Enhancing effect of macroporous adsorption resin on gamma-aminobutyric acid production by Enterococcus faecium in whole-cell biotransformation system

Article abstract of DOI:10.1007/s00726-020-02850-3

Gamma-aminobutyric acid (GABA) biosynthesis depended to a great extent on the biotransformation characterization of glutamate decarboxylase (GAD) and process conditions. In this paper, the enhancing effect of D101 macroporous adsorption resin (MAR) on the GABA production was investigated based on the whole-cell biotransformation characterization of Enterococcus faecium and adsorption characteristics of D101 MAR. The results indicated that the optimal pH for reaction activity of whole-cell GAD and pure GAD was 4.4 and 5.0, respectively, and the pH range retained at least 50% of GAD activity was from 4.8 to 5.6 and 4.0–4.8, respectively. No substrate inhibition effect was observed on both pure GAD and whole-cell GAD, and the maximum activity could be obtained when the initial L-glutamic acid (L-Glu) concentration exceeded 57.6?mmol/L and 96.0?mmol/L, respectively. Besides, GABA could significantly inhibit the activity of whole-cell GAD rather than pure GAD. When the initial GABA concentration of the reaction solution remained 100?mmol/L, 33.51 ± 9.11% of the whole-cell GAD activity was inhibited. D101 MAR exhibited excellent properties in stabilizing the pH of the conversion reaction system, supplementing free L-Glu and removing excess GABA. Comparison of the biotransformation only in acetate buffer, the GABA production, with 50?g/100?mL of D101 MAR, was significantly increased by 138.71 ± 5.73%. D101 MAR with pre-adsorbed L-Glu could significantly enhance the production of GABA by gradual replenishment of free L-Glu, removing GABA and maintaining the pH of the reaction system, which would eventually make the GABA production more economical and eco-friendly.

Full text of DOI:10.1007/s00726-020-02850-3

Amino Acids
https://doi.org/10.1007/s00726-020-02850-3
ORIGINAL ARTICLE
Enhancing efect of macroporous adsorption resin
on gamma‑aminobutyric acid production by Enterococcus faecium
in whole‑cell biotransformation system
Sheng‑Yuan Yang1 · Shu‑Min Liu1 · Min Jiang1 · Biao‑Shi Wang1 · Luo‑Hui Peng1 · Chan Zeng1
Received: 21 July 2019 / Accepted: 29 April 2020
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
Gamma-aminobutyric acid (GABA) biosynthesis depended to a great extent on the biotransformation characterization of
glutamate decarboxylase (GAD) and process conditions. In this paper, the enhancing efect of D101 macroporous adsorption
resin (MAR) on the GABA production was investigated based on the whole-cell biotransformation characterization of Ente-
rococcus faecium and adsorption characteristics of D101 MAR. The results indicated that the optimal pH for reaction activity
of whole-cell GAD and pure GAD was 4.4 and 5.0, respectively, and the pH range retained at least 50% of GAD activity was
from 4.8 to 5.6 and 4.0–4.8, respectively. No substrate inhibition efect was observed on both pure GAD and whole-cell GAD,
and the maximum activity could be obtained when the initial L-glutamic acid (L-Glu) concentration exceeded 57.6 mmol/L
and 96.0 mmol/L, respectively. Besides, GABA could signifcantly inhibit the activity of whole-cell GAD rather than pure
GAD. When the initial GABA concentration of the reaction solution remained 100 mmol/L, 33.51 9.11% of the whole-
cell GAD activity was inhibited. D101 MAR exhibited excellent properties in stabilizing the pH of the conversion reaction
system, supplementing free L-Glu and removing excess GABA. Comparison of the biotransformation only in acetate bufer,
the GABA production, with 50 g/100 mL of D101 MAR, was signifcantly increased by 138.71 5.73%. D101 MAR with
pre-adsorbed L-Glu could signifcantly enhance the production of GABA by gradual replenishment of free L-Glu, removing
GABA and maintaining the pH of the reaction system, which would eventually make the GABA production more economi-
cal and eco-friendly.
Keywords Macroporous adsorption resin · Gamma-aminobutyric acid · Glutamate decarboxylase · Enterococcus faecium
Introduction
(Abdou et al. 2006), and prevention of cardiovascular dis-
eases (Imam et al. 2014) and diabetic conditions (Hagiwara
et al. 2004). The role of GABA in the improvement of the
oxidative stress and function of thyroid (Xie et al. 2014) as
well as complex brain processes such as appetite regulation
and metabolism (Delgado 2013; Meng et al. 2016) are also
gradually acknowledged. Due to these proven various health-
promoting properties, GABA has attracted a lot of attention
in recent years and has a great potential to be used as a
bioactive compound in the food or nutraceuticals industries.
It was reported that GABA occurred in the polyamine
utilization pathway in E. coli after the glutamylation of a
polyamine putrescine (Kurihara et al. 2005). The results
suggested that this pathway might be another possibility
of GABA production by bacteria. However, biosynthesis
of GABA from L-glutamic acid (L-Glu), a cheap renew-
able source, is completely reasonable and valuable from an
industrial and economical point of view. GABA has been
Gamma-aminobutyric acid (GABA), a four-carbon non-pro-
tein amino acid, is ubiquitously present in various bacteria,
plants and vertebrates. It plays a metabolic role in the Krebs
cycle in plants and bacteria, and a potent neural signal trans-
mitter in vertebrates (Diana et al. 2014). GABA has diverse
healthy functions including anti-hypertension (Hayakawa
et al. 2004; Inoue et al. 2003; Liu et al. 2011), tranquilization
(Okada et al. 2000; Wu et al. 2014), immunity enhancement
Handling Editor: J. G. López.
* Sheng-Yuan Yang
yangsy1972@163.com
1
College of Chemistry and Chemical Engineering, Lingnan
Normal University, Cunjin Road 29, Chikan District,
Zhanjiang 524048, Guangdong, China
Vol.:(0123456789)
1 3

S.-Y. Yang et al.
biosynthesized by microorganisms such as Lactobacillus
rhamnosus (Lin 2013), Lactobacillus sakei (Sa et al. 2015),
Lactobacillus brevis (Villegas et al. 2016), Lactobacil-
lus plantarum (Lee et al. 2015), Lactobacillus paracasei
(Komatsuzaki et al. 2005), Lactococcus lactis (Lu et al.
2009), Enterococcus avium (Tamura et al. 2010), Streptococ-
cus salivarius subsp. thermophilus (Yang et al. 2008), and
Enterococcus faecium (Zhu et al. 2018) via decarboxylation
of L-Glu by glutamate decarboxylase (GAD, EC 4.1.1.15),
which is a pyridoxal 5′-phosphate (PLP) dependent enzyme.
However, the biosynthetic process is keeping at low produc-
tivity and high manufacturing costs.
expected if the H+ and L-Glu are timely replenished into the
extracellular solution and the GABA is eliminated from the
extracellular solution in time.
Solid acids have been mainly used as catalysts for various
reactions such as acetylation, alkylation, acylation, hydrol-
ysis, and dehydration. Application of heterogeneous solid
acid is an efcient method for environment-friendly pro-
cesses for its easy to be separated and recycled from reaction
system (Gupta and Paul 2014; Huang and Fu 2013; Shimizu
and Satsuma 2011). The adsorption efect of resins on the
product can facilitate the selective enrichment of product
at a relatively low cost, and improve the productivity by
alleviating feedback inhibition, mitigating the cytotoxicity
of product to cells and preventing the product degradation
(Phillips et al. 2013). Using ion-exchange resin as solid acid
instead of conventional homogeneous acids has been proved
to be an efcient method to counteract the pH increase dur-
ing GABA production catalyzed by GAD (Dinh et al. 2014;
Yang et al. 2018; Zhu et al. 2018). Macroporous adsorption
resin (MAR), which is synthetic porous crosslinked polymer
bead, has been widely used for the separation and purifca-
tion of natural products because of its advantages of large
adsorption capacity, high adsorption speed, good selectiv-
ity and excellent regeneration. However, to our knowledge,
there is no report whether D101 MAR can be used to regu-
late pH, L-Glu and GABA, and enhance GABA production
in whole-cell bioconversion.
In addition to the activity and stability of GAD, the com-
position and conditions of the conversion system are also
important factors afecting the production efciency and
manufacturing costs of GABA. In bacteria, GAD not only
meets the metabolic needs of amino acids but also plays a
key role in glutamate-based acid resistance system. By con-
suming a proton via incorporation into L-Glu, GAD can pro-
tect bacteria from extracellular acid stress (Foster 2004; Yu
et al. 2012). Because of this physiological function of GAD,
most bacterial GAD exhibits the highest activity only under
acid conditions and shows low activity at neutral and alka-
line pH values (Pennacchietti et al. 2009; Yu et al. 2012),
which is disadvantageous for industrial application of GABA
production. First, the solubility of L-Glu is very low under
acid conditions, which cannot initially provide or timely
replenish ample L-Glu to fully guarantee the biocatalytic
reaction under saturated substrates and keep the maximum
GAD activity. Second, pH of the biocatalytic reaction system
will keep elevating due to protons are eliminated during the
decarboxylation reaction of L-Glu. The pH increase result-
ing from the enzymatic reaction will eventually make the
enzyme inactivated, resulting in a low conversion. In addi-
tion, the impairment of cell mass transfer will reduce the
speed of proton and L-Glu to enter cells, remove GABA, and
aggravate the efects of pH, L-Glu and GABA on intracellu-
lar GAD activity in the whole-cell biotransformation system.
The velocity of cell mass transfer is closely related to the
concentration of intracellular and extracellular substances.
In theory, the concentration diference between intracellular
fuid and extracellular fuid is an important driving force for
substance permeation across lipid membranes. The higher is
the concentration of H+ and L-Glu in extracellular solution,
the more favorable is it for H+ and L-Glu to penetrate into
cells. These H+ and L-Glu will neutralize the intracellular
alkali and interact with the active center of GAD, which
eventually activity. If extracellular GABA concentration is
low, intracellular GABA tend to be secreted toward extracel-
lular increases the enzyme environment, which will reduce
the intracellular pH and release active sites of GAD, thus
enhancing the catalytic activity of GAD. From the above
analysis, the improvement of GABA production could be
In the present study, the efects of commercial D101
MAR on the GABA-production in the whole-cell biotrans-
formation system of E. faecium GDMCC60203 were inves-
tigated for the frst time.
Materials and methods
Materials
D101 MAR was purchased from Tianjin Haoju Resin Tech-
nology Co., Ltd. (Tianjin, China). L-Glutamic acid (mini-
mum 98% purity) and GABA (minimum 99% purity) were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The
pure GAD of E. faecium GDMCC60203, which gave a sin-
gle band on sodium dodecyl sulphate–polyacrylamide gel
electrophoresis (SDS–PAGE) (as shown in Fig. 1), was puri-
fed by our laboratory. All the other reagents used were of
analytical or chromatographic grade unless otherwise stated.
Microorganism, medium, culture conditions
and cells suspension
Enterococcus faecium GDMCC60203 was isolated from
Chinese traditional fermented pickles and preserved in
Guangdong Microbial Culture Center (Guangzou, China).
1 3

Enhancing efect of macroporous adsorption resin on gamma‑aminobutyric acid production by…
GABA analysis
The transformation solution of pure GAD or whole-cell
GAD was frstly appropriately diluted with distilled water,
and then the GABA content of the dilution was assayed
according to the method of Rossetti and Lombard (1996) by
HPLC (Agilent 1200 Series, United States) with ZORBAX.
Eclips XDB–C18 column (4.6 mm × 250 mm, 5 μm, i.d.
Agilent, Palo Alto, CA, USA) and an UV detector at 254 nm.
The column temperature was set at 25 °C and the injection
volume was 20 µL.
Determination of the biotransformation activity
of pure and whole‑cell GAD
Various pHs, concentration and constituents of L-Glu solu-
tion were used as the reaction substrate to meet the need of
research. The reaction mixture consisted of 1 mL L-Glu and
1 mL GAD liquid or cell suspension. The reaction mixture
of pure and whole-cell GAD were incubated at 40 °C for
30 min and 6 h respectively. After incubation, two millilitres
of absolute ethanol was added to terminate the reaction. The
suspension was centrifuged at 8000g (15 min, 4 °C) and the
GABA content of the supernatant was determined by HPLC.
One unit of GAD activity was defned as the amount of
enzyme that produced 1 μmol of GABA in 1 min. The high-
est GAD activity was defned as 100% for the calculation of
relative activity, and the data were expressed as mean from
three independent experiments.
Fig. 1 SDS-PAGE electrophorogram of GAD. The concentration
of stacking gel and separating gel was 5% and 8%, respectively. The
thick of slab gel was 1.0 mm. L1 marker, L2 crude GAD, L3 and L4
purifed GAD
The strain was grown on de Man–Rogosa–Sharpe (MRS)
slant at 37 °C for 36 h and maintained at 4 °C. Preculture was
prepared by inoculating a loop of E. faecium GDMCC60203
lawn from MRS slant into 50 mL Peptone-Sucrose-Beef
extract (PSB) medium supplemented with 15 g/L peptone,
10 g/L beef extract, 12.5 g/L sucrose, 5.0 g/L sodium acetate
and 1.0 g/L Tween 80 with initial pH 6.8. The preculture
was incubated statically at 37 °C for 12 h. Then, 1 mL of
preculture was inoculated into 100 mL PSB medium and
incubated statically at 37 °C for 18 h as starter. Two millili-
tres of starter were inoculated into 100 mL PSB medium and
incubated statically at 37 °C for 48 h. Fermentation broth
was centrifuged at 8000g for 20 min at 4 °C and the cells
were harvested and washed twice with normal saline. The
resulting cells were collected and resuspended in normal
saline at a fnal concentration of 100 mg wet cell/mL.
Efects of pH, substrate and reaction product
on the GAD activity of pure enzyme and whole‑cell
To investigate the pH-dependent activity profle of pure and
whole-cell GAD, 0.25 mol/L of L-Glu with various pHs
ranging from 4.0 to 5.8 were used to evaluate the efects of
pH on biotransformation reaction. The efects of substrate
on the biotransformation activity of pure and whole-cell
GAD were performed with a diferent initial concentration
of L-Glu in the reaction mixture within a range from 3.6
to 115 mmol/L. For the efect of reaction product (GABA)
on biotransformation activity of pure and whole-cell GAD,
exogenous GABA was added to the initial reaction mixture,
and the biotransformation was carried out with GABA at
an initial concentration of 0 and 100 mmol/L, respectively.
Except for the pH-dependent activity experiments, the initial
reaction pH of the pure and whole-cell GAD biotransforma-
tion was conducted at 5.0 and 4.2, respectively. In addition
to these descriptions, other determination procedures were
performed according to the statement of ‘Determination of
the biotransformation activity of pure and whole-cell GAD’.
Pretreatment of L‑Glu and D101 MAR
The L-Glu solution was prepared by 0.2 mol/L of acetate
bufer containing 0.1 mmol/L of PLP as dissolvent, and the
concentration and pH of the L-Glu solution were 0.25 mol/L
and 4.2 respectively unless otherwise stated. The newly
bought D101 MAR was washed with 95% (v/v) ethanol till
the efuent was not turbid when testing with distilled water,
and then the ethanol was removed by distilled water. The res-
ins used for whole-cell biotransformation experiment were
further balanced by L-Glu solution.
1 3

S.-Y. Yang et al.
where Y1 was the average GABA yield in the control group
and Y2 was the GABA yield in the experimental group.
Efect of D101 MAR on the pH stability of whole‑cell
biotransformation system
Efect of the amount of D101 MAR on GABA
The reaction mixture, consisted of 10 g pretreated D101
MAR, 10 mL L-Glu solution and 10 mL cell suspension,
was incubated in water bath shaker at 40 °C and 80 r/min
for 0, 3, 6, 9, 12 and 24 h, respectively. Then, the pH of
the reaction solution was determined. The operation of
the control group was the same as that in the experimental
group but the resins were not added.
production
To investigate the optimal amount of pretreated D101 MAR,
diferent amounts of the resins, ranging from 25 to 75 g,
were added to the bioconversion with 50 mL L-Glu solution
and 50 mL cells suspension. The reaction mixture was incu-
bated in a water bath shaker for 24 h at 40 °C with constant
shaking (80 r/min). Except the collected resin pellets were
eluted twice with 100 mL of 70% (v/v) ethanol, the GABA
of the reaction solution and the one absorbed on resins were
assayed according to ‘Efect of processing time of D101
MAR on GABA production’. The control group was carried
out the same as that of the experimental group but without
resins.
Adsorption characteristics of D101 MAR on amino
acids
L-Glu is gradually transformed into GABA during the bio-
transformation process, and the concentration of L-Glu
would be gradually decreased with the gradual increase
of GABA concentration. To investigate the adsorption,
desorption or substitution of L-Glu and GABA caused by
D101 MAR in this process, 10 g of pretreated D101 MAR,
which had been balanced by L-Glu solution, were added
to 50 mL of 0.2 mol/L GABA solution (using pH 4.2 and
0.2 mol/L acetate bufer as solvent), and incubated for
30 min at 40 °C maintained in a water bath shaker with the
rotation speed of 80 r/min. The L-Glu and GABA absorbed
on D101 MAR were eluted by 70% (v/v) ethanol after the
resins were washed with 20 mL of acetate bufer (pH 4.2,
0.2 mol/L) and assayed by HPLC.
Statistical analysis
All experiments were performed in triplicate. The results
were expressed as mean SD and the statistical analysis was
performed using SPSS software (version 19.0, Chicago, IL,
USA). Signifcance analysis between two levels of the same
group was performed by one-way analysis of variance mul-
tiple comparisons, and the diference between two groups of
the same level was analysed by independent sample t test.
The same letters marking in the tables or fgures meant there
was no signifcant diference between the two levels of the
same group (P>0.05), and the diferent letters meant there
was a signifcant diference between the two levels of the
same group (P<0.05). Furthermore, a single asterisk and
double asterisk marking in the tables or fgures meant there
was a signifcant diference (P<0.05) and extremely signif-
cant diference (P<0.01) between two groups of the same
level, respectively. All fgures were constructed through
Microsoft Ofce Excel 2007 version.
Efect of processing time of D101 MAR on GABA
production
The reaction mixture, consisted of 10 g pretreated D101
MAR, 10 mL L-Glu solution and 10 mL cells suspension,
was incubated in water bath shaker at 40 °C and 80 r/min
for 0, 3, 6, 9, 12 and 24 h, respectively. Resin pellets were
separated from cells and solution by passing through a
200 μm mesh screen. The fltrate was centrifuged at 8000g
for 15 min and 1 mL resulting supernatant was used to
analyze the concentration of GABA in the reaction solu-
tion. The collected resin pellets were eluted twice with
20 mL of 70% (v/v) ethanol, and the eluate was collected
and used for determination of GABA absorbed on resins.
The control group was managed as an experimental group
without the addition of resins. Relative yield (%) was cal-
culated using the following formula:
Results
Efects of pH on the GAD activity of pure enzyme
and whole‑cell
To investigate the optimal biotransformation conditions
for GABA production, GAD activities of pure enzyme and
whole-cell under various pHs were determined. As shown
in Fig. 2, with the increase of pH value, the GAD activi-
ties of both pure enzyme and whole-cell increased frst and
then decreased. For pure GAD, more than 50% activity of
pure GAD was maintained between pH 4.8 and 5.6 with
optimal activity at pH 5.0. However, for whole-cells GAD,
Y2
Relative yield (%) =
× 100
(1)
Y1
1 3

Enhancing efect of macroporous adsorption resin on gamma‑aminobutyric acid production by…
a
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
a
a
A
a
a
a
a
a
B
A
A
b
C
e
B
b
b
c
BC
C
d
D
c
D
D
E
d
pure GAD
whole-cell GAD
E
E
whole-cell GAD
pure GAD
F
F
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
0
10 20 30 40 50 60 70 80 90 100 110 120 130
Glutamate / mmol/L
pH
Fig. 2 Efects of pH on the biotransformation activity of pure GAD
Fig. 3 Efects of L-Glu on the biotransformation activity of pure
GAD and whole-cell GAD. The concentration of L-Glu was the fnal
concentration in the initial reaction mixture
and whole-cell GAD
the activity remained over 50% at the pH of 4.0–4.8 with
optimal activity at pH 4.4. The pH range of the reaction
system of whole-cells GAD was much lower than that of
pure GAD, indicating that there was a mass transfer barrier
in the whole cell, which required the pH of extracellular
environment to reach 4.4 to ensure the intracellular pH to
reach 5.0, i.e., a certain concentration diference of proton
donors and receptors between the internal and external cell-
microenvironment was needed to promote the exchange rate.
From these results, we hypothesized that the apparent GAD
activity of whole-cell could be greatly enhanced by timely
supplementation of H+ or elimination of alkaline factors.
Figure 2 showed that the pH range of whole-cell GAD
with reactivity was very small. When pH was higher than
4.4, there was a remarkable decrease in the activity of
whole-cells GAD, indicating that high pH value had a sig-
nifcant inhibition efect on enzyme activity. Therefore, it
is essential to maintain the acidic pH to keep the activity
of whole-cell GAD throughout the reaction so that higher
conversion is achieved. For pH value of the reaction sys-
tem would increase signifcantly during the decarboxylation
reaction and nearly 90% activity of whole-cell GAD could
be kept at pH 4.2 (Fig. 2), it was appropriate to choose 4.2 as
the initial reaction pH to ensure that whole-cell GAD could
maintain high activity in a longer conversion reaction time.
Therefore, the initial reaction pH for pure and whole-cell
GAD in follow-up experiments was 5.0 and 4.2, respectively.
Figure 3 showed that L-Glu did not have detectable sub-
strate inhibition efect on the GAD activity of pure enzyme
and whole-cell since the GAD activity of pure enzyme
and whole-cell both increased with the increase of L-Glu
concentration. However, the L-Glu concentration needed
to maintain the maximum GAD activity of whole-cell was
much higher than that of the pure enzyme, and the maximum
activity was obtained when the fnal L-Glu concentration
exceeded 57.6 mmol/L and 96.0 mmol/L, respectively. These
results implied that the transportation within the extracellu-
lar and intracellular L-Glu was limited by the barrier in the
whole cell, and higher fnal L-Glu concentration of extra-
cellular environment was required to overcome the obsta-
cles to ensure the L-Glu of intracellular microenvironment
could reach an optimal concentration. Based on this fact,
we hypothesized that the apparent GAD activity of whole-
cell could be greatly enhanced by timely supplementation
of substrate.
Efects of reaction product on the GAD activity
of pure enzyme and whole‑cell
Product of enzymatic reactions has feedback inhibition on
the activity of enzymes in some cases. As a catalytic reaction
product of GAD, the efects of GABA on the GAD activity
of pure enzyme and whole-cell were investigated. As shown
in Fig. 4, when the initial GABA concentration of the reac-
tion solution was 100 mmol/L, the activity of pure GAD was
almost unafected by GABA, while 33.51 9.11% of GAD
activity of the whole-cell was inhibited. The results inferred
that the intracellular GABA could not be discharged in time
due to the mass transfer barrier in the whole cells, which
resulted in a high concentration of intracellular GABA and
hindered the timely exchange of GABA and L-Glu in the
Efects of substrate on the GAD activity of pure
enzyme and whole‑cell
As a substrate of GAD, L-Glu is an essential compound for
GABA synthesis in biotransformation. However, superfu-
ous or insufcient addition of L-Glu would afect the GAD
activity and decrease the GABA production in some cases.
1 3

S.-Y. Yang et al.
140
120
100
80
the conversion reaction was carried out in 0.2 mol/L acetate
bufer, pH values of the reaction system in the experimental
group (coupled with D101 MAR) and control group (with-
out D101 MAR) both increased gradually with the reaction
proceeding. And in 24 h, the pH values of the reaction sys-
tem in the experimental group and control group increased
from 4.2 to 4.65 0.09 and 5.17 0.06 respectively, while
the pH of the experimental group increased more slowly
than that of the control group. The pH of the experimental
group during the conversion reaction process was extremely
signifcant lower to that of the control group (P<0.01). The
results indicated that D101 MAR could stabilize the pH of
the conversion reaction system to a certain extent.
0 mmol/L of exogenous GABA in initial reaction mixture, control group
100 mmol/L of exogenous GABA in initial reaction mixture, experimental group
A
a
a
B
60
40
20
0
Whole-cell GAD
Pure GAD
Fig. 4 Efects of GABA on the biotransformation activity of pure
GAD and whole-cell GAD. The concentration of GABA was the fnal
concentration in the initial reaction mixture
Adsorption characteristics of D101 MAR on amino
acids
To achieve timely supplementation of L-Glu and elimination
of free GABA of the reaction solution, adsorption properties
of D101 MAR on L-Glu and GABA were investigated via
simulating the changes of L-Glu and GABA in the trans-
formation reaction. As shown in Table 1, 0.53 0.04 mmol
of L-Glu could be absorbed by per gram of D101 MAR
balanced with L-Glu solution. When the D101 MAR pre-
adsorbed with L-Glu was mixed with 0.2 mol/L GABA solu-
tion (pH 4.2), 0.24 0.03 mmol of L-Glu could be desorbed
from each gram of resins, and 0.21 0.03 mmol of GABA
could be absorbed by per gram of resins. The results indi-
cated that the adsorption of D101 MAR to L-Glu and GABA
seemed to be weak. However, the concentration of L-Glu
in the reaction broth decreased while the concentration of
GABA increased in the process of biotransformation, indi-
cating the adsorption properties of D101 MAR to L-Glu and
GABA could fully meet the expectation of controlling the
concentration of free substrates and products in the conver-
sion reaction solution.
enzyme activity centre, thus reducing the activity of GAD.
Based on this, we hypothesized that the apparent GAD activ-
ity of whole-cell could be greatly enhanced by reducing the
GABA concentration in the reaction solution in time and
accelerating the excretion of intracellular GABA.
Efect of D101 MAR on the pH stability of whole‑cell
biotransformation system
It has been demonstrated previously that the pH of the reac-
tion system could signifcantly afect the activity of whole-
cell GAD, and was an important biotransformation condition
afecting the yield of GABA. To fnd a green method to
control the pH of the reaction system in time, the efect of
D101 MAR on the pH stability of whole-cell biotransforma-
tion system were investigated. As shown in Fig. 5, although
5.5
a
Efect of D101 MAR on GABA production
5.0
4.5
4.0
3.5
3.0
b
c
c
**
A
According to the aforementioned results, D101 MAR could
regulate the pH value, concentration of free L-Glu and
GABA of the conversion reaction system. However, the
efects of D101 MAR on GABA production by whole-cell
biotransformation were still unclear. Figure 6 showed that
**
d
**
B
**
**
e
AB
B
BC
C
Co-reaction with D101 MAR
Without D101 MAR, control group
Table 1 Adsorption characteristics of amino acids by D101 macropo-
rous adsorption resins (MAR)
MAR L-Glu (mmol/gresins
)
GABA (mmol/
resins) (new
adsorption)
0
3
6
9
12
15
18
21
24
27
g
Reaction time / h
Initial adsorp- Residual
Desorption
tion
adsorption
Fig. 5 Efect of D101 macroporous adsorption resins (MAR) on the
D101 0.53 0.04
0.29 0.03
0.24 0.03 0.21 0.03
pH stability of whole-cell biotransformation system
1 3

Enhancing efect of macroporous adsorption resin on gamma‑aminobutyric acid production by…
3.0
2.5
2.0
1.5
1.0
0.5
0.0
160
140
120
100
80
to 50 g/100 mL) in the reaction solution. Thereafter, the
GABA production began to decrease when the resin con-
tent was higher than 50 g/100 mL. The highest total GABA
production (11.86 0.49 mmol/L) was obtained when the
resin content was 50 g/100 mL, in which the GABA rela-
tive yield increased by 138.71 5.73% compared with the
control group (without D101 MAR) (P<0.05).
A
B
BC
A
CD
D
**
a
D
B
*
C
*
b
60
c
D
d
Co-reaction with D101 MAR
40
Discussion
Without D101 MAR, control group
Relative yield
E
e
20
F
f
GABA is synthesized through the decarboxylation of L-Glu
by GAD. GAD is localized in the cytoplasm, and L-Glu
and GABA can permeate through membrane and exchange
with each other in lactic acid bacteria (Higuchi et al. 1997).
However, L-Glu and GABA cannot permeate freely due to
the semi permeability of cell membranes. The permeation
rate has a signifcant relationship with the concentration of
intracellular and extracellular solute. Therefore, there may
be great diferences in the bioconversion characteristics
between whole-cell and free enzyme. Figures 2, 3 and 4
in this paper demonstrated that the efects of pH value, the
concentration of L-Glu and GABA on the biotransforma-
tion of whole-cell were signifcantly greater than those on
pure enzymes. Based on the results of Figs. 2, 3 and 4, we
hypothesized that the GABA production could be improved
by timely supplementation of H+ and L-Glu, and elimination
of alkaline factors and free GABA in the reaction solution.
Thus, we frst investigated the ability of D101 MAR to regu-
late the pH value, the concentration of L-Glu and GABA of
the reaction system, and then applied the resins to GABA
production by whole-cell biotransformation. Results showed
that GABA production of whole-cell biotransformation was
11.86 0.49 mmol/L, which was 1.39-fold compared to the
group without resins.
0
0
3
6
9
12
15
18
21
24
27
Reaction time / h
Fig. 6 Efect of D101 MAR on the GABA production of biotransfor-
mation system
16
12
8
150
120
90
60
30
0
A
a
GABA yield
Relative yield
C
AB
ab
AB
ab
B
b
c
d
4
D
0
CK
25
37.5
50
62.5
75
Resin content / g/100 mL
Fig. 7 Efect of resin content on GABA production. The CK was rep-
resented the control group, which was carried out the same as that of
the experimental group but without D101 MAR
the promotion of GABA production by D101 MAR became
more and more signifcant with the prolongation of whole-
cell biotransformation reaction time. After 9 h of reaction,
the GABA production of the experimental group (coupled
with D101 MAR) increased signifcantly compared with the
control group (without D101 MAR) (P< 0.05). When the
reaction reached 24 h, the GABA relative yield of the experi-
mental group was increased by 137.28 7.69% compared
with the control group (P<0.01). The results indicated that
D101 MAR could signifcantly enhance the GABA produc-
tion in the whole-cell biotransformation reaction system.
In general, to control the pH value within a certain range
and thus improve the productivity of GABA, the addition
of conventional acid/base such as hydrochloric acid and
sodium hydroxide to the reaction medium during the reac-
tion has been widely performed (Lammens et al. 2009; Lee
et al. 2013; Plokhov et al. 2000; Shi et al. 2017; Yang et al.
2008). Nevertheless, this method consumes large amounts
of acid/base solution and brings about the formation of a
signifcant amount of salts, which can eventually cause seri-
ous problems in separation and purifcation of GABA after
the reaction. To mitigate the impact of this problem, using
cation exchange resins as solid acid for GABA production
has been reported (Dinh et al. 2014; Yang et al. 2018; Zhu
et al. 2018). However, due to the complexity of ions in the
conversion reaction solution, it is difcult to achieve direc-
tional quantitative exchange between H ions in H-type cation
exchange resins and GABA, resulting in acidifcation of the
reaction solution and reduced GAD activity. For example,
Efect of the amount of D101 MAR on GABA
production
The efects of the amount of D101 MAR on GABA produc-
tion were shown in Fig. 7. It was found that the total GABA
production increased with the increase of resin content (up
1 3

S.-Y. Yang et al.
during bioconversion, IRP-69 cation exchange resins can
decrease the pH of the reaction solution from 4.5 to 4 or
less when the addition of resins exceeded 20 g/100 mL (Zhu
et al. 2018), and 732 cation exchange resins can decrease
the pH of 0.2 mol/L acetate bufer from 4.2 to 3.32 when
50 g/100 mL of resins were incubated with acetate bufer for
1 h at 40 °C (Yang et al. 2018). However, this phenomenon
was not observed in the experiment of D101 MAR.
to reduce the product feedback inhibition and increase the
GABA production.
As a weak acid, L-Glu gradually replenished from D101
MAR could act as acid titrate during the process of sub-
strate consumption, and thereby maintained the pH of the
reaction system. Though GABA is a neutral substance with
an isoelectric point of 7.30, it is still an alkaline factor com-
pared with pH 4.2 of the reaction system and has a certain
alkalization efect on the reaction system. The adsorption of
GABA by D101 MAR can decrease the concentration of free
GABA in the reaction solution, and thereby reduce its efect
on the pH of the reaction system. Therefore, D101 MAR,
like cation exchange resins, can use as solid acid. Since it
has no large quantities of H+ release, D101 MAR does not
acidify the reaction solution appreciably, which means that it
has more advantages than ion exchange resins in the GABA-
production via whole-cell biotransformation.
The solubility of L-Glu in water was just 0.864% at
25 °C (Shi et al. 2017). The L-Glu used in this paper was
nearly saturated at a concentration of 0.25 mol/L dissolved
in 0.2 mol/L acetate bufer (pH 4.2), and the theoretical
fnal concentration of L-Glu in the initial reaction mixture
was 125 mmol/L. Figure 3 showed that only when the fnal
concentration exceeded 96.0 mmol/L, L-Glu could keep the
maximum reaction activity in the whole-cell biotransforma-
tion. The results signifed that the reaction activity would
decrease with the gradual consumption of L-Glu. Table 1
proved that L-Glu could be adsorbed by D101 MAR at pH
4.2 when the concentration of L-Glu was 0.25 mol/L. Nev-
ertheless, the adsorption was relatively weak. When the con-
centration of L-Glu was decreased, it could be desorbed from
D101 MAR again. Thus, the L-Glu adsorbed on D101 MAR
could be replenished into the reaction solution in time, so as
to improve the conversion activity. Meanwhile, it was also
presented in Table 1 that D101 MAR could adsorb GABA.
Therefore, GABA produced by the bioconversion reaction
could bind to the resins timely, and thereby reduced the
concentration of free GABA in the reaction solution, so as
From the above analysis, the roles of D101 MAR on the
promotion of GABA production were via gradual replenish-
ment of free substrate concentration, reducing free product
concentration and maintaining the pH of the reaction system.
The roles of D101 MAR in promoting GABA production
were briefy illustrated in Fig. 8.
It is well established that in-situ product recovery
(ISPR) can yield great benefts for processes limited by
inhibitions, product toxicity, degradation, an unfavorable
position of the thermodynamic equilibrium, or kinetic
control (Bechtold and Panke 2009; Woodley et al. 2008).
Biocatalysis constitutes an efective tool for the production
Fig. 8 Sketch map of the role of
D101 MAR in GABA produc-
tion by whole-cell biotransfor-
mation
1 3

Enhancing efect of macroporous adsorption resin on gamma‑aminobutyric acid production by…
of fne chemicals. In recent years, the application of ISPR
in the bioprocess has attracted more and more attention of
researchers (Bechtold and Panke 2009; Van Hecke et al.
2014). Application of resins in whole-cell biocatalysis can
be advantageous not only due to its ability to improve the
yield and volumetric productivity and to overcome the
inhibitory efect of product but also due to its ability to
enrich the product leading to reduced downstream pro-
cessing steps and subsequently reducing the cost (Phillips
et al. 2013; Van Hecke et al. 2014). In the resin-coupled
whole-cell bioconversion of this study, D101 MAR with
pre-adsorbed L-Glu gave an advantage to the replenish-
ment of free substrate gradually, remove the product and
maintain the pH of the reaction system. Therefore, this
study potentially provided an alternative method of green
GABA production and ISPR technology.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
Research involving human participants and/or animals This article
does not contain any studies with human participants or animals per-
formed by any of the authors.
Informed consent This article does not contain individual participants
requiring informed consent.
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