Continuous enzyme-coupled assay for microbial transglutaminase activity

Article abstract of DOI:10.1016/j.ab.2013.07.014

Transglutaminases (protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13) are a family of calciumdependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a γ-glutamyl-ε- lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG's promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K_M = 3 mM) than the commonly used Cbz-Gln-Gly (K_M = 58 mM), attesting to its potential for application in biocatalysis and bioconjugation.

Full text of DOI:10.1016/j.ab.2013.07.014

Analytical Biochemistry 441 (2013) 169–173
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Continuous enzyme-coupled assay for microbial transglutaminase
activity
⇑
Samuel K. Oteng-Pabi, Jeffrey W. Keillor
Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6H5, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2013
Received in revised form 27 June 2013
Accepted 10 July 2013
Transglutaminases (protein-glutamine:amine c-glutamyltransferase, EC 2.3.2.13) are a family of calcium-
dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of pri-
mary amines. When a lysine residue acts as the acyl-acceptor substrate, a -glutamyl- -lysine isopeptide
c
e
bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to sev-
eral biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase
family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Further-
more, mTG’s promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The
acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine res-
idues dramatically affect substrate specificity and activity. Here, we have developed and optimized a
modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides.
This direct continuous assay presents significant advantages over the commonly used hydroxamate
assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48
(WALQRPH), a high-affinity peptide that shows greater affinity with mTG (KM = 3 mM) than the com-
monly used Cbz-Gln-Gly (KM = 58 mM), attesting to its potential for application in biocatalysis and
bioconjugation.
Available online 19 July 2013
Keywords:
Microbial transglutaminase
Glutamate dehydrogenase
Enzyme-coupled assay
Microtiter plate
Transamidation
Hydroxamate assay
Ó 2013 Elsevier Inc. All rights reserved.
Transglutaminases (TGs,1 protein-glutamine:amine
transferase, EC 2.3.2.13) are a family of calcium-dependent enzymes
that catalyze an acyl transfer between glutamine residues and a
wide variety of primary amines [1]. When a lysine residue acts as
c-glutamyl-
Moreover, the bacterial enzyme is expressed as a zymogen and
activated through N-terminal cleavage to the resulting 38-kDa
mTG [11–13]. Isolated from Streptomyces mobaraensis, mTG has
been characterized and shown to have very little sequence similar-
ity to any mammalian TG, whose molecular weights are greater
than 70 kDa [14]. Convergent evolution may explain why mTG
shares a similar function as its mammalian counterparts but exhib-
its more robust catalytic properties, including high stability
throughout a broad range of pH and temperatures in comparison
with other TGs [15–17].
the acyl-acceptor substrate, a
c-glutamyl-e-lysine isopeptide bond
is formed between two proteins [2] (Fig. 1). TG-mediated protein
cross-linking is critical to several biological processes and diseases,
including extracellular matrix stabilization [3], blood coagulation
[4], formation of cataracts [5], type 2 diabetes [6], neurodegenerative
diseases [7], and certain cancers [8].
Several TGs have been identified in mammalian tissues;
although they play many diverse physiological roles, the require-
ment of calcium as a cofactor is pervasive with all mammalian
members [9]. Microbial transglutaminases (mTGs) are a distinct
variant of the transglutaminase family by virtue of their calcium-
independent catalysis of the isopeptide bond formation [10,11].
The broad substrate specificity of mTG makes it amenable as a
tool for conjugation and opens up its potential as a biocatalyst.
For instance, mTG cross-linking has been exploited in the conjuga-
tion of biodegradable polymers. Hydroxyethyl starch (HES) is a
polymer that has been used as an effect agent for therapeutic drug
delivery [18]. The conjugation of the polymer to drugs and proteins
is normally executed chemically; however, mTG-mediated cross-
linking presents a selective, less expensive alternative [18].
mTG’s promiscuity with respect to its acyl-acceptor substrate
highlights its biocatalytic potential. The acyl-donor substrate, how-
ever, is more limited in its scope; the amino acid sequences flank-
ing glutamine residues dramatically affect their activity. For
example, it has been shown that the most likely sequences for
mTG specificity have similarities at residues –3, –1, +1, and +2
⇑
Corresponding author.
E-mail address: jkeillor@uottawa.ca (J.W. Keillor).
Abbreviations used: TG, transglutaminase; mTG, microbial transglutaminase; SPPS,
1
solid-phase peptide synthesis; DMF, dimethylformamide; HBTU, O-benzotriazolyl-
N,N,N0,N0-tetramethyluronium hexafluorophosphate; HPLC, high-performance liquid
chromatography; FA, formic acid; EDTA, ethylenediaminetetraacetic acid; NADH,
nicotinamide adenine dinucleotide (reduced form); GDH, glutamate dehydrogenase;
a
-KG, a-ketoglutarate.
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2013.07.014

170
Microbial transglutaminase activity assay / S.K. Oteng-Pabi, J.W. Keillor / Anal. Biochem. 441 (2013) 169–173
O
O
H
N
H
N
mTG
R"
+
+
R
R
NH2
N
H
NH3
H2N R"
O
R'
O
R'
α-KG
NADH
NAD+
GDH
L-Glu
Fig.1. Glutamate dehydrogenase (GDH)-coupled microbial transglutaminase (mTG) activity assay. mTG catalyzes the acyl-transfer reaction from glutamine to a primary
amine. (With Cbz-Gln-Gly, R = Cbz and R0 = Gly. With 7M48, R = WAL and R0 = RPH. R00-NH2 = Gly-OMe). Released ammonia acts as a substrate in the GDH-catalyzed reductive
amination of
a-ketoglutarate (a-KG). This reaction results in the disappearance of the reduced form of nicotinamide adenine dinucleotide (NADH), monitored by its
absorbance at 340 nm.
relative to the position of the glutamine residue, namely aromatic,
leucine, arginine, and proline residues [19]. However, very few spe-
cific peptide sequences encompassed by these general preferences
have been studied in enough detail to provide quantitative infor-
mation regarding their affinity for mTG. For example, although
Cbz-Gln-Gly is used as a standard acyl-donor substrate, its affinity
for mTG is relatively low [20], requiring that it be used at relatively
high concentrations. This illustrates the importance of identifying a
high-affinity acyl-donor substrate for mTG if its full potential as a
bioconjugation catalyst is to be realized.
Healthcare). The purified activated mTG was dialyzed against
50 mM phosphate buffer (pH 8.0). The average yield was 75 mg
of activated mTG per liter of culture, with greater than 80% purity
as estimated through evaluation of 10% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) followed by
staining with Coomassie blue. Aliquots were snap-frozen and
stored at –80 °C in 15% glycerol.
Peptide synthesis
In turn, the identification of a high-affinity acyl-donor substrate
requires a rapid and convenient activity assay that can be applied
to the screening of multiple peptides. However, the assay most
commonly used for measuring mTG activity is the hydroxamate as-
say [21,22], a discontinuous method that requires high quantities
of reagents and substrates in order to achieve reliable results. A
sensitive, continuous, and rapid assay for mTG activity, therefore,
is urgently required.
To that end, we have developed an enzyme-coupled assay in or-
der to measure the activity of mTG with any Gln-containing pep-
tide sequence. Based on a microtiter plate scale, this assay uses
little material and is amenable to high-throughput screening appli-
cations. We applied it here to quantitatively measure the kinetic
parameters for several high-affinity peptide sequences.
Peptides were synthesized using an automated peptide synthe-
sizer (Liberty CEM Microwave Peptide Synthesizer) by conven-
tional Fmoc-based solid-phase peptide synthesis (SPPS).
Synthesis was carried out on a scale of 200 mg of Knorr amide resin
with a loading capacity of 0.93 mmol/g (0.25 mmol scale). Each
round of peptide elongation consists of (i) Fmoc deprotection with
20% piperidine in dimethylformamide (DMF) for 5 min three
times; (ii) washing with DMF; (iii) coupling with O-benzotriazol-
yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU),
N,N-diisopropylethylamine (DIEA), N-methylpyrrolidinone (NMP),
and the appropriate amino acid in DMF for 11 min; and (iv) wash-
ing with DMF. The molar ratio of the amino groups on the resin/
building block/HBTU was 1:4:3.6. When the peptide was complete,
acetylation at the native N terminus (to protect primary amine)
was carried out using 20% pyridine in DMF. Peptide was then
cleaved from the resin using 90:5:5 trifluoroacetic acid (TFA)/
dichloromethane (DCM)/thianisole for 120 min. After cleavage,
the resin was filtered and washed three times under vacuum using
1:1 cylcohexane/acetone. The peptide was dissolved in a minimal
amount of acetone and precipitated from the solution using cold
Et2O with sonication. The precipitated peptide was then recovered
through centrifugation at 1100g for 30 min.
Materials and methods
Expression and purification of mTG
The plasmid pDJ1-3 was kindly provided by Joelle Pelletier
(Université de Montréal). pDJ1-3 encodes the proenzyme of mTG
from S. mobaraensis inserted between the NdeI and XhoI restriction
sites of the vector pET20b [23]. mTG was expressed and purified
according to a previously published protocol [24] with the follow-
ing amendments. The plasmid encoding for mTG bearing a C-ter-
minal hexahistidine tag was transformed into Escherichia coli
HPLC analysis
High-performance liquid chromatography (HPLC) was per-
formed on a Waters 2996 instrument equipped with a photodiode
array detector. Ultraviolet (UV) absorbance of peptides was mea-
sured at 254 and 274 nm. Analytical HPLC was performed on a
BL21-Gold (DE3) in the presence of 100 lg/ml ampicillin. mTG
was expressed according to the autoinduction protocol [25]. The
culture was centrifuged at 3000g for 30 min at 4 °C; the superna-
tant was discarded and the pellet was resuspended in 50 mM phos-
phate buffer (pH 8.0) with 300 mM NaCl. Cells were disrupted by
sonication over ice (three cycles of 30-s pulse at 20% intensity/
1 min pause) using a Branson sonicator. mTG was then activated
by cleavage of the proenzyme alpha sequence through incubation
in a 1:10 ratio (w/v) of trypsin (1 mg/ml) to unpurified mTG for
75 min at 37 °C. The activated mTG was purified using a 1-ml Hi-
sTrap Ni-NTA column (GE Healthcare) equilibrated in 50 mM phos-
phate buffer (pH 8.0) with 300 mM NaCl and eluted with an
imidazole gradient (0–140 mM) on an Åkta FPLC instrument (GE
Waters C18 column (5
l
m, 3.8 ꢀ 150 mm) with a run time of
30 min, a flow rate of 0.5 ml/min, and a linear gradient of 50–
95% of 0.1% formic acid (FA) in methanol (eluent B) in 0.1% aqueous
FA (eluent A).
Hydroxamate assay
The previously reported hydroxamate assay [22,26] was
adapted to use with mTG as follows. First, 0.75 ml of 0.2 M acyl-do-
nor substrate was mixed with 0.25 ml of 2 M hydroxylamine,

Microbial transglutaminase activity assay / S.K. Oteng-Pabi, J.W. Keillor / Anal. Biochem. 441 (2013) 169–173
171
0.25 ml of 0.02 M ethylenediaminetetraacetic acid (EDTA), and
1 ml of 1.0 M Tris–acetate and adjusted to pH 6.0. Next, a 400-
aliquot of this solution was incubated with 100 l of enzyme solu-
tion (at varying concentrations) at 37 °C for 10 min. After incuba-
tion, 500
0.8 M HCl was added. The solution was centrifuged for 5 min at
10,000g (to clear any precipitated protein), and the absorbance of
the supernatant fraction was measured at 525 nm. Activity was
0.10
0.08
0.06
0.04
0.02
0.00
l
l
l
l
l of 2.0 M FeCl3ꢁ6H2O, 0.3 M trichloroacetic acid, and
determined using the extinction coefficient of Cbz-L-glutamyl-(c-
hydroxylamine)-glycine, determined to be 340 M–1 for a 1-cm path
length at 525 nm [26], using authentic synthetic material [27].
GDH-coupled enzyme assay
0
2
4
6
The optimized assay was performed using 200 mM MOPS buffer
(pH 7.2), 1 mM EDTA, 500 lM reduced form of nicotinamide adenine
GDH (U)
dinucleotide (NADH), 10 mM ketoglutarate, 2 U of glutamate dehy-
drogenase (GDH), varying concentrations of acyl-donor substrate
(1–66 mM), and 10 mM glycine methyl ester as an acyl-acceptor
Fig.2. Rates of reaction of 0.1 U of mTG as a function of added GDH. Rate values
were measured in triplicate in a 200- l reaction volume by monitoring the decrease
of NADH over time. Error bars represent the standard errors of the mean values.
l
substrate [28] in a final volume of 180
was preincubated for 5 min at 37 °C prior to initiation of the reaction
by the addition of 0.10 U of mTG (in a volume of 20 l) to give a final
volume of 200 l. For the corresponding blank solution, the mTG
ll. The reaction solution
l
presence of the reagents required for the mTG reaction. Table 1
shows the negligible effect of the presence of mTG on the GDH
reaction. The rate of the positive control reaction of GDH on the
addition of 0.1 mM ammonium chloride is also shown in Table 1.
When this reaction was repeated in the presence of 40 mM added
Cbz-Gln-Gly and 10 mM Gly-OMe, the reaction rate was found to
be the same within experimental error. When the reaction was re-
peated in the presence of added mTG, the reaction rate was simi-
larly unaffected. Finally, when the reaction was run in the
presence of mTG and its substrates but without GDH, only a slight
background disappearance of NADH was observed, as expected
(Table 1).
Second, the activity of mTG was determined using the well-
known hydroxamate assay in the absence and presence of added
reagents required for the GDH-coupled assay. Specifically, the rate
of mTG-mediated hydroxamate formation was determined to be
0.412 0.021 nmol/min over a 10-min period in the presence of
l
solution was replaced with water. The assays were performed in a
96-well plate, and the decrease in absorbance due to the oxidation
of NADH was followed against a blank at 340 nm in a Synergy HT
Multi-Mode Microplate Reader thermostated at 37 °C. After a brief
lag during which sufficient ammonia is produced to saturate GDH,
linear slopes of absorbance versus time were measured. This lag
phase is intrinsic to all continuous enzyme-coupled assays, for
which it is difficult to measure true initial rates. However, the brev-
ity of this lag in this optimized assay allows for the accurate mea-
surement of approximate initial rates, which are still a valid
indicator of substrate turnover. These approximate initial rates were
corrected for path length and blank using the instrument software.
Results and discussion
0.8 U of mTG in a 500-
was repeated in the presence of the additives required for the
GDH reaction (i.e., 500 M NADH, 3.34 mM -KG, and 2 U of
ll reaction volume. When the reaction
Relative rates of coupled enzymes
l
a
GDH catalyzes the reductive amination of
a-ketoglutarate (a-
GDH), the rate was determined to be 0.397 0.019 nmol/min.
The similarity of these results shows that mTG and GDH, as well
as their respective substrates, have a negligible effect on each
other’s activity.
KG) with concomitant consumption of the coenzyme NADH. In
the past, we have coupled this reaction to the catalytic activity of
tissue transglutaminase in the development of an assay [29] that
has since been applied to kinetic and inhibition studies [30]. The
adaptation of this assay for application to mTG requires that sev-
eral conditions be met.
Limit of detection
Most importantly, for this assay to report on mTG activity, it is
crucial that the GDH-mediated consumption of NADH and ammo-
nia be faster than the mTG-mediated release of ammonia during
acyl transfer (Fig. 1). To establish this, the effect of GDH concentra-
tion on the observed mTG reaction rate was studied at a high con-
centration of Cbz-Gln-Gly as acyl-donor substrate. A concentration
of 58 mM was used, which is near the solubility limit of Cbz-Gln-
Gly under the assay conditions. As shown in Fig. 2, the observed
reaction rate (using 0.1 U of mTG) was found to increase linearly
with added GDH up to the addition of 2 U. When the concentration
of GDH exceeds 2 U, no further increase in reaction rate was
observed, indicating that the reaction rate is limited by mTG-
mediated release of ammonia under these conditions.
Under the conditions of the optimized activity assay, 0.1 U of
mTG were used. However, to explore the full scope of our
microtiter plate-based assay, its sensitivity and lower limits of
detection were also determined. As shown in Fig. 3, the observed
rate of reaction decreases linearly in the presence of decreasing
Table 1
Reaction rates of GDH assay in absence and presence of added reagents required for
mTG reaction.
Added reagent(s)
Ratea (nmol/s)
Ammonia
Cbz-Gln-Gly, GlyOMe + ammonia
mTG + ammonia
0.170 0.007
0.165 0.006
0.164 0.006
Substrate concentration optimization
mTG, Cbz-Gln-Gly, GlyOMe + ammonia (no GDH)
0.00190 0.00072
Another condition that must be established is that the reactants
and products of either enzyme have no effect on the activity of the
other. First, the reaction of GDH was monitored in the absence and
Note. Rates were measured in triplicate, and reported errors are standard deviations
of the mean values.
a
Measured in a 200-ll reaction volume.

172
Microbial transglutaminase activity assay / S.K. Oteng-Pabi, J.W. Keillor / Anal. Biochem. 441 (2013) 169–173
amounts of added mTG. Remarkably, the assay was found to have a
linear dynamic range of more than 100-fold in mTG concentration.
However, in the presence of less than 0.02 U of mTG, it is difficult
to discern the rate of mTG-mediated ammonia release.
pH
0
2
4
6
8
10
0
-1
-2
-3
-4
-5
-6
pH Dependence
The effect of pH was then studied to determine the sensitivity of
the enzyme-coupled assay. Using the same concentrations of sub-
strates and enzymes as in the optimized assay (see Materials and
Methods), the pH of the reaction buffer was varied between 2.5
and 9.0. As shown in Fig. 4, negligible differences in reaction rates
were observed between pH 6.0 and pH 9.0, suggesting that neither
of the coupled enzymes is greatly affected by variations in pH over
this broad range centered around physiological pH. This is consis-
tent with a previous report that mTG has an optimal activity be-
tween pH 6.0 and pH 9.0 [16].
Fig.4. pH Rate profile of the GDH-coupled assay. Shown is pH versus Log(rate)
(where rate is reported in units of nmol/s) of mTG-mediated transamidation,
measured by GDH-coupled assay, over the pH range 2.5–9.0, with 0.10 U of mTG,
50 mM Cbz-Gln-Gly, and 10 mM Gly-OMe in a 200-ll reaction volume.
Application to KM measurement
tides named M42 and M48 were found to be among the most reac-
tive of all the peptides screened in their phage-displayed library.
Initially, we designed pentapeptide variants from the –2 to +2 posi-
tions of these sequences relative to the central reactive Gln residue.
These peptides, named 5M42 (Ac-ELQRP-NH2) and 5M48 (Ac-
ALQRP-NH2), were synthesized according to standard Fmoc-based
SPPS protocols as described in Materials and Methods. Their kinetic
parameters as acyl-donor substrates were then determined by
Michaelis–Menten analysis (see Materials and Methods); surpris-
ingly, their affinity for mTG was found to be as low as that of
Cbz-Gln-Gly (data not shown). Reasoning that this may be due to
therelative importance of the –3 and +3 residues [14], we then de-
signed heptapeptide variants from the –3 to +3 positions. Specifi-
cally, peptides 7M42 (Ac-YELQRPY-NH2) and 7M48 (Ac-
WALQRPH-NH2) were prepared by SPPS and characterized kineti-
cally. In Table 2, it can be seen that the KM value of 7M42 was
determined to be several-fold lower than that of Cbz-Gln-Gly,
whereas the KM value of 7M48 was found to be nearly 20-fold low-
er (Fig. 5). The increased mTG affinity of the heptapeptide sub-
strates may be due to the introduction of an aromatic residue at
the –3 position, as observed previously by Hitomi and coworkers
[19]. Analysis of the active formof mTG reveals a hydrophobic re-
gion localized in the binding site of the enzyme [19]. N-terminal
aromatic and hydrophobic residues of the donor substrate that
are able to interact with this region may account for the significant
increase in affinity of our heptapeptides. This experiment illus-
trates the potential for the development of simple peptidic sub-
strates that react much more efficiently with mTG than Cbz-Gln-
As shown above, the optimized GDH-coupled assay is a conve-
nient, rapid, sensitive, and robust method for measuring mTG
activity. As such, it offers several significant advantages over the
widely applied hydroxamate assay. Namely, it is a continuous as-
say, rather than a discontinuous assay, that requires less material,
can be carried out quickly and conveniently in microtiter plate for-
mat, and is readily amenable for high-throughput screening. Fur-
thermore, because it is based on the release of ammonia from
the glutamine-donor substrate, it is independent of the precise
peptide sequence of the glutamine sequence and the identity of
the acyl-acceptor substrate. The hydroxamate assay does not have
such generality because it is based [21] on the extinction coeffi-
cient of a specific hydroxamate product formed between Cbz-
Gln-Gly as acyl-donor substrate and hydroxylamine as a nucleo-
philic acceptor substrate, excluding the wide variety of primary
amines that could be studied with this GDH-coupled assay. As a
simple demonstration of this advantage, the GDH-coupled assay
was applied to the facile kinetic characterization of alternative
Gln-donor peptide substrates, allowing their comparison with
Cbz-Gln-Gly, the standard acyl-donor substrate used in many
mTG studies.
The sequences of these alternative peptide substrates were
based on the high-affinity peptides discovered by Hitomi and
coworkers through their phage display studies [19]. Notably, pep-
0.20
Table 2
0.15
Kinetic values obtained for transamidation and deamination (hydrolysis), mediated
by mTG, of high-affinity peptides and Cbz-Gln-Gly (acquired by both GDH assay and
hydroxamate assay).
0.08
0.06
0.10
Substrate
KM (mM)
Vmax (mM/s)
Vmax/KM (s–1
)
0.04
Transamidationa
Cbz-Gln-Glyb
58.9 2.4
55.1 3.3
15.3 1.4
3.07 0.70
0.546 0.077
0.420 0.051
0.428 0.031
0.435 0.098
(9.27 0.41) ꢀ 10–3
(7.62 0.22) ꢀ 10–3
(2.80 0.06) ꢀ 10–2
0.142 0.014
0.02
0.05
0.00
7M42
7M48
0.0
0.1
0.2
[mTG] (U)
0.3
0.4
0.00
Deamination
Cbz-Gln-Gly
7M42
1610 103
245 34
157 28
0.024 0.005
0.041 0.002
0.036 0.003
(1.47 0.23) ꢀ 10–5
(1.66 0.04) ꢀ 10–4
(2.28 0.03) ꢀ 10–4
0
2
4
6
8
[mTG] (U)
7M48
Note. Results obtained were performed in triplicate using a 96-well microplate
Fig.3. Monitoring the lower limit of detection of GDH-coupled assay. Initial rate
was measured by the GDH assay as a function of units of mTG (from ꢂ0.01 to ꢂ 6 U;
lowest points also shown in inset) in the presence of 25 mM Cbz-Gln-Gly, over
reader. Reported errors are standard errors of the mean values.
a
Measured in the presence of 10 mM Gly-OMe as acceptor substrate.
Values in italics were measured using the hydroxamate assay for comparison.
b
20 min, performed in triplicate on a microplate reader (200-ll reaction volume).

Microbial transglutaminase activity assay / S.K. Oteng-Pabi, J.W. Keillor / Anal. Biochem. 441 (2013) 169–173
173
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Fig.5. Michaelis–Menten plot of mTG activity, measured by the GDH-coupled assay
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presence of 10 mM Gly-OMe as acyl-acceptor substrate, carried out in triplicate
using a 96-well microplate reader (200-
l
l reaction volume). Error bars represent
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Gly while demonstrating the convenience of the application of the
enzyme-coupled assay to detailed kinetic studies.
High-affinity substrate peptide sequences discovered in this
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a
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high-affinity peptide sequence could be modified using mTG as a
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We have developed and optimized an assay that can be used to
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microbial transglutaminase. This direct continuous assay allows
for sensitive detection of the reaction of a broad range of different
substrates. Using this assay, two acyl-donor substrates were iden-
tified that demonstrated significantly greater affinity with mTG
relative to the commonly used substrate Cbz-Gln-Gly. These sub-
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lytic and bioconjugation applications mediated by mTG.
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Acknowledgments
The authors thank Joelle Pelletier (Université de Montréal) for
helpful discussion and thank the Natural Sciences and Engineering
Research Council of Canada for financial support.
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