Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Enhancing secretion of polyethylene terephthalate

hydrolase PETase in Bacillus subtilis WB600 mediated by the
SPamy signal peptide
N. Wang1,2,†, F. Guan2,†, X. Lv1, D. Han3 , Y. Zhang2, N. Wu2, X. Xia1 and J. Tian2
1 School of Biotechnology, Jiangnan University, Jiangsu Wuxi, China
2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China
3 Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China

Significance and Impact of the Study: High-level expression of polyethylene terephthalate hydrolase
(PETase) facilitates biodegradation of PET. In this study, the expression elements, signal peptide and
promoter, in the secretory expression system, were optimizing for maximizing secreted expression of
PETase in Bacillus subtilis. The constructed strains yielded the greatest bis-(2-hydroxyethyl) terephthalate
degradation and PET-film etching activities.

Keywords
PETase, Bacillus subtilis, signal peptide,
promoter, biodegradation.
Correspondence
Xiaole Xia, School of Biotechnology, Jiangnan
University, Jiangsu Wuxi 214122, China.
E-mail: xiaolexia@jiangnan.edu.cn
Jian Tian, Biotechnology Research Institute,
Chinese Academy of Agricultural Sciences,
Beijing 100081, China.
E-mail: tianjian@caas.cn
†
These authors contributed equally to this
work.
2020/0289: received 16 February 2020,
revised 28 April 2020 and accepted 4 May
2020
Abstract
The polyethylene terephthalate hydrolase (PETase) has been proved to have a
high activity to degrade polyethylene terephthalate (PET), but few studies have
been carried on its secretion in Bacillus subtilis. In this study, the coding gene
of PETase, which was isolated from the Ideonella sakaiensis, was synthesized
and expressed in B. subtilis. Then, we evaluated the ability of five Bacillus
signal peptides to enhance PETase secretion by B. subtilis. The results indicated
that the SPamy-induced secretion of PETase was the highest, and its activity
against p-Nitrophenyl palmitate was about fourfold that of the natural signal
peptide SPPETase. The weak promoter P43 provided sufficient time for
translation and folding of PETase, resulting in increased extracellular
expression. Use of P43 and SPamy in combination yielded the greatest bis-(2-
hydroxyethyl) terephthalate degradation and PET-film etching activity due to
maximized secretion of PETase by B. subtilis. Our findings will facilitate
biodegradation of PET plastic.

doi:10.1111/lam.13312

seafood and other means, posing a threat to human

Introduction
Thermoplastics have been produced from fossil raw mate-
rials since the 1940s and are widely used in packaging
and textiles (Fecker et al. 2018). Polyethylene terephtha-
late (PET), a polymer of diethylene terephthalate that has
high heat resistance and toughness (Taniguchi et al.
2019), is used in, for instance, PET film and bottles.
Moreover, a large amount of PET plastics flow into the
ocean every year, harming marine animals and forming
particles (Horton et al. 2017) that enter the body through
health and the environment (Son et al. 2019). The cur-
rently used chemical treatments are costly (Han et al.
2017) and produce toxic secondary pollutants (Austin
et al. 2018). In contrast, biological methods are more
environmentally friendly and thus show promise for
reducing PET pollution (Parisi et al. 2019).
The biodegradation of PET has been investigated exten-
sively (Chen et al. 2018; Kawai et al. 2019). Bacterial
hydrolases such as cutinases, esterases and car-
boxylesterases hydrolyse PET (Perz et al. 2016; Joo et al.

Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology 235

SPamy enhances secretion of PETase
2018). In 2016, Yoshida et al. (2016) identified a new
PET-degrading bacterium, Ideonella sakaiensis, which pro-
duced two a/b hydrolases, PETase and MHETase, and
they could hydrolyse PET to terephthalic acid and ethy-
lene glycol. Subsequently, Han et al. (2017) and Joo et al.
(2018) analysed the crystal structure of PETase and
revealed the mechanism of its degradation. In 2019, Seo
et al. (2019) confirmed that PETase hydrolyses PET at a
rate 5
5-fold to 120-fold higher than other enzymes, and
the mutant (S121E/D186H/R280A) designed by them had
13
9-fold increased degradation activity after incubation
for 72 h (Son et al. 2019).
The above studies focused on the mechanism of
PETase. Few engineered bacteria or enzyme preparations
can directly degrade PET film because it is a high-molec-
ular-weight polymer and cannot penetrate the cell mem-
brane. Moreover, PETase secretion by Ideonella sakaiensis
is very low (Huang et al. 2018). Bacillus subtilis is a well-
known biosafety strain with clear genetic background and
mature genetic manipulation methods. It secretes proteins
directly into the culture supernatant and is widely used in
industrial production of various heterologous enzymes
(van Dijl and Hecker 2013), such as pullulanase (Zhang
et al. 2018) and b-mannanase (Song et al. 2017). Also,
the spores have low water content, high temperature
resistance and tolerance to environmental factors. These
advantages make B. subtilis useful for exogenous expres-
sion (Gu et al. 2018), in which the promoter (Hirooka
and Tamano 2018) and signal peptide (Liu et al. 2019)
are important factors. In 2018, Huang et al. used six sig-
nal peptides, including the natural signal peptide of
PETase, to mediate the exogenous expression of PETase
in B. subtilis 168. The natural signal peptide SPPETase, a
twin-arginine signal peptide, showed the highest amount
of secretion in a manner not dependent on the twin-argi-
nine translocation pathway (Huang et al. 2018). SPPETase
is not native to B. subtilis, resulting in limited exogenous
expression of PETase.
In this study, B. subtilis WB600 was used to exoge-
nously express PETase. The B. subtilis WB600 strain is a
variant derived from B. subtilis 168 with 6 protease genes
be knocked out, which can significantly improve the sta-
bility of secreted proteins. SPPETase and the Bacillus-
derived signal peptides SPAprE, SPapr, SPBprA, SPSacC and
SPamy were screened to overcome the low yield and activ-
ity of PETase in B. subtilis. SPamy-mediated PETase secre-
tion was the highest, and the p-Nitrophenyl palmitate
hydrolysis activity in the supernatant was about fourfold
higher than that of SPPETase. The weak promoter P43 pro-
vided sufficient time for translation and folding of
PETase, resulting in increased extracellular expression.
HPLC and SEM showed that greater secretion of PETase
was associated with higher bis-(2-hydroxyethyl)
236 Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology
1472765x, 2020, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1111/lam.13312 by National Taiwan University, Wiley Online Library on [06/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
N. Wang et al.
terephthalate (BHET) degradation and PET-film etching
activity. Our data show that it is possible to engineer
PET-degrading bacteria using B. subtilis WB600, and this
will facilitate the biodegradation of PET plastic.
Results and discussion
Determination of the optimum temperature of the
engineered bacteria
We investigated the growth and PETase secretion of
WB600-P43-SPamy at 22, 28 and 37°C. The OD600 of the
recombinant strain and the PETase activity in the super-
natant were assayed over 48 h. The recombinant strain
grew more rapidly at 37°C than at 28°C. However, at
37°C, PETase activity increased rapidly from 0 to 12 h,
peaking at 237
3 U ml 1 at 12 h. However, it slowly
decreased over the next 36 h and lowered to 50% of the
peak value at 32 h, possibly due to heat-induced degrada-
tion of PETase (Fig. 1). At 22°C, PETase activity
increased, but at a rate lower than that at 28°C (Fig. 1b).
At 28°C, PETase activity increased from 0 to 28 h, peak-
ing at 310
82 U ml 1 at 28 h; it did not decrease signifi-
cantly during the subsequent 20 h. Thus, 28°C was
regarded as optimum for the recombinant strain in terms
of growth and secreted PETase activity and was used for
culturing in subsequent experiments.
Effects of signal peptides on the secretory expression of
PETase
To maximize extracellular secretion, five Bacillus signal
peptide fragments, that is, SPAprE, SPapr, SPBprA, SPSacC
and SPamy, were fused to the N-terminus of PETase to
mediate its extracellular secretion under the control of the
P43 promoter. The five signal peptides belong to the Sec
pathway, which are different to the twin-arginine natural
signal of PETase. The natural signal peptide SPPETase was
used as the control (Table S3). The recombinant plasmid
was introduced into B. subtilis WB600, and transformants
were selected and named WB600-P43-SPAprE, WB600-
P43-SPapr, WB600-P43-SPBprA, WB600-P43-SPSacC,
WB600-P43-SPamy and WB600-P43-SPPETase (Table S2).
Next, we evaluated the effects of the signal peptides on
the activity and expression of PETase. The trend in
PETase activity over time was similar in the supernatants
of all engineered strains. The activity increased rapidly
from 0 to 12 h and then more slowly from 12 to 28 h.
After 28 h, the activity stabilized, and it began to decrease
at around 40 h. In addition, after 24 h, the PETase activ-
ity in the WB600-P43-SPamy supernatant was approxi-
mately fourfold that of SPPETase, and the secretion effects
of the five signal peptides selected in this study were
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N. Wang et al. SPamy enhances secretion of PETase

(a) (b) 350

1·0
300
0·8

PETase (U ml–1)
250

0·6 200
OD600

0·4 150

100
0·2
50
0·0
0
0 10 20 30 40 50 10 20 30 40 50
Time (h) Time (h)

Figure 1 Growth of WB600-P43-SPamy according to temperature and secreted polyethylene terephthalate hydrolase (PETase) activity. (a) Growth
and (b) secreted PETase activity of WB600-P43-SPamy according to temperature. ( ) 22°C; ( ) 28°C; ( ) 37°C. [Colour figure can be viewed
at wileyonlinelibrary.com]

significantly higher than that of SPPETase; among these,
the SPamy showed the greatest contribution to the PETase
secretion (Fig. 2a).
Western blotting showed that PETase was present in
the culture supernatants of all of the engineered bacteria.
SPamy had the greatest effect on secretion, followed by
SPSacC, SPapr, SPBprA, SPAprE and SPPETase (Fig. 2b). How-
ever, in Huang’s study, no production was obtained with
the signal peptide SPAmyE from B. subtilis. The signal pep-
tide of SPamy used in this study comes from the amylase
(a) 350
300
SPamy
PETase (U ml–1)
of B. Amyloliquefaciens. Although they are all amylases’
signal peptide, they belong to different strains and have
different coding sequences (Fig. S1), thus resulting in
such large secretion differences.
Effects of promoters on secretory expression of PETase
We used the constitutive promoters P43 and Pylb (Yu et al.
2015) to drive the expression of PETase in B. subtilis. The
engineered bacteria were named WB600-P43-SPamy and
WB600-Pylb-SPamy (Table S2). The trend in supernatant
did not differ according to fermentation duration. Although
Pylb showed activity about eightfold that of P43 (Yu et al.
2015), the activity of P43 was about 15% higher than that of

250 SPapr Pylb (Fig. 3a). Western blotting at 24 h confirmed that the
SPSacC
200 PETase expression induced by P43 was higher than that of
Pylb, consistent with the activity assay (Fig. 3b). Thus,
150 PETase secretion does not require a strong promoter, and
SPBprA
100 SPAprE WB600-P43-SPamy exhibited the highest PETase expression,
suggesting that P43 and SPamy are optimal for maximizing
50 SPPETase
secreted expression of PETase in B. subtilis.
0
10 20 30 40 50
(b) Activity of the secreted PETase against BHET
Time (h)
To evaluate the ability of the engineered bacteria to
degrade plastics, their hydrolysis of BHET, the monomer
of polyethylene terephthalate, was assayed. The engineered
bacteria (signal peptides, WB600-P43-SPAprE, WB600-
Figure 2 Activity and expression of the polyethylene terephthalate
hydrolase (PETase) according to signal peptide. (a) Activity of PETase
P43-SPapr, WB600-P43-SPBprA, WB600-P43-SPSacC,
according to signal peptide. (b) Quantity of PETase in supernatant WB600-P43-SPamy and WB600-P43-SPPETase; promoters,
according to signal peptide. 1–6: WB600-P43-SPPETase, WB600-P43- WB600-P43-SPamy and WB600-Pylb-SPamy) were cultured
SPAprE, WB600-P43-SPapr, WB600-P43-SPBprA, WB600-P43-SPSacC and at 28°C for 24 h, and the supernatant was used to hydrol-
WB600-P43-SPamy. [Colour figure can be viewed at wileyonlinelibra yse BHET at 30°C for 2 h. Two hydrolysates were
ry.com]

Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology 237

SPamy enhances secretion of PETase
(a)
300
250
PETase (U ml–1)
200
150
100
50
0 Materials and methods
10 20 30 40
Time (h)
(b)
Figure 3 Activity and expression of polyethylene terephthalate hydro-
lase (PETase) according to promoter. (a) PETase activity according to
promoter. (b) Quantity of PETase in the supernatant according to pro-
moter. 1–2: WB600-Pylb-SPamy and WB600-P43-SPamy, respectively.
[Colour figure can be viewed at wileyonlinelibrary.com]
detected after degradation of BHET for 2 h (Fig. S2). The
residual amount of BHET in the supernatant of WB600-
P43-SPPETase was about eightfold that of WB600-P43-
SPamy and WB600-P43-SPSacC, sevenfold that of WB600-
P43-SPapr, and 1
5-fold that of WB600-P43-SPBprA and
WB600-P43-SPAprE. Combining the previous results of p-
NPP degradation (Fig. 2a), SPamy resulted in the highest
secretion of PETase. Additionally, the signal peptides were
superior to the natural signal peptide of PETase (Fig. 4a).
The remnant BHET in the supernatant of WB600-P43-
SPamy was half that of WB600-Pylb-SPamy (Fig. 4b), prov-
ing that BHET was consumed more in WB600-P43-SPamy
fermentation broth and the activity of PETase was stron-
ger, further demonstrating that P43 was more suitable for
starting PETase to transcript in B. subtilis. Huang
reported that SPPETase resulted in the greatest promotion
of secretion by B. subtilis (Huang et al. 2018), but the five
signal peptides in this study were superior to SPPETase,
and SPamy resulted in the greatest secretion of PETase
from B. subtilis.
Degradation of PET film by secreted PETase
The engineered bacteria WB600-P43-SPamy and WB600-
P43-SPPETase were directly cultured in a shake flask at
238
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N. Wang et al.
28°C for 24 h and applied to a sterile PET film for 36 h.
P43 The surface morphology of the PET film was observed by
Pylb SEM. Compared with the control (Fig. 4c), WB600-P43-
SPamy caused morphological changes on the surface of the
PET film; furthermore, pores appeared, suggesting serious
corrosion (Fig. 4e). In contrast, WB600-P43-SPPETase
caused less corrosion on the surface of PET film (Fig. 4d).
The spots showed that the secreted PETase expressed in
B. subtilis could degrade PET plastics.
50
General
p-Nitrophenyl palmitate (p-NPP) was purchased from
Huaxia Yuanyang Technology Co., Ltd. (Beijing, China).
BHET (Sigma-Aldrich, St. Louis, MO, USA) was pur-
chased from Yinuokai Technology Co., Ltd. (Beijing,
China). PET films (0
0125-mm thickness) were purchased
from DuPont (France). Other commonly used reagents
and consumables are stored in the laboratory (Ding et al.
2019). The plasmids, strains, signal peptide sources and
primers are listed in Tables S1–S4, respectively.
Expression, purification and detection of PETase in
Escherichia coli
SignalP-5.0 software (Fecker et al. 2018) (http://www.cb
s.dtu.dk/services/SignalP/) was used to predict the signal
peptide of PETase (SPPETase), which comprises the first 27
amino acids at the N-terminus. The gene encoding
PETase without a signal peptide was synthesized and
ligated into pET27b (+) at the NcoI/HindIII sites to gen-
erate pET27b (+)-PETase by GenScript (Nanjing, China).
Next, the recombinant plasmid was transformed into
E. coli BL21(DE3) according to standard procedures (Rus-
sell et al. 2001), yielding BL21-PETase and then the
PETase was expressed and purified according to Yoshida
et al. (2016). Purified PETase (2 mg) was mixed with Fre-
und’s complete adjuvant (Sigma) and injected into a
healthy mouse three times per week. Antiserum was col-
lected 1 month later and used as a polyclonal antibody to
PETase (Wang et al. 2014) for Western blotting (Wang
et al. 2008).
Construction of the engineered strain and determination
of its optimum temperature
P43-SPamy (P43, promoter of the moderate-temperature
amylase from B. subtilis; SPamy, amy signal peptide from
Bacillus amyloliquefaciens; GenBank, CP041693.1) frag-
ment was amplified from a laboratory-preserved plasmid
using the primers P43-F(XbaI)/amy-R. The PETase-
Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology
 1472765x, 2020, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1111/lam.13312 by National Taiwan University, Wiley Online Library on [06/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
N. Wang et al. SPamy enhances secretion of PETase

(a) (b)
16 000 16 000

14 000 14 000

12 000 12 000
Peak area

Peak area
10 000 10 000

8000 8000

6000 6000
4000 4000
2000 2000
0 0
Con AprE apr BprA SacC amy SPPETase Con P43 Pylb

(c) (d) (e)

NONE SEI 10·0 kv X20,000 1 µm WD 6.1 mm NONE SEI 10·0 kv X20,000 1 µm WD 6.1 mm NONE SEI 10·0 kv X20,000 1 µm WD 6·1 mm

Figure 4 Bis-(2-hydroxyethyl) terephthalate (BHET) and polyethylene terephthalate (PET) film degradation activity according to signal peptide and
promoter. (a) Residual BHET after degradation by polyethylene terephthalate hydrolase (PETase) with different signal peptides. The peak areas
were 15 273
90  137
32 (Con), 9653
9  364
16 (AprE), 1881
85  107
27 (apr), 8745
00  314
38 (BprA), 1615
15  200
54 (SacC),
1645
15  259
01 (amy) and 13 449
5  655
49 (PETase), respectively. (b) Residual BHET after degradation by PETase with different promoters.
The peak areas were 15 273
90  137
32 (Con), 1600
80  95
18 (P43) and 3211
15  210
79 (Pylb), respectively, Con, control. Degradation
of PET film treated with (c) LB medium (d) WB600-P43-SPPETase (e) WB600-P43-SPamy, as visualized by SEM. [Colour figure can be viewed at wile
yonlinelibrary.com]

encoding gene was amplified from pET27b(+)-PETase
using the primers PETase-F/PETase-R. The primers
TER1-F/TER1-R (HindIII) were used to amplify TamyL1
(a terminator from amylase of B. subtilis) from the stored
plasmid. Finally, the primers P43-F(Xbal)/TER1-R (Hin-
dIII) were used to amplify the P43-SPamy-PETase-TamyL1
fragment by overlapping PCR, followed by ligation to
pUBC19 by XbaI and HindIII to generate pUBC19-P43-
SPamy-PETase. The recombinant plasmid was transferred
into competent B. subtilis WB600 cells to produce the
engineered strain WB600-P43-SPamy (Ding et al. 2019).
WB600-P43-SPamy was cultured at 22, 28 and 37°C in
LB medium at 200 rev min 1. The OD600 was determined
at 4, 6, 8, 10, 12, 14, 16, 24, 28, 32, 40 and 48 h to moni-
tor growth. The PETase activity in the supernatant at 4,
8, 12, 16, 24, 28, 32, 40 and 48 h was measured by the p-
NPP method. The enzyme reaction containing 1
8 ml of
Tris-HCl (50 mmol l 1, pH 8
0) reaction buffer mixed
with 100 ll of p-NPP (8 lmol l 1 ml 1) was preheated
at 37°C for 5 min, and 100 ll of PETase solution was
added for 10 min. Finally, the reaction was stopped with
500 ll of trichloroacetic acid (TCA) (M/V, 10%), devel-
oped with 500 ll of Na2CO3 (M/V, 10%), and the pro-
duct p-nitrophenol (p-NP) was continuously monitored
at a wavelength of 410 nm. A standard curve was gener-
ated based on the absorbance at 410 nm of 0, 5, 10, 15,
20, 30, 40, 60, 80 and 100 lmol l 1 p-NP (Fig. S3).
Signal peptide screening
The amy, SacC (from B. subtilis 168; GenBank,
NP_390581.1), AprE (from B. subtilis 168; GenBank,
NP_388911.2), apr (from B. licheniformis WX-02; Gen-
Bank, AKQ72288.1) and BprA (from B. licheniformis WX-
02, GenBank, AKQ72888.1) signal peptides and the natu-
ral signal peptide of PETase (SPPETase) were used. The
P43-sp (sp, signal peptide) fragments were amplified from
PHY300PLK-P-sp-pul-TamyL (Wang et al. 2019) using
the primers P43-F(XbaI)/sp-R. PETase-TamyL1 was
amplified from pUBC19-P43-SPamy-PETase using the pri-
mers PETase-F/TER1-R (HindIII). After homologous
recombination of P43-sp and PETase-TamyL1, the

Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology 239

SPamy enhances secretion of PETase
product was ligated to pUBC19 by XbaI and HindIII to
generate the plasmid pUBC19-P43-sp-PETase (pUBC19-
P43-SPSacC-PETase, pUBC19-P43-SPAprE-PETase,
pUBC19-P43-SPBprA-PETase and pUBC19-P43-SPapr-
PETase; Table S1). The encoding gene of PETase with
SPPETase fragment was constructed as follows: P43 and the
PETase fragment without a signal peptide but with the
terminator TamyL1 were amplified from pUBC19-P43-
SPamy-PETase using the primers P43-F (XbaI)/P43-SPPE-
Tase-R and SPPETase-PETase-F/TER1-R (HindIII), respec-
tively. The SPPETase-encoding gene was synthesized by
GenScript, and the P43-SPPETase-PETase fragment was
generated by overlapping PCR using the primers P43-F
(XbaI)/TER1-R (HindIII). The fragment was digested with
XbaI/HindIII and ligated to pUBC19 to generate
pUBC19-P43-SPPETase-PETase. All of the recombinant
plasmids were transferred into B. subtilis WB600 to gener-
ate WB600-P43-SPAprE, WB600-P43-SPapr, WB600-P43-
SPBprA, WB600-P43-SPSacC and WB600-P43-SPPETase
(Table S2). All of the engineered strains were cultured in
LB medium at 28°C and 200 rev min 1. The supernatants
were collected at 4, 8, 16, 20, 24, 28, 32, 40 and 48 h; the
expression level of PETase was assayed according to
‘Expression, purification and detection of PETase in
E. coli’ and PETase activity against p-NPP according to
‘Construction of the engineered strain and determination
of its optimum temperature’.
Promoter screening
The promoter Pylb (Yu et al. 2015) was amplified from
stored plasmid using the primers Pylb-F(Xbal)/Pylb-
(amy)-R, and the SPamy-PETase-TamyL1 sequence was
obtained from pUBC19-P43-SPamy-PETase using the pri-
mers amy-F/TER1-R (HindIII). The Pylb and SPamy-
PETase-TamyL1 fragments were ligated by homologous
recombination to generate the fragment Pylb-SPamy-
PETase-TamyL1. The plasmid pUBC19-Pylb-SPamy-PETase
was generated by ligation at Xbal/HindIII after both of the
above fragment and pUBC19 plasmid digested with XbaI/
HindIII. The recombinant plasmid was transferred into B.
subtilis WB600, producing WB600-Pylb-SPamy. The
amount and activity of PETase expressed by WB600-Pylb-
SPamy and WB600-P43-SPamy were compared as described
in ‘Expression, purification and detection of PETase in
E. coli’ and ‘Construction of the engineered strain and
determination of its optimum temperature’, respectively.
Evaluation of the BHET degradation activity of PETase
We assessed the BHET degradation activity of PETase in
culture supernatant. The total reaction system was 600 ll,
comprising (final concentrations) 0
9 mmol l 1 BHET,
240 Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology
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N. Wang et al.
40 mmol l 1 Na2HPO4-HCl (pH 7
0), 80 mmol l 1
NaCl, 20% (v/v) DMSO and 2% (v/v) crude enzyme
solution. The mixture was incubated at 30°C for 2 h, fol-
lowed by adding 9
6 ll of NaH2PO4-HCl (pH 2
5) and
120 ll of DMSO, and the reaction was stopped by incu-
bation at 85°C for 10 min. The reaction mixture was
passed through a 2
2-lm filter and analysed by HPLC
over a 15-min period. The mobile phase was 5% glacial
acetic acid, 0 min; 25% methanol, 5 min; 60% methanol,
15 min; and 100% methanol. A Zorbax Eclipse Plus C18
column (Agilent Technologies Inc. Palo Alto, California,
USA) was used with a flow rate of 0
8 ml min 1, column
temperature of 35°C, the maximum pressure of 400 bar.
Scanning electron microscopy
To evaluate their ability to degrade PET film, WB600-
P43-SPamy and the control strain WB600-P43-SPPETase
were inoculated into LB medium and cultured at 28°C
and 200 rev min 1 for 24 h. A 1 9 1-cm PET membrane
was washed in 1% SDS and sterile water and then added
to the bacterial suspensions for 36 h. The PET films were
removed, washed in 1% SDS and sterile water, and
observed by scanning electron microscopy (SEM) at 1 lm
to detect corrosion.
Acknowledgements
We are grateful to the Institute of Zoology, Chinese Acad-
emy of Sciences for helping us with antibody preparation
experiments.
Conflict of Interest
No conflict of interest declared.
References
Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns,
F.L., Silveira, R.L., Pollard, B.C., Dominick, G. et al.
(2018) Characterization and engineering of a plastic-
degrading aromatic polyesterase. Proc Natl Acad Sci USA
115, E4350–E4357.
Chen, C.C., Han, X., Ko, T.P., Liu, W.D. and Guo, R.T. (2018)
Structural studies reveal the molecular mechanism of
PETase. FEBS J 285, 3717–3723.
Ding, Z.D., Guan, F.F., Yu, X.X., Li, Q.B., Wang, Q., Tian, J.
and Wu, N.F. (2019) Identification of the anchoring
protein SpoIIIJ for construction of the microbial cell
surface display system in Bacillus spp. Int J Biol Macromol
133, 614–623.
Fecker, T., Galaz-Davison, P., Engelberger, F., Narui, Y.,
Sotomayor, M., Parra, L.P. and Ramirez-Sarmiento, C.A.

N. Wang et al.
(2018) Active site flexibility as a hallmark for efficient PET
degradation by I-sakaiensis PETase. Biophys J 114, 1302–
1312.
Gu, Y., Xu, X.H., Wu, Y.K., Niu, T.F., Liu, Y.F., Li, J.H., Du,
G.C. and Liu, L. (2018) Advances and prospects of Bacillus
subtilis cellular factories: from rational design to industrial
applications. Metab Eng 50, 109–121.
Han, X., Liu, W.D., Huang, J.W., Ma, J.T., Zheng, Y.Y., Ko, T.P.,
Xu, L.M., Cheng, Y.S. et al. (2017) Structural insight into
catalytic mechanism of PET hydrolase. Nat Commun 8, 6.
Hirooka, K. and Tamano, A. (2018) Bacillus subtilis highly
efficient protein expression systems that are
chromosomally integrated and controllable by glucose and
rhamnose. Biosci Biotechnol Biochem 82, 1942–1954.
Horton, A.A., Walton, A., Spurgeon, D.J., Lahive, E. and
Svendsen, C. (2017) Microplastics in freshwater and
terrestrial environments: evaluating the current
understanding to identify the knowledge gaps and future
research priorities. Sci Total Environ 586, 127–141.
Huang, X., Cao, L., Qin, Z., Li, S., Kong, W. and Liu, Y.
(2018) Tat-independent secretion of polyethylene
terephthalate hydrolase PETase in Bacillus subtilis 168
mediated by its native signal peptide. J Agric Food Chem
66, 13217–13227.
Joo, S., Cho, I.J., Seo, H., Son, H.F., Sagong, H.Y., Shin, T.J.,
Choi, S.Y., Lee, S.Y. et al. (2018) Structural insight into
molecular mechanism of poly (ethylene terephthalate)
degradation. Nat Commun 9, 12.
Kawai, F., Kawabata, T. and Oda, M. (2019) Current
knowledge on enzymatic PET degradation and its possible
application to waste stream management and other fields.
Appl Microbiol Biotechnol 103, 4253–4268.
Liu, H.L., Wang, S., Song, L.X., Yuan, H.B., Liu, K.Q., Meng,
W. and Wang, T.F. (2019) Trehalose production using
recombinant trehalose synthase in Bacillus subtilis by
integrating fermentation and biocatalysis. J Agric Food
Chem 67, 9314–9324.
Parisi, D., Riley, C., Srivastava, A.S., McCue, H.V., Johnson,
J.R. and Carnell, A.J. (2019) PET hydrolysing enzymes
catalyse bioplastics precursor synthesis under aqueous
conditions. Green Chem 21, 3827–3833.
Perz, V., Bleymaier, K., Sinkel, C., Kueper, U., Bonnekessel, M.,
Ribitsch, D. and Guebitz, G.M. (2016) Substrate
specificities of cutinases on aliphatic-aromatic polyesters
and on their model substrates. New Biotechnol 33, 295–304.
Russell, D., Sambrook, J., Fritsch, E., Maniatis, T., Russell,
D.W., Fritch, E., Russell, D. and Maccallum, P. (2001)
Molecular Cloning. A Laboratory Manual. New York, NY:
Cold Spring Harbor Laboratory Press.
Seo, H., Kim, S., Son, H.F., Sagong, H.Y., Joo, S. and Kim,
K.J. (2019) Production of extracellular PETase from
Ideonella sakaiensis using sec-dependent signal peptides in
E. coli. Biochem Biophys Res Commun 508, 250–255.
Son, H.F., Cho, I.J., Joo, S., Seo, H., Sagong, H.Y., Choi, S.Y.,
Lee, S.Y. and Kim, K.J. (2019) Rational protein
Letters in Applied Microbiology 71, 235--241 © 2020 The Society for Applied Microbiology
1472765x, 2020, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1111/lam.13312 by National Taiwan University, Wiley Online Library on [06/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SPamy enhances secretion of PETase
engineering of thermo-stable PETase from Ideonella
sakaiensis for highly efficient PET degradation. ACS Catal
9, 3519–3526.
Song, Y.F., Fu, G., Dong, H.N., Li, J.J., Du, Y.G. and Zhang,
D.W. (2017) High-efficiency secretion of beta-mannanase
in Bacillus subtilis through protein synthesis and secretion
optimization. J Agric Food Chem 65, 2540–2548.
Taniguchi, I., Yoshida, S., Hiraga, K., Miyamoto, K., Kimura,
Y. and Oda, K. (2019) Biodegradation of PET: current
status and application aspects. Acs Catal 9, 4089–4105.
van Dijl, J.M. and Hecker, M. (2013) Bacillus subtilis: from soil
bacterium to super-secreting cell factory. Microb Cell Fact
12, 6.
Wang, H.T., Pan, Y.Y., Hu, P.J., Zhu, Y.X., Li, J.Y., Jiang, X.J.
and Liu, G. (2014) The autophagy-related gene Acatg1 is
involved in conidiation and cephalosporin production in
Acremonium chrysogenum. Fungal Genet Biol 69, 65–74.
Wang, Q., Chu, X., Zhu, B. and Wu, N. (2019) Improving the
production of pullulanase in Bacillus licheniformis by
screening promoters and signal peptides. Rev China Agric
Sci Technol 21, 61–69.
Wang, X., Wu, N., Guo, J., Chu, X., Tian, J., Yao, B. and Fan,
Y. (2008) Phytodegradation of organophosphorus
compounds by transgenic plants expressing a bacterial
organophosphorus hydrolase. Biochem Biophys Res
Commun 365, 453–458.
Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji,
H., Maeda, Y., Toyohara, K., Miyamoto, K. et al. (2016) A
bacterium that degrades and assimilates poly(ethylene
terephthalate). Science 351, 1196–1199.
Yu, X., Xu, J., Liu, X., Chu, X., Wang, P., Tian, J., Wu, N. and
Fan, Y. (2015) Identification of a highly efficient stationary
phase promoter in Bacillus subtilis. Sci Rep 5, 18405.
Zhang, K., Su, L.Q. and Wu, J. (2018) Enhanced extracellular
pullulanase production in Bacillus subtilis using protease-
deficient strains and optimal feeding. Appl Microbiol
Biotechnol 102, 5089–5103.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. The sequence alignment between SPAmyE
and SPamy.
Figure S2. BHET degradation products. 1–2, degrada-
tion products.
Figure S3. The Standard curve of p-NP absorbance
value at 410 nm with its concentration.
Table S1. Plasmids used in this study.
Table S2. Strains used in this study.
Table S3. Relevant information of signal peptides
selected in this study.
Table S4. Primers used in this study.
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