bState Key Laboratory of Agricultural
Microbiology, College of Life Science and Technology, Huazhong
Agriculture University, Wuhan 430070, China.
*Corresponding author. E-mail address: aistar2000@hotmail.com
Abstract: In this study, we developed two new whole-cell
biocatalysts by immobilizing aminopeptidase (Aps) on the surface of
yeast cells, using N-terminal fusion and C-terminal fusion through
lectin mediated display system. After the two strains were cultured in
the medium with galactose as inducer for 48 hours, the activity of
expressing Aps was at a high level of 0.25 U/OD600/mL and 0.12
U/OD600/mL. The correct location of Aps was confirmed by
immunofluorescence analysis and flow cytometry. Afterwards two whole
cell catalysts could be reused with high stability as it retained more
than 70% of initial activity after ten repeated batch reactions. Using
β-cypermethrin (β-CP) as a substrate, the effectiveness of two new
whole-cell catalysts in the treatment of highly hydrophobic organic
pollutants was evaluated. The results showed that when the concentration
of β-CP was 200 mg·L-1, the hydrolysis rates of the
two whole cell catalysts were 33.16
μmol·L-1·day-1 and 28.99
μmol·L-1·day-1, and has the ability
to degrade a variety of pyrethroid pesticides. The β-CP residue in
lettuce and cherry tomatoes could be removed more than 70% under the
conditions of the Aga2N-Aps whole cell catalyst preparation dilution of
100 times. This is the first report on the development of surface
display Aps biocatalyst, which can be used as an effective and renewable
alternative for the treatment of highly hydrophobic organic pollutants.
Keywords: Biodegradation; Pyrethroids; Aminopeptidase; Yeast
cell surface display; Whole-cell biocatalyst
Introduction
Pyrethroid insecticide is a kind of synthetic bionic insecticide with
high efficiency and low toxicity, which is one of the most widely used
and difficult to be replaced pesticides in the next few decades (Lu et
al., 2021). At present, there are more than 20 kinds of commonly used
pyrethroids on the market, the most commonly used pyrethroids are
beta-cypermethrin, fenpropathrin, deltamethrin, cyhalothrin and
fenvalerate, etc. Beta-cypermethrin (β-CP), a major synthetic pyrethroid
insecticide, which is widely used to take precautions against and
control pests such as fruits, vegetables, grains, etc (Tiwary and Dubey,
2016). Because of its photothermal stability and high hydrophobicity, it
is easily adsorbed in soil and organic matter (Lu et al., 2021; Song et
al., 2015). Furthermore, insecticides residue in agricultural products
threaten human health through feeding behavior, and long-term human
contact would have the risk of carcinogenesis (Zhan et al., 2020),
teratogenesis and mutagenesis (Zhang et al., 2019). With the continuous
harmful pollution of β-CP to the ecological environment and human
health, the biodegradation of β-CP in the environment has attracted more
and consideration (Luo et al., 2018; Song et al., 2015). Of the various
means that are used to remediate β-CP environmental pollution, microbial
degradation is considered to be one of the most promising strategies
(Akbar et al., 2015).
In the current research work on the degradation of β-CP by
microorganisms, most of the reported functional enzymes are
intracellular enzymes, including pyrethroid-hydrolyzing esterase (EstP)
from Klebsiella sp. strain ZD112 (Saleem et al., 2008) andR. palustris PSB-S esterase (Est3385) (Luo et al., 2018). Due to
the high hydrophobicity and high relative molecular weight of β-CP, its
low efficiency into the cell will greatly affect its decomposition by
the cell (Tang et al., 2015). Therefore, how to adsorb it from the
environment to the surface of microorganisms and be ingested by
microorganisms is the first and key step in the detoxification and
metabolism of β-cypermethrin. It has been reported that the
extracellular carboxylesterase of Bacillus licheniformis B-1
(Zhang et al., 2021) and the extracellular aminopeptidase ofPseudomonas aeruginosa GF31 (A. X. Tang et al., 2017) can solve
the membrane barrier problem of enzyme and substrate to a certain
extent. However, the purification and recovery process of extracellular
free enzyme will reduce its activity, poor stability, low repetitive
utilization rate, increase production cost, and limit its large-scale
production and application (Gustavsson et al., 2014).
Microbial cell-surface display (CSD) is a powerful platform to present
and immobilize the protein of interest on microbial surface, it has been
effectively used in various fields, including the development of live
vaccines (Cheng et al., 2021), whole cell catalysis (Chordia et al.,
2021; Ye et al., 2021), biosensor development (Zhao et al., 2020),
environmental biosorption (Rangra et al., 2018) and so on. Among several
surface display systems, the yeast surface display (YSD) system has been
broadly applied as a stage for production of heterologous proteins, and
a well-studied yeast surface applied is Saccharomyces cerevisiae(S.cerevisiae ) (Chen et
al., 2016; Yang et al., 2019). S.cerevisiae has been granted
”Generally Recognized As Safe” (GRAS) approval by the Food and Drug
Administration, suitable for industrial and food use (Chun et al.,
2020). The foremost commonly utilized yeast display system were
a-agglutinin and α-agglutinin mediated systems. The a-agglutinin
mediated display system comprised two subunits: Aga1 and Aga2, of which
Aga1 has glycosylphosphatidylinositol (GPI) anchor function (Lozančić et
al., 2021; Tanaka et al., 2012). At that point Aga2 is connected with
Aga1 through two disulfide bonds, and target protein can be intertwined
with the N- or C-terminus of Aga2, and the ultimate target protein
realizes functional expression on the yeast cell surface (Wang et al.,
2015). Because the fusion site will affect the display quantity and
activity of fusion protein on the engineering bacteria cell surface,
steric hindrance effect between proteins must be considered (Kuroda and
Ueda, 2013). In the yeast surface display platform constructed by
selecting different fusion sites,
Endolysin LysSA11 fromStaphylococcal phage SA11 was displayed on the surface ofS.cerevisiae by N-terminal fusion (Chun et al., 2020), and Lim
has researched a new yeast display platform that involves linking two
diverse heterologous proteins to Aga2, one at the C-terminus and one at
the N-terminus (Lim et al., 2017).
At present, relevant studies have
proved that the use of surface display technology to anchor the target
protein on the outer membrane of microorganisms can solve the problem
that the limitation of high hydrophobic substrate transport barrier
across cell membrane (Ding et al., 2020), such as hydrolysis of
organophosphorus compounds (Song et al., 2019); biodegradation of
diisobutyl phthalate (DiBP) (Ding et al., 2020) and degradation of
highly crystallized polyethylene terephthalate (PET) (Chen et al.,
2020). This method allows the substrate to interact directly on the
surface of the microbial cell, eliminating the tedious process of
multi-step purification and recovery of free enzymes, and greatly
reducing the cost of preparation and application of the whole-cell
biocatalyst (Smith et al., 2015). Due to the unique chemical
selectivity, regioselectivity and enantioselectivity of enzymes, only a
few of the available functional enzymes were used for cell surface
display (An et al., 2014). Hence, the improvement of novel enzymes for
cell display systems an important investigate objective (Liang et al.,
2019). Aminopeptidase (Aps) is an
extracellular pyrethroid degrading
enzyme from Pseudomonas aeruginosa GF31, and different from the
widely reported intracellular pyrethroid degrading enzyme
(carboxylesterase) (Tang et al., 2015). As a new pyrethroid degrading
enzyme, Aps is a bifunctional enzyme that can hydrolyze pyrethroid and
protein (Tang et al., 2017). In previous studies, the display of Aps on
the cell surface and construction of whole-cell catalysts for
bioremediation have not been reported yet.
In this study, we aimed to fuse the Aps of Pseudomonas aeruginosaGF31 to the N-terminus and C-terminus of Aga2 subunits respectively and
display it on the surface of S.cerevisiae , constructing a novel
whole-cell biocatalyst. The environmentally friendlyS.cerevisiae engineering
strain was endowed with the capacity to degrade β-CP and could be
utilized for further environmental bioremediation. In addition, as a new
whole cell catalyst, displayed-Aps can be used as the basis of other
applied research of aminopeptidase, such as waste protein pollution
treatment, milk Debittering and so on.
Methods
Strains, media and chemicals
In this study, S.cerevisiae EBY100 strain (Invitrogen, Carlsbad,
CA) was selected as the host strain for the yeast surface display
system. Transformed yeast were cultured in SD-CAA media (synthetic
media, tryptophan auxotrophic, 2% D-glucose, 1 M sorbitol, pH 6.4, 25
μg/mL kanamycin). The expression of Aga2 fusion protein was induced by
galactose inducible promoter in SG-CAA medium (synthetic media,
tryptophan auxotrophic, 2% D-galactose, pH 6.75, 25 μg/mL kanamycin).
β-Cypermethrin (98.1%), fenpropathrin (91.6%), deltamethrin (98.6%),
alphacypermethrin (95.1%) and cyhalothrin (98.4%) were purchased from
Guangxi Plant Protection General Station (Nanning, China).
L-Leucine-p-nitroanilide (Leu-pNA) was purchased from Takara (Dalian,
China). All other chemicals are purchased from commercial sources and
have a purity of at least analytical grade.
Construction of displaying S.cerevisiae strain and
growth curve measurement
The Aps gene (KT735188) cloned from Pseudomonas aeruginosa strain
GF31 (Tang et al., 2017) was codon optimized for functional expression
according to the codon preference of the host strainS.cerevisiae. The first 72 nucleotides (encoding signal peptide)
and the stop codon of the Aps gene have been removed. To facilitate
protein characterization and visualization, human influenza
hemagglutinin (HA) was connected to the C-terminal or N-terminal of the
target protein Aps. Next, the modified Aps gene sequence was fused with
the N-terminus or C-terminus of the Aga2 subunit of a-agglutinin
anchored protein from the yeast display system, respectively, and
transformed and ligated into the pYD1 plasmid to construct the plasmids
pYD1-Aga2N-Aps and pYD1-Aga2C-Aps. The fusion protein cassette was shown
in Fig. 1B . The two vectors were transformed intoS.cerevisiae EBY100 cells by heat-shock transformation. The
engineered S.cerevisiae was cultured in SD-CAA medium at 30°C and
220 rpm for 36 h. After proliferation and culture, when the measured
OD600 reaches 2.5-3.0, the engineered yeast cells were collected after
centrifugation for 10 minutes under the condition of
5000×g at 4°C. After washing, they
were resuspended in SG-CAA medium to control OD600 to 1.0. Then the Aps
protein was induced by 2% galactose at 20°C.
The pYD1-Aga2N-Aps/pYD1-Aga2C-Aps transformants were inoculated into
SD-CAA medium and cultured at 30°C until the optical density 600 (OD600)
was 2.5 to 3.0. The proliferated yeast cells were centrifuged at 5000×g
for 15 min and then suspended in SG-CAA medium to make its OD600 in the
induction medium equal to 1.0, and cultivated at low temperature to
induce the appearance of the fusion protein.S.cerevisiae EBY100 was
used as a negative control. From 0 h to 60 h, OD600 was measured every 6
h and the growth curve was drawn. In order to explore the influence of
the induction time on the enzyme activity of Aps,
EBY100-pYD1-Aga2N-Aps/EBY100-pYD1-Aga2C-Aps were induced for 12, 18, 24,
30, 36, 48, 54 h for enzyme activity
assay.
Expression and detection of target protein Aps
After 48 h of induction, the yeast cells were collected by
centrifugation, washed with ddH2O more than three times, and resuspended
in SDS-PAGE loading buffer, adding dithiothreitol (DTT) (Li et al.,
2014). After the cells were boiled and centrifuged to extract proteins
(12000×g, 10 min), and the supernatant was collected and used for
Western blotting analysis (An et al., 2014). Soak the PVDF membrane with
TBST (blocking solution) containing 5% skimmed milk powder, and seal it
with a shaker at room temperature for 2 h. Dilute the corresponding
primary antibody with blocking solution, soak the PVDF membrane in the
primary antibody incubation solution, and incubate overnight at 4°C.
TBST thoroughly washes the PVDF membrane 5-6 times to remove excess
primary antibody. Dilute the corresponding HRP-labeled secondary
antibody (anti-HA Tag Monoclonal antibody) (ZEN-BIOSCIENCE, China) with
blocking solution 1:50000 dilution, soak the PVDF membrane in the
secondary antibody (IgG goat anti-mouse antibody and HRP) incubation
solution, and incubate for 2 h at 37°C on a shaker. Mix the enhancement
solution in the ECL reagent with the stable peroxidase solution in a
ratio of 1:1, drop the working solution on the PVDF membrane, and react
for a few minutes until the fluorescence band was obvious, then was used
for the visualization of the protein band.
Fluorescence microscopic assay
The strains identified as positive by PCR were inoculated into SD-CAA
medium and cultured overnight, the bacterial solution was centrifuged,
and the strain was replaced with SG-CAA medium to induce expression at
20°C, 200 rpm, 48 h. HA-tag (9A3) Mouse mAb (ZEN-BIOSCIENCE) was used as
the primary antibody for immunostaining. Centrifuge 200μl of the
bacterial solution, incubate with 1:200 concentration of Anti-Mouse IgG
(H&L) (ZEN-BIOSCIENCE, China, 1:100 diluted with 1% bovine serum
albumin) for 1 h in the dark, and then use 1:100 concentration of
fluorescent secondary antibody (Goat anti-Mouse IgG) (H+L) Secondary
Antibody, DyLight 488) incubate 30 min for 1 h. Finally, the incubated
yeast cells were washed more than three times with PBS, and by the
fluorescence microscope (IMAGER Z1).
Flow cytometry assay
Induced cells were collected and washed three more times with PBS buffer
three times (pH 7.4). The cell density was then adjusted to
approximately 107cell/mL and 200 μL of the adjusted
cells were repelled at 12,000×g for 3 min at 4°C. After washing samples
with PBSA buffer for 3-5 times, resuspend it in HA-tag (9A3) Mouse mAb
(ZEN-BIOSCIENCE) in PBSA diluted at 1:200 for 1 h. At this time, it was
at room temperature and kept in dark conditions. After being washed five
times with PBS, the samples were resuspended in an Anti-Mouse IgG (H&L)
(ZEN-BIOSCIENCE, China) and incubated on ice for 1 h. Then, the cells
were washed five times and analyzed. In order to observe and determine
the expression activity, the incubated yeast cells were collected and
resuspended in 300µL PBSA, and analyzed with a Guava flow cytometer
(Millipore).
Enzyme activity of surface-displayed Aps
The Aps activity was measured in 50 mM Tris-HCl buffer (pH 9.0) at 60
°C, using 2 mM L-Leucine-p-nitroanilide (Leu-pNA) as the substrate. The
reaction was initiated by adding S.cerevisiae suspension
(OD600=1.0) to the preincubated substrate solution. A spectrophotometer
(Beckman 600) was used to monitor the increase in absorbance at 405 nm
for 20 min at intervals of 2 min to calculate the initial hydrolysis
rate (Liang et al., 2019). One unit (U) of the Aps activity was defined
as the amount of enzyme that produced 1 μmol of p-nitroaniline/min under
standard conditions.
Effect of temperature and pH on enzyme activity and exploring
the reusability
Using Leu-pNA as the substrate, the impact of temperature and pH on the
activity of Aps were determined by spectrophotometry. Under standard
analysis conditions, the optimal temperature for Aps activity was
determined by measuring the reaction rate in the range of 10 to 80°C.
The optimum pH for enzyme activity was determined at 60°C for 20 min
from pH 5.0 to 10.0 in different buffers (50 mM): citrate/phosphate
buffer (pH 5.0 to 8.0), Tris/HCl (pH 7.0 to 9.0), and boric acid/borax
(pH 9.0 to 10.0).
The reusability of the cells used for measuring the enzyme activity was
evaluated by putting the whole cells in a 50 mM Tris-HCl buffer (pH 9.0)
and incubating at 60°C for 20 min. Stop the reaction with a mixture of
ice and water and centrifuge at 10,000×g for 3 min. The harvested cells
were resuspended in the same volume of reaction mixture and reused for
up to ten subsequent reaction cycles.
Degradation of pyrethroid pesticide by
surface-displayed Aps
The reaction rate was determined by measuring the decrease in a flask
containing 4 mL and 50-300 mg·L-1 β-CP. The
engineering yeast cells were inoculated into the pesticide reaction
system to obtain the final cell density of approximately OD600 = 1.0.
The reaction took place at 60°C and was incubated on a shaker at a speed
of 150 rpm, and then the reaction was stopped by adding 0.5 mL of 1 M
HCl. Remaining β-CP was extricated and detected by an Ultimate 3000
high-performance liquid chromatography system (Idstein, Germany)
prepared with ultraviolet detector. The specific detection method refers
to the previous research report (Tang et al., 2015). Flasks were
prepared as described above, and all experiments included a blank
control, including cells that did not contain or were not induced. All
experiments were performed at least three times.
Under the above conditions, the ability of Aps displayed by yeast to
degrade other pyrethroids was also investigated.
Removal residual β-CP in vegetables using
surface-displayed Aps
The stock solution of β-CP was diluted with tap water to the
concentration of 500 mg/L. Then, lettuce and cherry tomatoes were
immersed in this β-CP solution for 30 min to allow the β-CP to be
absorbed into or attached to the vegetables. Then collect them and put
them in a fume hood to dry naturally. The sample was immersed in the
washing solution of 1% whole cell catalyst or tap water at 45°C for 90
minutes, and then washed with tap water once. After the washing process,
the vegetable samples are placed in a cool place to dry. The control
sample was not treated after β-CP coating.
First, the two vegetables were homogenized for 2 min. Then, a 20 g
portion of the slurry sample was weighed to a 250 mL conical flask and
extracted in 50 mL of acetonitrile for 30 min. The homogenate was
filtered with a glass filter under reduced pressure and was shaken for 1
min followed by the addition of 4 g of sodium chloride. The liquid phase
layer was allowed to separate and 10 mL of acetonitrile extracts were
taken, evaporated dryness with a water bath at 60°C, and dissolved with
2 mL of acetonitrile. The final extract was filtered through a 0.22 µm
membrane to HPLC analysis. All treatments were performed in triplicate.
Results and discussion
Construction of Aps displaying
S.cerevisiae
strain.
The purpose of this study research is to use a-agglutinin as an
anchoring system to display Aps from Pseudomonas aeruginosa GF31
on S.cerevisiae cell surface, construct new whole cell
biocatalysts and explore its degradation ability of pyrethroid
pesticides. The subcloning of Aps and its heterologous expression inE.coli have been reported (Tang et al., 2017), considering that
Aps from Pseudomonas aeruginosa GF31 is an extracellular enzyme,
in comparison with the strain harboring complete enzyme encoding gene,
the transformant harboring Aps gene lacking signal peptide and precursor
peptides may be conducive to the expression of the enzyme. Taking into
account the codon bias of S.cerevisiae , the codons of the Aps
encoding gene were optimized. The traditional yeast surface display
strategy was to express the target protein as a fusion with the
C-terminus of Aga2 subunit, e.g. pCT (Tao et al., 2016) vector, or
N-terminus, e.g. pTMY (Jones et al., 2011), pYD5 (Wang et al., 2005) and
pCHA (Mata-Fink et al., 2013) vectors. In our study, Aps was taken as
the target protein, fused to the
N-terminus and C-terminus of Aga2 subunit respectively, considering that
the fusion site will affect the display quantity and activity of Aps on
yeast cell surface. Therefore, two
kinds of surface display vectors were constructed (called
pYD1-Aga2N-Aps and
pYD1-Aga2C-Aps). In order to detect the fusion protein in Western blot
and Flow cytometry, the epitope tag of human influenza hemagglutinin
(HA) was inserted into the downstream of Aps sequence. The fusion mode
between the N-terminal or C-terminal of the target protein Aps and the
anchoring motif Aga2 may be the main reason affecting the enzyme
activity. The expression process was regulated by the GAL1 promoter.
Beneath the induction of galactose, the Aps melded with HA epitope tag
and Aga2 was expressed, and the N-terminal secretion signal sequence of
Aga2 actuates the conveyance of the fusion protein to the exterior of
the cell. At long last, the fusion protein was automatically immobilized
on the surface of yeast cells through two disulfide bonds. The disulfide
bond covalently binds Aga2 and Aga1, and Aga1 was fixed on the cell wall
via the GPI anchor (Fig. 1 ).
Expression of Aps in the recombinant S.cerevisiae
strain
The expression pattern of fusion protein was detected by twelve alkyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results
showed that there was a band corresponding to Aga2N-Aps and Aga2C-Aps at
64 kDa and 62 kDa, respectively, which appeared on S.cerevisiaecells containing pYD1-Aga2N-Aps (Fig. 2A , Lane 3 , arrow) and
pYD1-Aga2C-Aps (Fig. 2A ,
Lane 4, arrow). These results indicate that the synthesized fusion
protein appeared correctly expressed in S.cerevisiae engineering
strains, and the corresponding protein size was consistent with the
calculated molecular weight.
For the recombinant plasmids, pYD1-Aga2N-Aps and pYD1-Aga2C-Aps, the HA
tag was fused to the C-terminus or N-terminus of the displayed-Aps.
Western blot examination with anti HA monoclonal antibody appeared that
Aga2N-Aps and Aga2C-Aps fusion proteins may well be recognized in yeast
cells after induction, and the molecular weight of the protein band was
steady with that expected (Fig. 2B , Line 2, 4, arrow), which
means that the Aps of N-terminal fusion and C-terminal fusion were
successfully expressed in S.cerevisiae recombinant strains
(Fig. 2B , Line 2, 4, arrow). Moreover, the protein bands were
also detected by Western blot analysis of DTT treated protein solution
(Fig. 2B , Line 5, 6, arrow), which indicated that the fusion
proteins Aga2N-Aps and Aga2C-Aps were exactly expressed on the surface
of S.cerevisiae cells, because DTT can break the disulfide bond,
the protein displayed on the surface can be eluted. Interestingly, no
matter in the results of SDS-PAGE or Western blot, similar protein bands
with the fusion proteins Aga2N-Aps and Aga2C-Aps were also observed in
the samples of uninduced S.cerevisiae engineering strains
(Fig. 2A , Lane 1, 2; Fig. 2B , Lane 1, 3), but the
degree of expression was weaker than that after induction, which
indicated that the fusion protein had background expression even in the
uninduced state (Detzel et al., 2013).
Surface localization analysis of Aps
The presence of Aga2N-Aps and Aga2C-Aps on the surface ofS.cerevisiae cells was further confirmed by indirect
immunofluorescence (Fig. 3 ). When observed under the
fluorescence microscope, the cells carrying pYD1-Aga2N-Aps and
pYD1-Aga2C-Aps plasmids showed green fluorescence on the cell surface
after induction (Fig. 3 ), which confirmed the correct position
of the protein on the cell surface. The induced cells carrying the
plasmid pYD1-Aga2N-Aps showed stronger fluorescence intensity than those
carrying the plasmid pYD1-Aga2C-Aps (Fig. 3B and D ), which
preliminarily indicated that the N-terminal fusion of Aga2 with the
target protein Aps was more conducive to its successful expression and
display on the cell surface.
Then, the recombinant yeast strains were investigated by flow cytometry,
and the recombinant protein Aga2-Aps displayed on the surface was
characterized (Fig. 4 ). The primary antibody that recognizes
the HA epitope and the Goat Anti-Mouse IgG (H&L) secondary antibody
were used to detect and label the fusion protein. In the flow cytometry
data, the area under the peaks indicates the number of gated cells, and
the fluorescence (FL) intensity reflects the number of fluorescent
labels on a single cell. In this case, the relative density of Aps was
represented on the surface. In the given population, the proportion of
yeast cells Aga2N-Aps displaying (Fig. 4C ) accounted for
76.87%, and the proportion of yeast cells Aga2C-Aps displaying
(Fig. 4B ) accounted for 41.70%. The proportion of yeast cells
Aga2N-Aps displaying was about 35% higher than that showing Aga2C-Aps,
which confirmed that the fusion target protein Aps at the N-terminal of
Aga2 was indeed more conducive to the successful expression on the
surface of S.cerevisiae than that at the C-terminal.
Characteristics of
surface displayed
Aps
The optimal temperature and pH of two whole-cell biocatalysts in the
reaction system were investigated. As shown in Fig. 5A , the
activity of Aps enzyme of yeast carrying plasmids pYD1-Aga2N-Aps and
pYD1-Aga2C-Aps increased linearly from 10°C to 60°C and the maximal
activity was detected at 60°C. When the temperature reaches 80°C, the
enzyme activity of EBY100-pYD1-Aga2N-Aps still reaches 73.3% of the
highest activity, it shows that the Aps displayed on the surface has
good heat resistance, which is beneficial for subsequent environmental
remediation. Although the maximum enzyme activity of the yeast cells
expressing Aga2C-Aps fusion protein was only 53.57% of that of the
yeast cells expressing Aga2N-Aps, the enzyme activity of the two
engineering yeasts reached the maximum at 60°C, and maintained good
enzyme activity in the range of 40 to 80°C. The optimal pH for the two
entire cell biocatalysts were decided at pH 9.0 and more than 50% of
enzymatic activity was kept at pH values extending from pH 7.0 to 10.0
(Fig. 5B and C ). Under the two optimal conditions, the Aps
activities of whole cells expressing Aga2N-Aps and Aga2C-Aps were 0.25
U/OD600/mL and 0.12 U/OD600/mL.
For surface expression approach, the growth inhibition of cell should be
taken into consideration. To determine whether the surface display of
Aga2N-Aps and Aga2C-Aps fusion protein inhibits growth of the cell,
growth profile of S.cerevisiae strain carrying
pYD1-Aga2N-Aps、pYD1-Aga2N-Aps and without vector was compared. The
final cell density of the three strains was not much different after 60
h of induction (the final OD600 value only differed by 0.3), and no
growth inhibition was observed for the cells expressing the Aga2-Aps
fusion protein (Fig. 5D ). During the cultivation of the
recombinant EBY100 strain, the inducer galactose could induce the
surface display of Aps. Some scholars pointed out that the induction
time has a significant impact on the biological activity of the
displayed enzyme. The optimal induction time depends on the target
protein, vector and host strain used in the surface display system (Chen
et al., 2020). Fig. 5E shows that the enzyme activity of Aps
increases with time from 0-48 h after induction, and the Aps activity
reaches its maximum value after 48 h induction. Then, a significant
decrease in Aps activity was observed after induction for more than 48
h. The results showed that the induction time had an effect on the
expression level of Aps on the surface of yeast. It was not difficult to
speculate that during the growth period of the cell, the displayed-Aps
accumulated continuously as the amount of cells increases, as shown inFig. 5D. Hence, we chose 48 h as the induction time for
producing the whole-cell biocatalyst within following studies.
The reusability of surface displayed Aps
In order to test the operational stability of two biocatalysts, which
express Aga2N-Aps and Aga2C-Aps fusion proteins respectively, during
repeated use, we investigated the activity of two biocatalysts in
multiple reaction cycles. As shown in Fig. 6 , the biocatalyst
activity of Aps displayed on the surface of yeast well maintained ten
consecutive reactions. After five consecutive reactions, the whole cell
catalyst expressing Aga2N-Aps maintained more than 80% of the activity,
and after ten consecutive reactions, the activity still reached more
than 70% of the initial activity. Although the overall enzyme activity
of the whole cell catalyst expressing Aga2C-Aps was lower than that of
the whole cell catalyst expressing Aga2N-Aps, it still maintains more
than 70% of the initial activity after ten consecutive reactions,
indicating that both whole-cell biocatalysts have good properties of
reusability. Generally speaking, when complex protein purification
procedures (protein ultrafiltration or column chromatography, etc)
cannot be carried out, it is difficult to separate a variety of soluble
functional enzymes from the system, and the recovery rate is low, which
will affect the reusability of free enzymes (Chen et al., 2016). While,
the biocatalyst constructed on yeast host cells to express the target
protein could be considered as 5-10 μm particles, coated with a
functional coating, and could be separated by simple centrifugation and
washed with buffer and reused.
Degradation of pyrethroid by recombinant S.cerevisiae
strain
This study also explored the effect of substrate concentration on the
biodegradation of whole-cell catalysts when β-CP, the most commonly used
among highly hydrophobic pyrethroid pesticides, was used as the
substrate. As shown in Fig. 7A , the whole-cell catalysts
expressing Aga2N-Aps and Aga2C-Aps could degrade 200
mg·L-1 β-CP at a maximum rate of 33.16
μmol·L-1·day-1 and 28.99
μmol·L-1·day-1. The degradation rate
catalyzed increased as the substrate concentration increased from 50 to
200 mg·L-1, however, the degradation rate decreased if
the substrate concentration continues to increase. Although the
substrate β-CP concentration was as high as 300
mg·L-1, the whole-cell catalysts expressing Aga2N-Aps
and Aga2C-Aps still has the degradation ability of 25.39
μmol·L-1·day-1 and 19.65
μmol·L-1·day-1 . Therefore, the
results show that in our research, the surface display catalysts with
two different fusion modes constructed based on cell surface display
technology have a wide range of substrate concentration during the
hydrolysis process with β-CP as the substrate. And high substrate
concentration tolerance, provides a new possibility for effective
restoration of water and soil contaminated by β-CP in the future.
In order to investigate the ability to degrade various pyrethroids, five
commonly pyrethroids were used to evaluate the substrate specificity of
displaying Aps (respectively: β-cypermethrin, fenpropathrin,
deltamethrin, cyfluthrin, alphacypermethrin). As shown in Fig.
7B , the two whole cell catalysts had obvious degradation effects on
five commonly used pyrethroid pesticide substrates. And that, the two
whole cell catalysts show the best degradation ability to
β-CP, followed by fenpropathrin.
We also found that there was no significant difference in the enzyme
activity of Aps when Aps fused to the N-terminus and C-terminus in the
reaction system with pesticide as substrate compared to the reaction
system with Leu-pNA as substrate. It is speculated that the mass
transfer ability of substrate may play a major role in the reaction of
pesticide degradation. The two whole cell catalysts can hydrolyze five
different pyrethroid pesticides, indicating that the displayed Aps is a
broad-spectrum pyrethroid hydrolase. Since the residual pesticide was a
mixture in the environment, the broad-spectrum pyrethroid-degrading
whole cell catalyst would have a wide range of application prospects in
practical applications (Hu et al., 2019).
Removal residual β-CP in vegetables using
surface-displayed Aps
Washing vegetables with whole cell catalyst solution was more effective
than washing with tap water in reducing β-CP residues (Figure
8 ). In the process of removing high concentration pesticide residues,
after washing in two whole cell catalyst solutions of Aga2N-Aps and
Aga2C-Aps, the β-CP residues on cherry tomato decreased from 278.41
mg·kg-1 to 86.31 mg·kg-1 and 105.82
mg·kg-1 respectively, and the β-CP residues on lettuce
decreased from 337.10 mg·kg-1 to 138.22
mg·kg-1 and 157.99 mg·kg-1respectively (Figure 8A ). However, only by washing with tap
water, the residue removal rates were 2.91% and 7.70%, respectively.
In most cases, pesticide will remain on the surface of vegetables and
fruits. Usually, the pesticide residues are removed only by washing with
tap water, and the high hydrophobicity of β-CP makes it difficult to be
removed directly by tap water. Frying and frying are more effective in
reducing residues. However, some vegetables, such as cucumber, lettuce
and fresh fruit, are usually eaten raw without frying or frying.
Therefore, it is very important to develop a new washing solution to
remove pesticide residues from vegetables. It is found in the report
that purified and concentrated extracellular enzyme solution is
effective for the removal of β-CP. In this study, the two whole-cell
catalysts only need a simple cell collection process to achieve the same
purpose, omitting the complex and cumbersome enzyme purification
process, which has more practical significance.
Countries all over the world have set strict standards for pesticide
residues in vegetables, especially edible vegetables and fruits with
skin, which lead to the inability to apply pesticides before picking
these vegetables and fruits, affecting the yield and quality of
agricultural products. Pyrethroids are commonly used as pesticides for
vegetable and fruit insecticides. Because of their high hydrophobicity,
it is difficult to remove them by simple tap water washing. When the
pesticide residue concentration in cherry tomato and lettuce is
relatively low, tap water washing can only remove 7.39% and 10.38%.
Through the further exploration of two whole cell catalysts Aga2N-Aps
and Aga2C-Aps, it is found that these two whole cell catalysts not only
have obvious removal effect on vegetables and fruits with high
concentration of pesticide residues, but also maintain good degradation
ability in vegetables and fruits with low concentration of pesticide
residues. After washing cherry tomato and lettuce treated with low
concentration of β-CP in two whole cell catalyst solutions, the
pesticide residues on cherry tomato decreased from 4.87
mg·kg-1 to 1.36 mg·kg-1 and 1.51
mg·kg-1, and on lettuce from 5.59
mg·kg-1 to 1.84 mg·kg-1 and 1.81
mg·kg-1 respectively (Figure 8B ). The
residual concentration of β-CP in cherry tomato and leaf lettuce treated
with whole cell catalyst has been lower than the Chinese pesticide
residue limit standard (GB2763-2021), showing a good pesticide treatment
effect. Interestingly, this phenomenon is different from that previously
reported. The strains reported by Karpouzas and Walker (Karpouzas et
al., 2001), showed significant differences in degradation rates when
degrading high concentration pesticide substrates and low concentration
pesticide substrates. In addition, the whole cell catalyst displayed on
the surface of yeast has a good practical application prospect because
of its good biosafety and lower price compared with purified enzyme. The
yeast surface display whole cell catalyst constructed in this paper has
good treatment effect on vegetables and fruits with high and low
concentrations of pesticide residues. It has application potential both
in agricultural production enterprises and as a household vegetable and
fruit cleaning agent.
Conclusions
In this study, a-agglutinin was used as the anchor protein, and Aps was
successfully displayed on the surface of S.cerevisiae cells by
N-terminal fusion and C-terminal fusion respectively. Immunofluorescence
analysis and flow cytometry analysis showed that Aps was anchored on the
surface of yeast. Under the optimal reaction conditions of
pH=9, 60°C, the enzyme activities of
the two whole-cell catalysts reached 0.25 U/OD600/mL and 0.12
U/OD600/mL, respectively. In addition, the two whole-cell catalysts can
still maintain more than 70% activity after repeated use for 10 times,
and have a good reusability. It was found that the two whole-cell
catalysts can also remove β-CP residues in vegetables and might be used
for wash solutions in the future. Moreover, displayed-Aps is a
bifunctional enzyme, which can degrade multiple pyrethroid pesticides
and has the potential to remediate waste protein pollution. The broad
spectrum of the substrate, good thermal stability and pH adaptation
range also illustrate its high potential in restoring complex
environmental scenes.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (No. 21966007), and the Natural Science Foundation of Guangxi
Province (No. 2018GXNSFAA281278). The authors thank other members of our
laboratory for providing scientific assistance and helpful discussion.