)Glc(α1 → 4), but methylation analysis
showed the absence of branching. Additionally, the presence of
terminal, 4-substituted, and 6-substituted glucopyranose
residues, in molar percentages of 9, 48, and 43%, was
confirmed by methylation analysis.
The combined data show that Geobacillus GtfC is a
thermostable 4,6-α-glucanotransferase with a unique product
specificity, cleaving off predominantly maltose units from
amylose V and reattaching them with an (α1 → 6)-linkage.
Geobacillus GtfC thus synthesizes a unique linear polymer with
alternating (α1 → 6)/(α1 → 4)-linked glucoses (see model
structure; Figure 8). Importantly, terminal Glc(α1 → 6) and
→6)Glcα/β were not detected in NMR spectra. It is the
absence of branches that mainly distinguishes the reaction
specificity of Geobacillus GtfC from that of the L. reuteri NCC
2631 GtfB, Azotobacter chroococcum NCIMB 8003, and
Paenibacillus beijingensis GtfD types of 4,6-α-glucanotransferases,
which produce heavily branched reuteran-like polymers.
13,18,19 Besides, these reuteran-producing enzymes were
found to transfer malto-oligosaccharides of different degrees of
polymerization (from 2 to 7), whereas the Geobacillus GtfC
exclusively cleaves and reattaches maltosyl units. Despite the
wide product specificity of the GH70 family, the structure of
the Geobacillus GtfC product is unique and clearly differs from
other α-glucans synthesized by GSs and 4,6-α-glucanotransferases.
Due to its linear structure and alternating (α1 → 4)/
(α1 → 6)-linkage pattern, the Geobacillus GtfC resembles
more to the pullulan polymer produced from starch by the
fungus Aureobasidium pullulans and used as a food additive.
However, pullulan is built up from maltotriose elements linked
by (α1 → 6) linkages instead of maltose units.
Geobacillus GtfC Is an Exo-Acting Enzyme. Amylopectin
was incubated with Geobacillus GtfC. TLC analysis showed
the formation of small quantities of maltose and other
oligosaccharides (mainly DP4) (Supporting Information
Figure S2A), though clearly less than formed with incubation
of amylose V with GtfC (Figure 2B, lane 2). This
demonstrated that GtfC only has a minor activity toward
amylopectin, which was confirmed by comparing the 1D 1H
NMR spectra of treated and untreated amylopectin (Supporting
Information Figure S2B). The spectra show the presence of
→4,6)Glc(α1 → 4) branching by anomeric proton signals at δ
5.36. A minimal difference is detected by the appearance of α/
β-anomeric protons (δ 5.22/4.65) and a H-2 signal (δ 3.27)
stemming from reducing-terminal →4)Glcα/β, indicating the
formation of very small amounts of malto-oligosaccharides.
Importantly, the treatment with Geobacillus GtfC did not
destroy the high-molecular-mass nature of amylopectin, with
the molecular mass remaining too high to be determined by
HPSEC analysis (data not shown). This indicates that
Geobacillus GtfC cannot bypass branches within amylopectin,
confirming the exo-acting nature of the GtfC enzyme.
Geobacillus GtfC 4,6-α-Glucanotransferase Has Antistaling
Activity. The only GH70 4,6-α-glucanotransferase
that has been tested in bread baking is the S. thermophilus GtfB,
slightly improving the textural properties and increasing the
fraction of slowly digestible starch of bread.20 The S.
thermophilus enzyme is rather different from the Geobacillus
GtfC, converting amylose in branched α-glucans,21 whereas
Geobacillus GtfC forms linear (α1 → 4)/(α1 → 6)-alternating
α-glucans. Moreover, the S. thermophilus GtfB enzyme has no
activity above 50 °C21 and thus only modifies the limited
soluble fraction of the starch during dough proofing. This is
because, in dough, starch is in its native physically inaccessible
semicrystalline granule form, going in solution at temperatures
above 65 °C40 (starch gelatinization), and only then the
enzymes can effectively attack the starch. The high thermostability
of Geobacillus GtfC, unfolding at 68 °C in the absence
of a substrate (Figure 4D), now allowed a proper evaluation of
4,6-α-glucanotransferase for antistaling activity in bread baking,
in analogy with the maltogenic amylases (e.g., Novamyl) used
within the bakery industry.41 Note that both maltogenic
amylases and Geobacillus GtfC are exo-acting enzymes cleaving
off maltose units from the nonreducing ends of linear (α1 →
4)-glucan chains. The difference being that maltogenic amylase
catalyzes hydrolysis forming maltose, whereas GtfC transfers
the maltose to an acceptor forming (α1 → 6)-glycosidic
linkages. The question arose whether Geobacillus GtfC also has
antistaling activity. The potential antistaling activity of
Geobacillus GtfC was first evaluated in a regular wheat bread,
including 7.1 kU of enzyme activity per kg of starch within the
recipe. Geobacillus GtfC did not alter the visual appearance of
the baked breads nor did it change the specific volume or the
crumb appearance (Figure 9). Nevertheless, the texture of the
bread improved, with the hardness of the Geobacillus GtfC
breads increasing slowly over time and being softer at each
time point (Figure 9). Thus, Geobacillus GtfC has a clear
antistaling activity in wheat breads.
Subsequently, Geobacillus GtfC was evaluated in a glutenfree
recipe. Typically, gluten-free breads suffer from very fast
staling since they lack the elasticity provided by the gluten
network and thus more heavily rely on the elasticity provided
by the starch. Because the gluten-free recipe tested in this
study was based on potato starch, which gelatinizes at a lower
temperature than wheat starch,40 there is a wider temperature
operation window for the enzyme to act on the gelatinized
starch during baking. The recipe contained 9.6 kU of GtfC
activity per kg starch, resulting in gluten-free breads with a
good visual appearance similar to the control (Figure 10).
Geobacillus GtfC improved the crumb structure of the bread,
having a clearly finer structure compared to the control.
Moreover, the hardness of the GtfC containing gluten-free
breads increased far slower, thus maintaining the favorable soft
crumb structure over a longer period (Figure 10). Geobacillus
GtfC displays a pronounced antistaling effect in a gluten-free
bread recipe.
The question remaining for future research is to unravel how
and to what extent GtfC is changing the molecular structure of
starch during bread baking. Our current hypothesis is that the
enzyme forms small quantities of the same type of (α1 → 4)/
(α1 → 6)-alternating oligosaccharides in bread as generated
from low concentrations of amylose and malto-oligosaccharides.
Most likely, the Geobacillus GtfC enzyme acts on both the
amylose fraction and the side chains of the amylopectin
molecules during bread baking. This will result in shorter
exterior side chains and/or exterior side chains that have
alternating (α1 → 4)/(α1 → 6) glycosidic linkages at their
nonreducing end, both reducing the process of retrogradation.
The previously characterized E. sibiricum and B. coagulans
DSM 1 GtfC types of 4,6-α-glucanotransferases both transfer
glucose units.17,35 Unexpectedly, the Geobacillus GtfC transferred
maltose units. Evidently, the donor subsite region of this
enzyme is spacious enough to accommodate two glucose
moieties. In addition, the observation that the Geobacillus GtfC
enzyme formed small amounts of short oligosaccharides when
acting on amylopectin (Supporting Information Figure S2),
though far less than from amylose (Figure 2), suggests that its
donor subsites cannot accommodate branched donor substrates.
The Geobacillus GtfC enzyme only forms linear
products, suggesting that the acceptor subsite region is quite
narrow, preventing the formation of branched products. In
contrast, the 4,6-α-glucanotransferase of A. chroococcum
NCIMB 8003 and L. reuteri NCC 2613 converts linear
malto-oligosaccharides into branched reuteran-like α-glucans.
13,19
Only the three-dimensional (3D) structure of the 4,6-α-
glucanotransferase GtfB protein of L. reuteri 121 is currently
available.21 This enzyme also acts on linear substrates to form
linear products but transfers glucose instead of maltose units. A
structural explanation for the distinct product specificity of the
Geobacillus GtfC is currently under investigation.