Biochemical Engineering Journal 176 (2021) 108205

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Nano-immobilization of PETase enzyme for enhanced polyethylene

terephthalate biodegradation
Yunpu Jia a, b, Nadia A. Samak a, b, c, e, Xuemi Hao a, b, Zheng Chen a, b, Gama Yang a, b,
Xuhao Zhao a, Tingzhen Mu a, Maohua Yang a, Jianmin Xing a, b, d, *
a
CAS Key Laboratory of Green Process and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences,
Beijing 100190, PR China
b
College of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, PR China
c
Environmental microbiology and biotechnology, Aquatic microbiology, University of Duisburg-Essen, 4141 Essen, Germany
d
Chemistry and Chemical Engineering Guangdong Laboratory, Shantou 515031, PR China
e
Processes Design and Development Department, Egyptian Petroleum Research Institute, Nasr City 11727 Cairo, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: PET hydrolase (PETase), discovered in Ideonella sakaiensis, is a promising agent for the biodegradation of
Plastic biodegradation polyethylene terephthalate (PET) capable of PET decomposition under mild reaction conditions with limited
Immobilization of biocatalyst stability and productivity. Here, the immobilization of His-tagged PETase was achieved by synthesizing enzyme-
Biomimetic mineralization
inorganic nanoflowers, PETase@Co3(PO4)2, which was designed based on the principle of biomimetic miner­
alization. Immobilization of PETase onto nanostructured Co3(PO4)2 enjoys high enzyme loading and low mass
transfer inhibition due to large specific surface area, high movement speed, and large surface curvature caused
by small particle size. The nano-effect of inorganic carriers materialize the 10 ◦ C optimum temperature swelling
of the immobilized PETase with enhanced pH tolerance (6.0–10.0) than the free counterpart. The long-duration
reaction showed that the productivity of terephthalic acid (TPA) was 3.5 times higher than that of the free
enzyme. PETase@Co3(PO4)2 still retained 75% of the initial activity after 12 days compared with the free en­
zymes, which showed almost no activity. The excellent and stable catalytic performance of PETase@Co3(PO4)2
with low cost demonstrates the synthetical usefulness of immobilization via biomimetic mineralization in the
enzyme utilization in industrial PET depolymerization.

1. Introduction from incineration.

Biodegradation has been considered an effective method to control
Plastics have been massively produced over the past few decades and PET pollution profit for its eco-friendly and cost-effective characteristics
become essential to modern society, driven by their durability, plas­ [4–6]. The biodegradation of PET could be performed by the whole-cell
ticity, and chemical stability. However, poor management and disposal organism or by the produced enzymes. Several enzymes that catalyze
methods engendered explosive accumulation worldwide [1]. The envi­ PET hydrolysis have been discovered in the past few years and identified
ronmental threat of plastic pollution, especially in marine ecosystems, is as promising and environmental biocatalysts to alternate chemical or
further recognized for the ultra-long lifetimes of plastics [2–4]. Espe­ physical recycling [7–12]. For example, after extracting the cutinase
cially for PET, with only two simple monomers, TPA and ethylene glycol named LCC from a fosmid library of a leaf-branch compost metagenome
(EG), connected by an ester bond, is relatively solid and difficult to [12], Tournier et al. improved its hydrolysis capacity that ultimately
degrade naturally. The current treatment methods of PET waste like achieved a minimum of 90% PET depolymerization into monomers over
recycling, landfill treatment, and incineration treatment, have apparent 10 h [10]. Polyester hydrolase TfCut2 from Thermobifida fusca KW3 is
limitations, for example, the considerable investment in PET recycling, another candidate with a high-PET hydrolytic activity [11]. Roth and his
land pollution causing by landfill treatment, and air pollution resulting co-workers [13] structurally characterized TfCut2 and proposed a model

* Corresponding author at: CAS Key Laboratory of Green Process and Engineering & State Key Laboratory of Biochemical Engineering, Institute of Process En­
gineering, Chinese Academy of Sciences, 1, Zhongguancun Bei-er-tiao, Haidian District, Beijing 100190, PR China.
E-mail address: jmxing@ipe.ac.cn (J. Xing).

https://doi.org/10.1016/j.bej.2021.108205
Received 13 June 2021; Received in revised form 14 August 2021; Accepted 3 September 2021
Available online 10 September 2021
1369-703X/© 2021 Elsevier B.V. All rights reserved.
Y. Jia et al. Biochemical Engineering Journal 176 (2021) 108205

for the binding of the enzyme towards its polymeric substrate. In 2016, a tagged PETase gene was transformed to E. coli BL21 (DE3) cells. Suc­
PET-specific hydrolase named PETase (EC: 3.1.1.101, PDBID:5XJH), cessful transformants were cultured overnight (1%, v/v) in 50 mL Luria-
identified from bacterium Ideonella sakaiensis strain 201-F6, exhibits Bertani (LB) medium containing 50 mg/L kanamycin at 37 ◦ C. 0.6 mM
high depolymerization activity against PET films under mild reaction isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce the
conditions [14]. However, its instability during the long-term storage His-tagged PETase expression after the culture OD600 value reached 2.4.
and plastic depolymerization process hinders its application in industrial After induction at 21 ◦ C for 36 h, the mixture was centrifuged for 5 min
large-scale PET biodegradation. at 4 ◦ C. The collected cells were resuspended in a Lysis buffer composed
Immobilization of PETase using the facile methods is a suitable so­ of sodium phosphate (20 mM), imidazole (20 mM), and NaCl (0.5 M)
lution to the abovementioned problems. In the past few decades, with pH 7.4. After that, the resuspended cells were disrupted by an ul­
immobilized enzymes influenced by carrier choices have demonstrated trasonic homogenizer at 4 ◦ C. The lysate containing the PETase enzyme,
superior performance due to their higher stability, better reusability, was collected via centrifugation at 4 ◦ C for 20 min after sonication.
and easier separation from the reaction mixture than their free forms Subsequently, the clear lysate was loaded onto the His-tagged nickel
[15]. Traditional immobilization approaches mainly include three column (Cytiva, America) for purification. A wash buffer (sodium
groups, binding to supports, carrier-free insolubilized enzyme aggre­ phosphate (20 mM), imidazole (20 mM), NaCl (0.5 M), pH 7.4) was used
gates, and entrapment in polymer materials [31]. Compared with to wash the column. Elution buffer (sodium phosphate (20 mM), imid­
traditional materials, nanostructures can stabilize enzymes for a long azole (500 mM), NaCl (0.5 M), pH 7.4) was used to elute the bound
time in different systems with nano-effects such as the small size and the proteins. The purification process was conducted according to the pre­
large surface curvature of the nanoparticles [16]. vious literature [22]. Sodium dodecyl sulfate polyacrylamide gel elec­
Recently, the biomimetic mineralization process has caught many trophoresis (SDS-PAGE) checked the purity of the purified PETase
eyeballs with its ease operation, ultrahigh enzyme activity recovery rate, enzyme.
mild reaction conditions, and the specially enhanced stabilization effect
of enzyme− inorganic hybrid nanoflowers on enzymes component [17]. 2.2.2. Preparation of cobaltous phosphate nanoparticles
Unlike any traditional immobilization method, the biomimetic miner­ Cobaltous phosphate nanoparticles were synthesized according to
alization process is a new immobilized enzyme technology accompanied the previous reports [21]. Briefly, Co(NO3)2.6 H2O was added to 10 mM
by inorganic salt precipitation. A low-concentration buffer is used to phosphate-buffered saline (PBS) solution (10 mM, pH 7.4) with 10 mM
slow down the rate of inorganic salt formation, with enzyme molecules Co2+ as a final concentration. After complete oscillation, the mixture
introduced in the reaction to allow inorganic salt precipitation to adhere turned purple and pink floccules began to appear. Subsequently, the
to the enzyme slowly [24]. Then, embedding was carried out to mixture was incubated for 36 h at room temperature. After washing 3
immobilize of the enzyme. The method utilizes the specific chelation of times using ultrapure water, the cobaltous phosphate nanoparticles
the imidazole group in his-tag exposed on the protein surface and metal were obtained by suspension with a certain amount of ultrapure water.
ions to immobilize the protein, whose affinity then be purified. The
inorganic nanoparticles as carriers reduced the steric hindrance and 2.2.3. Preparation of PETase-inorganic hybrid nanoflowers
substrate diffusion limiting. Following the protein-inorganic PETase@Co3(PO4)2 nanoflowers were synthesized by mixing crude
(Cu3(PO4)2) hybrid system by Ge et al. [18], and subsequently, some enzyme (10 mL), PBS (90 mL, 10 mM), and Co(NO3)2.6 H2O (0.291 g),
scientists synthesized several other hybrid nanoflowers using different then incubated at 25 ◦ C for 60 h. After that, they were collected by 3
inorganic or organic components for the efficient immobilization of cycles of centrifugation (6500 rpm) for 5 min, and subsequently, the
hydratase, lipase and transaminase [19–21]. Encouraged by these early produced nanoflowers were washed with ultrapure water.
achievements, a biomimetic mineralization strategy that introduces an
affinity tag at the N-terminal of the PETase to realize the purification 2.2.4. Tryptophan fluorescence emission of PETase-inorganic hybrid
and immobilization of the enzyme was adopted. PETase flower-like nanoflowers and free PETase
cobalt phosphate nanoparticles were produced by the nucleation reac­ The free PETase and PETase-inorganic hybrid nanoflowers were
tion of the cobalt ions and PETase enzyme that was assembled on the characterized by tryptophan fluorescence measurements. The fluores­
surface of particles. Catalytic activities and stabilities of the free and cence spectrum was recorded at the same enzyme concentration. The
immobilized PETase toward PET films were studied. The reusability excitation wavelength was 230 nm and emission fluorescence were
efficiency and storage performance of the immobilized PETase enzyme measured from 280 nm to 600 nm using Microplate Reader (Infinite
were also examined. M200, Tecan Japan Co., Ltd., Kawasaki, Japan).

2. Experimental
2.3. Enzyme activity assay
2.1. Materials
2.3.1. Enzyme assays for 4-nitrophenyl butyrate
The complete gene sequence of PETase from Ideonella sakaiensis A mixture of 49 mL phosphate buffer (100 mM, pH 7.5) and 1 mL of
strain 201-F6 (Genbank GAP38373.1) was codon-optimized and syn­ p-nitrophenyl butyrate(p-NPB) stock solution (4 mM, 0.084 g in abso­
thesized, then inserted into the target vector pET-30a (+) for the lute ethanol) was reacted well, then 50 μL of the enzyme solution was
expression in Escherichia coli BL21 (DE3). For immobilized PETase, a 6x added to a cuvette containing 1 mL of p-NPB solution and mixed by
His tag was added into the N-terminal of its sequence. The sequence of pipetting. The absorbance at 400 nm was measured at 35 ℃ using
the resulting plasmids was confirmed using Sanger sequencing at Gen­ HITACHI U-2910 spectrophotometer (Japan) with a measured absor­
Script China, Inc. Acetonitrile (HPLC grade) was purchased from bance that can be availed to calculate units of lipase activity (U) ac­
Shanghai Macklin Biochemical Co., Ltd. (China). All other chemicals of cording to Eq. (1). One unit of PETase activity was defined as the amount
analytical grade were purchased from Sigma-Aldrich Trading Co. Ltd. of enzyme required to hydrolyze 1 μmol of substrate per minute [32].
(Shanghai, China) unless otherwise stated. For the activity measurement of the enzyme, the final enzyme activity
(Ufinal ) is defined as the absorbance change caused by the addition of
2.2. Preparation of PETase@Co3(PO4)2 enzyme minus the absorbance change caused by the same reaction
system without enzyme. In case of PETase@Co3(PO4)2, the final enzyme
2.2.1. Expression and purification of His-tagged PETase activity is defined as the absorbance change caused by the addition of
The recombinant plasmid pET-30a(+) (Scheme S1) carrying the His- the enzyme minus the absorbance change caused by the reaction system

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Y. Jia et al. Biochemical Engineering Journal 176 (2021) 108205

with the same concentration of Co3(PO4)2 Eq. (2).

(A400nm (t1 ) − A400nm (t0 ) ) × Vtotal
Uenzyme = (1)
ε × Venzyme × (t1 − t0 ) × l

A400nm (t1 )= absorbance at 400 nm measured after the enzymatic reac­

tion is finished.
A400nm (t0 )= absorbance at 400 nm measured before the enzymatic
reaction is started

t1 = time point when the reaction is finished

t0 = time point when the reaction is started, usually 0 min
Vtotal = volume of reaction sample plus enzyme sample (Venzyme )
ε = molar extinction coefficient [mmol− 1 dm3 cm− 1]
l = light path length [cm]
Ufinal = Uenzyme − Ucontrol (2)

Ucontrol = activity of inorganic components or reaction buffer.

2.3.2. Optimum temperature and pH

The free and immobilized PETase were incubated for 1 h in acetate
buffer (pH 4–6), phosphate buffer (pH 7.0–10.0), or borate buffer (pH
10–12) with different pH values and at different temperatures
(20–60 ◦ C). The 100% maximum activity or initial activity was followed
by the relative activity results.

2.3.3. Stability and reusability assay

Thermal stability of the immobilized PETase@Co3(PO4)2 and free
PETase were studied. Samples were incubated in Tris-HCl buffer (pH Fig. 1. SDS-PAGE analysis of crude cell lysates containing His-tagged PETase
8.0, 50 mM) for 30 min at 40 or 45 ◦ C, separately with the pH stabilities (lane 1) and purified by Ni-NTA affinity chromatography (lane 2) or bionic
of the immobilized PETase@Co3(PO4)2 and PETase investigated by mineralization process (lane 3). Lane M: protein marker; Lane 4: eluted frac­
incubating them in phosphate (pH 10.0, 50 mM) or acetate buffer (pH tions from the PETase@Co3(PO4)2 using 500 mM imidazole. The target protein
was marked by a red arrow.
4.0, 50 mM) at room temperature over different time intervals. Then,
their residual activity to 4-nitrophenyl butyrate was measured.
Reusability of PETase@Co3(PO4)2 nanoflowers were studied for ten The degradation products were measured by High-performance liquid
cycles by hydrolyzing 4-nitrophenyl butyrate during repeated usages at chromatography, HPLC (Agilent Technologies).
optimized conditions. After each catalytic cycle of reaction, the immo­
bilized enzymes were collected by centrifugation, washed twice, and 2.4. Synthesis of MHET substrate
resuspended using phosphate buffer (50 mM, pH 7.5) three times for the
next reaction. The catalytic activity in the first cycle was defined as Mono(2-hydroxyethyl) terephthalate (MHET) was synthesized from
100%. the hydrolysis of bis(2-hydroxyethyl) terephthalate (BHET) with KOH.
Briefly, 2 g (8.74 mmol) BHET was reacted with 0.42 g (8.4 mmol) KOH
2.3.4. Enzyme assays for highly crystallized PET (hcPET) in 20 g MgSO4-dried and filtered ethylene glycol for 2.5 h at
PET from a conventional bottle (NONGFU SPRING, 2 L, specific 110− 130 ◦ C. The mixture of the 30 mL H2O and the reaction system was
polymer characteristics unspecified) was cut into small pieces of 0.017 g extracted three times with 3–5 mL CH2Cl2 with the CH2Cl2-phase dis­
about 1 cm2 square in area. The 5 mL unpurified crude enzyme from carded. The precipitation of MHET was on the heels of the aqueous
100 mL of cell culture was reacted with PET film in 30 mL of buffer phase pH adjusted to 3 with 25% hydrochloric acid (HCl). The extraction
containing 50 mM Na2HPO4-HCl (pH 8.0). Controls were performed procedure was conducted with 30 mL boiled water with three times
using buffer or buffer containing Co3(PO4)2 without enzyme. The filtration at 4 ◦ C and dried precipitation at 60 ◦ C.
experiment was stopped by removing the PET fragments from the flasks.
After washing and drying, PET films were processed for scanning elec­ 2.5. HPLC analysis
tron microscopy (SEM) analysis as following: the enzyme-treated PET
films were washed successively with 1% SDS, ultrapure water, and The degradation products such as MHET, TPA and BHET were
ethanol. Finally, the PET film was dried before coated with Au, and Field separated using HPLC with the aid of C18 column on an Agilent 1200 LC
Emission Scanning Electron Microscopy (FE-SEM) observed the degra­ system with a G1315D diode array detector (DAD). Samples and stan­
dation with a JSM-7800 or a JSM-6700 F (Japan Electronics). dards were injected at a volume of 20 μL onto a ChromCore C18 column
5 µm, 4.6 × 250 mm column (NanoChrom, China). A mixture of 0.1%
2.3.5. Enzyme assays for amorphous PET films and PET powder (v/v) formic acid (A) and acetonitrile (B) was used as the mobile phase
degradation at a flow rate of 0.8 mL min− 1 to separate the analytes of interest. The
The materials used to test PET degradation products were PET mobile phase B was changed gradually from 1% to 5% for 5 min, 5–44%
powder and amorphous PET films purchased from Goodfellow GmbH for 7 min, 44–70% for 3 min, with the constant ratio for 10 min. The
(Product number 048–768–25 and product number 029–198–54, sepa­ concentration of TPA, MHET and BHET was detected at a wavelength of
rately). The 5 mL unpurified crude enzyme (or 5 mL nanoflowers solu­ 241 nm and quantified from the areas of the absorption peaks by stan­
tion with the same enzyme concentration) was incubated with 0.15 g dard curves.
amorphous PET films and 0.2 g PET powder in a 30 mL total reaction
system, with the reaction processes the same as that in Section 2.3.4.

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Y. Jia et al. Biochemical Engineering Journal 176 (2021) 108205

Fig. 2. SEM image of Co3(PO4)2 nanoflowers (a) and PETase@Co3(PO4)2 (b).

3.2. Characterization of the immobilized PETase@Co3(PO4)2

The biomimetic mineralization process of PETase and Co3(PO4)2 was

initiated by the Co2+ - protein molecules complexes [18] through the
coordination facility of amide groups in the protein backbone [23]. SEM
images showed that cobalt phosphate crystals had a unique flower-like
shape and an elegant structure with a diameter of about 10 µm
(Fig. 2a). Unlike Co3(PO4)2 nanoflowers, the rough surface morphology
of PETase@Co3(PO4)2 with a beautiful rose-like structure and a diam­
eter of about 10 µm was rough due to the incorporation of PETase
enzyme (Fig. 2b). Furthermore, the visibly aggregated enzyme particles
on the nanoflowers indicated a successful bond between PETase and the
Co3(PO4)2 crystals.
Tryptophan, an important intrinsic fluorescent, can be used to esti­
mate the nature of the tryptophan microenvironment. Tryptophan
fluorescence emission experiments studies revealed the differences in
structural rearrangements of the enzyme. Compared with the free
PETase, cobalt phosphate mineralization caused a λmax blue shift in
PETase, with significant swelling of the fluorescence intensity (Fig. 3).
This phenomenon is attributed to the significantly altered polar envi­
Fig. 3. Fluorescence studies of soluble PETase, PETase@Co3(PO4)2 ronment of the aromatic residues (phenylalanine and tryptophan, Phe
and Co3(PO4)2. and Trp) caused by cobalt phosphate molecules. These alterations
indicate essential changes in the tertiary structure of PETase as it formed
3. Results and discussion part of cobalt phosphate biological mineral [24]. However, the preser­
vation of the catalytic properties of the soluble PETase in enzyme may be
3.1. Specificity of cobalt phosphate to His-Tagged PETase from cell due to the void critical domains of catalysis in the nucleation points.
Lysates Meanwhile, the PETase@Co3(PO4)2 nanoflowers were characterized
by X-ray Diffraction (XRD) (Fig. S1). The pleasant match between the
Cell lysates containing PETase were mixed directly with Co(NO3)2 result and the diffraction standard card for Co3(PO4)2.8H2O (JCPDS file
and PBS solution without any purification process to prepare PETa­ no. 410375) indicated that the incorporation of the PETase did not affect
se@Co3(PO4)2. Ni-NTA column witnessed the unsatisfactory effect in the the crystal morphology of Co3(PO4)2. Additionally, the surface func­
enzyme purification, as shown in Fig. 1, lane 2. This may be attributed to tional groups of Co3(PO4)2 and PETase@Co3(PO4)2 were characterized
Ni2+ can bind to some non-His-tagged proteins, resulting in poor by Fourier transform infrared (FT-IR) spectrometry. The peak at
selectivity and affecting the purification effect [33,34]. In contrast, Co2+ 1550 cm− 1, 1750 cm− 1, 2971 cm− 1, and 3321 cm− 1 corresponding to
enjoys a higher purity target protein with the weaker non-specific the − NH bending vibration, − C− N stretching vibration, − CH3 stretch­
binding ability to His-tagged proteins [35]. To verify the selectivity of ing vibration, and N-H stretching vibration (Fig. S2), are considered as
the immobilization process, SDS-PAGE was used to analyze PETase different characteristic absorption peaks that were invisible in
purity and concentration with the molecular weight of PETase, 29 kDa, Co3(PO4)2. Furthermore, Thermogravimetric Analysis (TGA) was also
according to the calculation. The intense band between 28 and 38 kDa in conducted to verify the existence of the PETase in nanoflowers. As
line 3 (Fig. 1) elucidated that the used method efficiently enriched and shown in Fig. S3, the two samples with different relative gravities
purified the His-tagged PETase from the crude enzyme when compared decomposed starts from 100 ◦ C and finished around 170 ◦ C, resulting in
with the conventional one. crystalline water loss. The extra weight loss from 200◦ to 400◦ C in
PETase@Co3(PO4)2 as the PETase decomposed further elucidated the
immobilization of PETase in PETase@Co3(PO4)2.

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Y. Jia et al. Biochemical Engineering Journal 176 (2021) 108205

Fig. 4. Optimum temperature(a) and pH (b) of PETase@Co3(PO4)2 and free PETase. Thermal stability(c) and pH stability(d) of PETase@Co3(PO4)2 and free PETase.

X-ray photoelectron spectroscopy (XPS) was performed to investi­ temperature and pH indicated the higher tolerance of the PETase@­
gate the surface element composition and chemical bonds of PETa­ Co3(PO4)2 towards changed of temperature and pH with more excellent
se@Co3(PO4)2, whose survey scan displayed five characteristic peaks at stability than its free form. These phenomena might be ascribed to the
782, 531, 400, 285 and 134 eV, with their corresponding to Co, O, N, C specific His-tags affinity to Co2+ that failed to affect protein stability
and P, respectively (Fig. S4) suggesting the organic-inorganic hybrids of with favorable changes in secondary structure, climbed PETase rigidity,
particles. Then, high-resolution XPS spectra were obtained for Co, O, and protection on the conformation of PETase during extreme envi­
and C, chimed with the previous literature [20]. The N2 ronmental conditions. The maximum enzyme activity of PETase@­
adsorption-desorption isotherms for both PETase@Co3(PO4)2 and Co3(PO4)2 and free enzyme measured under optimal temperature and
Co3(PO4)2 nanoflowers were type III curves (Fig. S5), which indicated pH conditions were 4102 U/mL and 4411 U/mL respectively. The
that the holes in the materials are slit holes formed by accumulated of higher steric hindrance between the immobilized PETase and the sub­
flake particles [25]. The pore-size distribution plot was calculated by the strate than that of the free enzyme led to the slightly lower enzyme
Barrett-Joyner-Halenda (BJH) absorption method, revealed the average activity of the immobilized enzyme than that of the free one [30].
pore size of Co3(PO4)2 and PETase@ Co3(PO4)2 15.1 nm and 16.0 nm,
respectively. The specific surface area and cumulative volume of pores
3.4. . Stability and reusability assay
were 32.5 m2g− 1 and 0.13 cm3g− 1 for Co3(PO4)2, and 42.4 m2g− 1 and
0.17 cm3g− 1 for PETase@ Co3(PO4)2, respectively. The variation of
The reaction rate usually increases as temperature climbs within a
these parameters of PETase@Co3(PO4)2 compared with Co3(PO4)2
specific range, but higher temperature values can quickly destroy the
confirmed the successful immobilization of PETase.
structure and active site of the enzyme. Therefore, its ability to resist
high temperatures is a crucial parameter for performance evaluation of
3.3. Optimum temperature and pH the immobilized enzyme. Fig. 4c showed the thermal stabilities of free
and immobilized PETase. After being incubated at 40 ◦ C for 3 h, the
The effects of temperature and pH on the activity of PETase and immobilized PETase@Co3(PO4)2 still held 94.4% primary activity while
immobilized PETase@Co3(PO4)2 were investigated. Fig. 4a indicated the free PETase kept 57.3% only. After incubation at 45 ◦ C for 3 h, the
the optimum pH for both free PETase and immobilized PETase@­ residual activity of free PETase maintained 17.2% primary activity,
Co3(PO4)2, 7.0, with PETase@Co3(PO4)2 surprisedly presenting a more compared to 82.8% for the immobilized PETase@Co3(PO4)2. The pH
extensive pH range of 6.0–10.0 than that of free PETase. The biomineral stability results of free PETase and immobilized PETase@Co3(PO4)2 are
surface could buffer the microenvironment near PETase by maintaining shown in Fig. 4d. At pH values of 4.0 and 10.0, the activity of free en­
neutral pH even under extreme pH conditions [26]. The optimum zymes was dropped sharply compared with that after 5 h of incubation.
temperature of the immobilized PETase@Co3(PO4)2, 45 ◦ C, was 10 ◦ C In contrast, the PETase@Co3(PO4)2 retained 67.6% of the original ac­
higher than that of the free enzyme. The shifted of the optimum tivity at pH 10.0% and 62.3% at pH 4.0 after incubation for 4 h. Clearly,

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Y. Jia et al. Biochemical Engineering Journal 176 (2021) 108205

Fig. 5. Storage and reusability results. (a) Storage stability studies of free PETase and PETase@Co3(PO4)2 nanoflowers. (b) Reusability of immobilized PETase.

Fig. 6. SEM photographs of bottle-grade PET surface. Untreated (a), treated with PETase@Co3(PO4)2 (b) and Co3(PO4)2 for 48 h (c), treated with free PETase for
48 h (d), and treated with free PETase for 7 days (e).

Fig. 7. The hydrolysis products concentration after reacting PETase enzyme and immobilized PETase with PET particles (a) and amorphous PET film (b) for 48 h
at 30 ◦ C.

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Y. Jia et al. Biochemical Engineering Journal 176 (2021) 108205

PETase@Co3(PO4)2 showed significant improvement in thermal stabil­ CRediT authorship contribution statement
ity and pH tolerance of the enzyme compared to that of the free PETase.
The limitedly maintained enzyme structure by the high nanopore cur­ Yunpu Jia: Conceptualization, Methodology, Validation, Data
vature stably immobilized the enzyme, keeping high catalytic activity curation, Writing – original draft, Writing – review & editing. Nadia A.
even in extreme environments [27]. Samak: Methodology, Writing – review & editing. Xuemi Hao: Writing
To test the storage stability, free PETase or immobilized PETase@­ – review & editing, Supervision. Zheng Chen: Validation. Gama Yang:
Co3(PO4)2 nanoflowers were incubated in PBS solution at room tem­ Validation. Xuhao Zhao: Validation. Tingzhen Mu: Validation. Mao­
perature. Fig. 5 indicated that the immobilized PETase@Co3(PO4)2 only hua Yang: Validation. Jianmin Xing: Conceptualization, Writing –
decreased 25% of its residual activity after storage at room temperature review & editing, Supervision, Project administration.
for 12 days compared with the almost inactive free enzymes. For the
reusability, PETase nanoparticles minimizing alterations in structure
Declaration of Competing Interest
and enzyme active sites enabled 70.2% retained residual activity to 4-
nitrophenyl butyrate of the immobilized PETase@Co3(PO4)2 after 10
The authors declare that they have no known competing financial
cycles of use [17].
interests or personal relationships that could have appeared to influence
In order to compare the degradation ability of PETase@Co3(PO4)2
the work reported in this paper.
and free PETase on PET, post-consumed bottle-grade PET was used as
the substrate. The PET degradation reaction was terminated by
Acknowledgment
removing the PET material from the enzymatic decomposition reaction
system after 48 h at 35 ◦ C with the PET films washed and dried, and
The authors gratefully acknowledge the financial support provided
their surface morphology examined by SEM (Fig. 6). The control group,
by the National Natural Science Foundation of China (grant numbers
without any enzyme, did not induce any detectable change for the PET
31961133017, 31961133018, 31961133019). These grants are part of
substrate surface morphology, whereas the free PETase caused grooves
“MIXed plastics biodegradation and UPcycling using microbial com­
and folds, chiming with the previously reported literature [14,22,28,
munities” MIX-UP research project, which is a joint NSFC and EU H2020
29]. Surprisingly, PETase@Co3(PO4)2 could cause more severe erosion
collaboration. In Europe, MIX-UP has received funding from the Euro­
on the PET surface than the free PETase with obviously eroded PET film
pean Union’s Horizon 2020 research and innovation program under
surface. To achieve the same degradation effect as PETase@Co3(PO4)2,
grant agreement No 870294.
free PETase needed a more prolonged time, as shown in Fig. 6e, that
slowly decreased the free PETase activity until it was lost. On the other
hand, the immobilized PETase@Co3(PO4)2 with high activity was Appendix A. Supporting information
consistent with the previous results shown in Fig. 5. The discovered
enzyme image attached to the PET surface with a degradation reaction Supplementary data associated with this article can be found in the
marked the occurred degradation (Fig. S6). online version at doi:10.1016/j.bej.2021.108205.
The degradation products were analyzed with PET particles and
amorphous PET film as the substrate, as indicated in Fig. 7. The amor­ References
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