Study on oligomerization of glutamate decarboxylase from Lactobacillus brevis using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques
Study on oligomerization of glutamate decarboxylase from Lactobacillus brevis using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques
Jaeyeong Choi 0 1 2
Seungho Lee 0 1 2
Javier A. Linares-Pastén 0 1 2
Lars Nilsson 0 1 2
0 Department of Food Technology, Engineering and Nutrition, Lund University , 22100 Lund , Sweden
1 Division of Biotechnology, Department of Chemistry, Lund University , Naturvetarvägen 16, 22362 Lund, Skåne , Sweden
2 Department of Chemistry, Hannam University , 1646 Yuseong-daero, Yuseong-gu, Daejeon 34054 , Republic of Korea
3 Lars Nilsson
In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.
Glutamate decarboxylase (GAD); Oligomerization; Asymmetrical flow field-flow fractionation (AF4); Multi-angle light scattering (MALS); Lactobacillus brevis (L; brevis); Probiotic
Glutamate decarboxylase (GAD) catalyzes
decarboxylation of glutamic acid giving γ-aminobutyric acid
(GABA) (Fig. 1). GAD uses pyridoxal phosphate (PLP)
as the co-factor and H+ as the co-substrate. This enzyme
is present in a variety of organisms, from bacteria to
humans. GABA is one of the main neurotransmission
inhibitors in the central nervous system. In addition,
evidence exist that GABA can lower blood pressure in
patients with mild hypertension and that it has other potential
beneficial health effects, although mechanisms are not
known yet [
]. While GABA is an attractive potential
functional ingredient for food, chemically synthesized
GABA is not accepted for use in food .
GABA is found in some fermented food, as for instance in
kimchi . Some strains of Lactobacillus brevis (L. brevis)
were identified as GABA producers . Due to its potential
health effects in humans, L. brevis is recognized as a putative
probiotic . Thus, the potential production of GABA at
industrial levels using L. brevis as whole cell or its glutamate
decarboxylase (LbGadB; notice that BGAD^ represents the
general name of glutamate decarboxylases present in a variety
of organisms, while BLbGadB^ represent specifically the
glutamate decarboxylase from L. brevis) enzyme is very
attractive. Therefore, there is great interest to understand the
structure and function of LbGadB. It was previously reported that
the highest level of activity is reached when GAD is in the
hexameric form, while the lowest was observed when GAD
was present as a dimer [6, 7]. Thus, oligomerization plays an
important role in the GAD mechanism.
In general, size-exclusion chromatography (SEC), gel
electrophoresis, and field-flow fractionation (FFF) are used to
observe oligomerization of proteins.
SEC is commonly used for measurement of hydrodynamic
size (or more commonly molecular weight, MW) based on
size-based separation of analytes and their size distribution
from calibration curve of standard samples or the
multiangle light scattering (MALS) [8, 9]. However, difficulties
are frequently encountered when SEC is applied to high MW
analytes as they may undergo degradation by shear or they
may be trapped in the SEC columns [
analytes may reach the column exclusion limit or the
permeation limit leading to underestimation or overestimation of
MW, in addition to blockage of the column [11, 12].
Asymmetrical flow field-flow fractionation (AF4) also
provides separation of analytes based on their hydrodynamic
sizes. In AF4, an open channel without packing material is
used  and AF4 thus has several advantages over SEC. Due
to the relatively gentle separation conditions in AF4 (i.e., low
pressure and shear), degradation of analytes is prevented
during separation. AF4 has been successfully used for the
separation and characterization of high MW analytes including
proteins, DNA, viruses, and polysaccharides [14–17]. In
addition, AF4 provides means to determine some physical
properties, without calibration, such as MW, size, molecular
density, and conformation when it is coupled online with the
The stability of the active hexameric form of GAD from
L. brevis depends on several factors, including temperature,
pH, salt concentration, and type of salt. Thus, the study of the
effect of these factors on the GAD oligomerization is
fundamental to optimize the reaction conditions. In this work, the
use of diverse techniques, such as AF4 coupled online with
MALS (AF4-MALS), dynamic light scattering (DLS),
differential scanning fluorimetry (DSF), and molecular modeling
methods, allows the integrated characterization of the main
physicochemical factors that determine the stability of the
oligomeric enzyme GAD.
Material and methods
Citric acid (C6H8O7), sodium hydrogen phosphate anhydrous
(Na2HPO4), sodium chloride (NaCl), potassium chloride
(KCl), calcium chloride (CaCl2), ammonium sulfate
((NH4)2SO4), and sodium azide (NaN3) were purchased from
Sigma-Aldrich (St. Louis, USA).The carrier liquid for AF4
was prepared with water purified through a Milli-Q
purificat i o n s y s t e m ( M i l l i p o r e C o . L t d . , B i l l e r i c a , U S A ,
resistance = 18.2 MΩ/cm).
L. brevis DSM 1269 was purchased from the Leibniz Institute
DSMZ-German Collection of Microorganisms and Cell
Cultures. The strain was cultured in a MRS (Man, Rogosa,
and Sharpe) medium at 30 °C overnight after which genomic
DNA was extracted using E.Z.N.A Genomic Isolation Kit
(Omega Bio-Tek, USA). PCR primers, specific for GAD
gene, were designed based on the genomic sequence of strain
ATCC 367: Forward 5′-ATG ACG ACT ATC ATA TGA ATA
AAA ACG ATC AGG AAA C-3′ and reverse 5′-GTC AGC
TGC CCC TCG AGA CTT CGA ACG GTG GTC-3′ with
restriction sites (underlined in the sequences) for NdeI and
XhoI, respectively. The amplified gene was inserted in the
protein expression vector pET21b giving the construct
pET21b::LbGadB, which was introduced in Escherichia coli
(E. coli) Origami 2 (DE3) (Novagen brand, Merck KGaA,
Darmstadt, Germany). Recombinant E. coli was grown in
500 mL of LB (Luria Bertani) medium supplemented with
100 μg/mL ampicillin, at 37 °C. Recombinant LbGadB
production was induced when the culture optical density (OD) at
λ = 600 nm reaches 0.6, with isopropyl
β-D-1-thiogalactopyranoside, at 30 °C during 6 h. Finally, the cell pelleted was
harvested by centrifugation at 8000×g per 10 min in a Sorvall
refrigerated centrifuge (RC5C, USA) for the recombinant
Recombinant LbGadB was purified by ion-metal affinity
chromatography. E. coli cell pellet was washed twice with
binding buffer pH 7.4 (50 mM sodium phosphate, 0.5 M
NaCl, and 20 mM imidazole). Cell lysis was performed
suspending 1 g of cell pellet in 5 mL BugBuster® Protein
Extraction Reagent (San Diego, CA, USA) with 5 μL
Lysonase™ Bioprocessing Reagent and incubated for
30 min at 25 °C. Next, the suspension was centrifuged at
14,000×g, the precipitate was discarded, and the supernatant
was injected in a 5-mL HisTrap column FF (GE Healthcare,
Uppsala, Sweden) previously equilibrated with binding
buffer. After injection, the column was washed with binding buffer
and the recombinant protein eluted with elution buffer pH 7.4
(50 mM sodium phosphate, 0.5 M NaCl, and 0.5 M
imidazole). Finally, the excess of NaCl was removed by dialysis in
50 mM sodium phosphate buffer at pH 7.4. Protein purity was
determined by SDS-PAGE and the concentration was
quantified spectrophotometrically at 280 nm.
Hybrid homology model of LbGadB was constructed
using the YASARA software . Crystallographic
structures of other glutamate decarboxylases deposited in the
Protein Data Bank (PDB codes: 3HBX, 1XEY, 1PMM,
and 3MAD, with amino acid sequence identities of 38,
39, 38, and 23%, respectively) were used as templates.
The modeled structure was refined by molecular dynamic
simulation using the AMBER03 force field . The
solvent was simulated with explicit molecules of water.
Analysis of the modeled structure was done using UCSF
Chimera v1.11.2 .
Asymmetrical field-flow fractionation
The asymmetrical flow field-flow fractionation (AF4) used in
this work was an Eclipse 3+ system (Wyatt Technology,
Dernbach, Germany) coupled online with a UV detector
(UV-975, Jasco Corporation, Japan) set at 280 nm, a
multiangle light scattering (MALS) detector (DAWN HELEOS ΙΙ,
Wyatt Technology), and a differential refractive index (dRI)
detector (Optilab T-rEX, Wyatt Technology). The AF4
channel was trapezoidal with the tip-to-tip length of 26.5 cm and
the width at the inlet and outlet of 2.2 and 0.6 cm, respectively,
and was equipped with a 350-μm-thick Mylar spacer and a
regenerated cellulose (RC) membrane (molecular weight
cutoff of 10 kDa, Millipore, Bedford, USA).The AF4 carrier
liquid with various pH (3 to 8) was 10 mM citrate-phosphate
buffer and was pumped into the AF4 channel using an Agilent
1200 HPLC pump equipped with an auto-sampler and an
inline vacuum degasser (Agilent Technologies, Waldbronn,
Germany). The channel flow rate was kept constant at
0.5 mL/min, while the cross flow rate was kept constant at
4.5 mL/min for the first 20 min; after which, it was
exponentially decreased from 4.5 to 0.1 mL/min with the half-life time
of 2 min and then kept constant at 0.1 mL/min for 30 min. The
channel was washed with the carrier liquid for 10 min without
cross flow at the end of each run. All AF4 experiments were
performed at room temperature. The collection and processing
of AF4 data were performed using the ASTRA software
(Version 6.1.1, Wyatt Technology) with the dn/dc value of
0.185 mL/g. In all cases, the Berry method was used to fit
the light scattering data [21, 22]. From AF4 retention time,
the hydrodynamic diameter (dH) of a sample was calculated
by the AF4 theory using the FFFHydRad 2.0 software [23,
Determination of thermal stability of LbGadB
Thermal stability of LbGadB in different pH values was
det e r m i n e d u s i n g t h e P r o m e t h e u s N T 4 8 n a n o D S F
(NanoTemper Technologies, GmbH, Munich Germany).
Protein samples were prepared in pH 4, 5, 6, 7, 8 (McIlvaine
buffer system, 100 mM), and 9.6 (glycin-NaOH buffer,
100 mM). The capillaries were directly filled with 10 μL of
every sample. Intrinsic fluorescence at emission wavelengths
of 330 and 350 nm was monitored in a temperature gradient
from 20 to 90 °C. All data analysis was performed using the
PR control software (Version 2.0, Munich Germany).
Dynamic light scattering
The dynamic light scattering (DLS) analysis was performed
using DynaPro Plate Reader ΙΙ (Wyatt Technology) equipped
with a laser with the wavelength of 830 nm as the light source.
The analysis conditions were as follows: temperature = 25, 37,
45, and 60 °C; accumulation time = 100 s; and number
accumulation = 10. The LbGadB was prepared using various salt
types (NaCl, KCl, CaCl2, and (NH4)2SO4) at concentrations
(0, 0.3, 0.6, 0.9, and 1.2 M). Each sample of 60 μL was put in
the supported 384-well plate before DLS analysis.
Results and discussion
Glutamate decarboxylase gene from L. brevis DSM 1269 was
cloned and the recombinant protein was successfully
produced in E. coli. The gene was sequenced and deposited in
the GenBank (accession code: KX417371). The protein
sequence was obtained by theoretical translation of the gene.
The protein sequence was identical to the LbGadB of the
strain ATCC 367. The MW of the monomer was determined
experimentally by SDS-PAGE (see Fig. 2) and theoretically
from the protein sequence, giving the consistent value of
The molecular model was obtained in the hexameric form,
which is a trimer of dimers. The overall structure has two
layers, where every dimer contributes with one subunit to each
layer. Ramachandran plot analysis showed that 94.5% of the
amino acids were in the preferred regions, 5% in the allowed
regions, and 0.5% were outliers; this suggests that the model is
acceptable. The dimer dimension was 6.4 × 7.6 nm, while the
hexamer diameter was 13.1 nm and the width was 7.6 nm
Fig. 3 Molecular models of
LbGadB: a dimer and b hexamer
Effect of the pH in the oligomerization of LbGadB: AF4
Figure 4 shows AF4-UV-MALS fractograms of the
recombinant LbGadB obtained at various pH. Figure 4a shows the LS
responses (measured at 90°) and the molecular weight
distributions (MWD). Figure 4b shows the same fractograms as
those in Fig. 4a at the retention time of 0~20 min. The UV
responses are shown in Fig. 4c. As shown in Fig. 4a–c, at pH 7
and 8, the dimers of LbGadB are eluted at ~ 7 min. At pH 6,
hexamers are formed, and thus the elution time was increased
(~ 9 min). Also a broad band was observed at pH 6 at around
30 min, probably due to elution of LbGadB aggregates. When
pH was further lowered down to 5 and then 4, the intensity of
the broad band at around 30 min increases, due to more
aggregation, with no distinct hexamer peak observed. It is noted,
at pH 3, the intensity of the broad band was decreased, and a
tailed band was observed at the elution time of around 5 min,
which is due to large aggregates eluting in the steric/
hyperlayer mode [25, 26]. As shown in Fig. 4b, the molecular
weight decreases with increasing time for the tailed band,
which confirms the aggregates are eluted by the steric/
hyperlayer mode. The average MW of the tailed band at
5 min was 5.7 × 108 Da by determined by MALS, which is
much higher than the average MW (1.1 × 105 Da) of the 7-min
peak observed at pH 8.
The MW of the dimer and the hexamer of recombinant
LbGadB were determined to be 110 and 350 kDa at 10 mM
citrate-phosphate buffer of pH 8 and 6, respectively, by
AF4MALS. These MW values are in agreement with those
shown in a. c UV fractogram at 280 nm and dH from AF4 theory. d
Relative amount of dimer, hexamer, and aggregates of LbGadB
measured from peak area of deconvoluted UV fractograms
measured for the LbGadB monomer, 55 kDa by SDS-PAGE
and molecular modeling.
The radius of gyration (rg) of the dimer and the hexamer of
the LbGadB could not be measured by MALS as the analytes
behaved as isotropic scatterers, and hence, no angular
dependence in the scattered light is observed. The dH was determined
using AF4 theory [23, 24] and were compared with results
obtained from molecular modeling for the dimer and the hexamer
of LbGadB. From the AF4 theory, the dH of the dimer and the
hexamer of LbGadB were determined to be 6 and 10 nm,
respectively, and were in reasonable agreement with those from
molecular modeling, which were 6.4 × 7.6 nm for the dimer and
7.6 × 13.1 nm for the hexamer. The sizes determined by AF4
theory and modeling are not expected to be identical, as they
have different physical meanings, i.e., the dH determined by
AF4 is the molecular size obtained from the diffusion coefficient
(through the Stokes-Einstein equation) . While, the sizes
calculated from homology model are based on its atomic
three-dimensional structure built using as templates
crystallographic structures of homologous proteins .
n.d = no data
a Molecular modeling result
Table 1 shows the MW and size determined by various
methods for recombinant LbGadB. In Table 1, the MW and
rg were determined by MALS, dH were determined by AF4
theory, and molecular dimensions by modeling, respectively.
In general, proteins show UV absorption at 280 nm due to
UV absorbing amino acid, such as tryptophan, tyrosine, and
phenylalanine. However, the large aggregates cause scattering
effects, so correction of the results is necessary such as
deconvolution for quantitative analysis. Thus, all results of
UV detector were deconvoluted to dimer, hexamer, and
aggregates for semi-quantitative analysis of oligomerization of
LbGadB. Figure 4d and Table 2 show the relative percent
concentration of the dimer, hexamer, and aggregates of
recombinant LbGadB obtained by deconvoluting the UV
fractograms in Fig. 4c using the PeakFit software (ver. 4.0,
Systat Software Inc., San Jose, USA) with the Savitsky-Golay
Consistent with previous studies of plant (Arabidopsis
thaliana) glucatamate decarboxylase (AtGAD1) , it
seems that LbGadB from L. brevis is stable in a dimer
form at pH 7 or higher. In Fig. 4d and Table 2, only 18%
of LbGadB are present in a hexamer form at pH 7. The
LbGadB is present mostly as hexamer at pH 6. Then at
pH 5 or lower, most of LbGadB are present as aggregates
(98% at pH 5 and 100% at pH 4), indicating the hexamer
of LbGadB is not stable at pH below about 5. The
isoelectric point (pI) of LbGadB calculated by the molecular
modeling was 5.15 in the present study and aggregation of
LbGadB will be promoted at pH below pI. The hexamer
concentration changes drastically from 18% at pH 7 to
98% at pH 6.
It has been reported that AtGAD1 is present mostly as
hexamer at pH below 7 ; in this line, our results show
that the hexameric form of LbGadB is more stable at
pH 6 than pH 7. Both enzymes, AtGAD1 and LbGadB,
share 38% of amino acid sequence identity (query cover
of 94%, BLAST search). The thermal stability of
LbGadB in different pHs shows the maximum stability
at pH 6 (Tm 57 °C) and the lowest at pH 4 (Tm 46 °C)
(Fig. 5). At pH 6, the main form is the hexamer, which
means that this oligomeric form is the most stable form,
while at pH 4 almost all of the protein aggregate (Fig.
4d). At pHs 7 and 8, the predominant form is the dimer,
which is less stable than the hexamer since the thermal
unfolding transition midpoint (Tm 54 °C) is lower in both
pHs (Fig. 5). The dimer and hexameric forms are
stabilized by hydrogen-bonding interactions; therefore, the
thermal stability is increased [28, 29]. While, in the case
of the aggregates, it is expected that is not thermally
stable because it is mostly formed physically with low
or without hydrogen-bonding interactions. Thus, the
results of AF4 and thermal stability mean LbGadB has
most stable form occurs at pH 6. It is new discovery
for oligomerization of LbGadB.
Effect of type and concentration of salt and temperature
on oligomerization of LbGadB
Figure 6 shows the dH of LbGadB determined by DLS
with four different types of salts (NaCl, KCl, CaCl2, and
(NH4)2SO4) added at five different concentrations (0,
0.3, 0.6, 0.9, and 1.2 M) at four different temperatures
(25, 37, 45, and 60 °C). These salts are generally
recognized as safe (GRAS) according to Food and Drug
Administration (FDA). All results show a similar trend
of an increase in dH with an increase of salt
concentration or temperature. At the lower temperatures (25 and
37 °C), however, no significant changes in dH were
observed by a change in the salt concentration. Similarly, at
the lower salt concentrations (0 and 0.3 M), no
significant changes in dH were observed by a change in the
temperature. The effect by the salt type was not clear.
B a s e d o n t h e r e s u l t s a t 3 7 ° C ( s e e E l e c t r o n i c
Supplementary Material (ESM) Fig. S1 for more details),
the hexameric form of LbGadB is most stable in NaCl or
KCl and is not affected by the salt concentration in the
The MWs and sizes of dimer and hexamer of LbGadB
determined by AF4-UV-MALS-dRI were in good agreements with
those from molecular modeling. The effects of temperature,
salt type, and salt concentration on oligomerization of
LbGadB were investigated using DLS. The hexamer content
of LbGadB determined by deconvolution of UV/Vis detector
response was 98%, indicating the hexamer form is most stable
at pH 6. The hexamer also showed high thermal stability (up
to about 57 °C). This seems to be a new finding on
oligomerization of LbGadB. Results prove that AF4-UV-MALS-dRI is
a powerful tool for separation of dimer, hexamer, and
aggregates of LbGadB and also for monitoring of oligomerization
Acknowledgements The authors would like to thank Dr. Teresia
Hallström for her assistance in using the instrument Prometheus NT.48
nanoDSF in a Demo and Prometheus NT.48 nanoDSF, Munich,
Germany, for allowing us to use their instrument. The authors
acknowledge the support provided by the National Research Foundation (NRF) of
Korea (NRF-2013K2A3A1000086 and NRF-2016R1A2B4012105) and
the Swedish Foundation for International Cooperation in Research and
Higher Education (STINT).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest. There are no competing interests present and there are no patents,
products in development, or marketed products to declare. Furthermore,
we agree with the policies on sharing data and materials, as guide for
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