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Frontiers in Microbiology 01 frontiersin.org
Polyurethane biodegradation by
Serratia sp. HY-72 isolated from
the intestine of the Asian mantis
Hierodula patellifera
Jong-Hoon Kim
1, Seung Hoon Choi
1, Min Gu Park
2, Dong
Hwan Park
2, Kwang-Hee Son
1
* and Ho-Yong Park
1
*
1 Microbiome Convergence Research Center, Korea Research Institute of Bioscience and
Biotechnology, Daejeon, Republic of Korea, 2 Department of Agricultural Biotechnology, College of
Agriculture & Life Science, Seoul National University, Seoul, Republic of Korea
Polyurethane (PU), currently replacing existing synthetic materials worldwide,
is a synthetic polymer derived from polyols, isocyanates, and a chain extender
added by condensation reactions. PU wastes which are dicult to recycle, are
commonly discarded in landfills and flow into ecosystems, thereby causing
serious environmental problems. In recent years, insect-associated microbes
have become a promising, eco-friendly strategy as an alternative to plastic
recycling. This study aimed to evaluate the potential of Serratia sp. HY-72
strain isolated from the intestine of the Asian mantis (Hierodula patellifera) for
PU degradation. The 65 kDa family I.3 lipase which degrades PU was identified
and characterized, with a specific activity of 2,883 U mg−1. The bacterial filtrates
and the recombinant lipase degraded Impranil (a colloidal polyester-PU
dispersion, 100 g l−1) by 85.24 and 78.35% after 72 h incubation, respectively.
Fourier transform infrared spectroscopy analysis revealed changes in Impranil
functional groups, with decreased C=O functional group and aliphatic chain
signals, and increased N-H bending with C-N stretching and C-O stretching.
The current study also revealed that the HY-72 strain biodegraded the
commercial PU foams (polyester- and polyether- PU) with 23.95 and 10.95%
weight loss after 2 weeks, respectively with changes in surface morphology
and structure such as cracks, roughness, and surface roughening. Altogether,
this is one of the few studies reporting biodegradation of PU by the insect-
associated microbe. These findings suggest that the insect-associated
microbe could bea promising resource for biodegradation and recycling of
plastic waste.
KEYWORDS
polyurethane, polyurethanase, biodegradation, insect-associated microbe, Serratia,
plastic waste management
TYPE Original Research
PUBLISHED 19 December 2022
DOI 10.3389/fmicb.2022.1005415
OPEN ACCESS
EDITED BY
Jie Wang,
China Agricultural University,
China
REVIEWED BY
Martin Koller,
University of Graz,
Austria
Vasudeo Zambare,
University of Technology Malaysia, Malaysia
*CORRESPONDENCE
Kwang-Hee Son
sonkh@kribb.re.kr
Ho-Yong Park
hypark@kribb.re.kr
SPECIALTY SECTION
This article was submitted to
Microbiotechnology,
a section of the journal
Frontiers in Microbiology
RECEIVED 29 July 2022
ACCEPTED 02 December 2022
PUBLISHED 19 December 2022
CITATION
Kim J-H, Choi SH, Park MG, Park DH, Son
K-H and Park H-Y (2022) Polyurethane
biodegradation by Serratia sp. HY-72
isolated from the intestine of the Asian
mantis Hierodula patellifera.
Front. Microbiol. 13:1005415.
doi: 10.3389/fmicb.2022.1005415
COPYRIGHT
© 2022 Kim, Choi, Park, Park, Son and
Park. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
Kim et al. 10.3389/fmicb.2022.1005415
Frontiers in Microbiology 02 frontiersin.org
1. Introduction
Plastics are composed of chains comprising numerous organic
subunit linked by dynamic covalent bonds and are widely used
owing to their stability, ease of manufacturing, economic
eciency, and convenience (Alabi etal., 2019; Atanasova etal.,
2021; Roy etal., 2021). Plastic production has grown exponentially,
with a very large number of plastics produced since 1950 (Bilal
et al., 2021). Plastic disposal systems, such as incineration,
recycling, and landlls, are ineective in plastic waste management
(Seneviratne etal., 2006). Plastic decomposition is extremely slow
because of the chemical structure of most plastics and poor
degradation capabilities of natural decomposition processes.
Furthermore, it generates small particles, known as microplastics,
which are more likely to cause serious ecological and biological
concerns. In particular, the accumulation of plastic waste
continues to have harmful eects on the ecosystem (Barnes etal.,
2009; Mukherjee etal., 2011; Osman etal., 2018; Amobonye etal.,
2021). Hazardous contaminants are eluted from plastic waste,
causing the biological accumulation of harmful chemicals in the
food chain and leading to problems such as endocrine disorders
and reduced species diversity (Liaqat etal., 2020).
Polyurethane (PU) is a type of plastic with increasing usage
over the decades because of its diverse properties and is currently
replacing existing synthetic materials (Liu etal., 2021). PU was
discovered and produced by Dr. Otto Bayer in 1937. Since then, it
has become ubiquitous in modern life and is extensively used in
medicine and industries (Howard, 2002). PU has a high melting
point and tensile strength, improving its durability (Bayer, 1947).
It is a polymer derived from the condensation of polyisocyanates
and polyols with carbamate ester bonds (-NHCOO-; Nakajima-
Kambe etal., 1999) and is classied into four main types according
to the polyol composition (Vargas-Suárez et al., 2019). PU is
commonly discarded in landlls or incinerated for heat production
owing to its highly complex polymer structure (Ignatyev etal.,
2014; Utomo etal., 2020). e decomposition of PUs is slow and
produces serious pollutants (Liu et al., 2021). Furthermore,
improper incineration generates toxic gases such as carbon
monoxide and hydrogen cyanide that negatively aect human
health and ecosystems (McKenna and Hull, 2016; Alshehrei,
2017). Physical and chemical degradation are inecient in
eliminating PU waste. erefore, the widespread and increasing
use of PU in modern society makes biodegradation as important
as manufacturing these plastics.
Since the 1960s, several studies have conrmed that
microorganisms and their enzymes can degrade PUs (Schmidt
etal., 2017; Magnin etal., 2020). ese microorganisms either use
PUs directly as nutrient sources or indirectly degrade them with
several enzymes and metabolites. Esterases, lipases, proteases, and
ureases produced by microorganisms can biodegrade polyester-PU
(Nakajima-Kambe etal., 1999; Howard, 2002; Loredo-Treviño
et al., 2012) and proteases and esterases can directly disrupt
urethane binding sites (Loredo-Treviño etal., 2012). Among the
hydrolysis enzymes, lipases derived from bacteria are considered
more suitable for industrial environments due to their large-scale
substrate specicity (Javed et al., 2018). Despite continuous
research and notable success, PU-degrading enzymes remain a
challenge and require extensive research. erefore, there is a
pressing need for scientic and technological advances in context
of bacterial hydrolytic enzymes for PU biodegradation.
Insects have developed symbiotic interactions with numerous
microorganisms to overcome environmental limitations
(Sudakaran etal., 2015). Recently, the plastic-degrading capability
of insect-associated microorganisms is drawing attention. Insect
intestinal microbiota has evolved to produce eective degradative
enzymes and utilize various substrates as nutrients to overcome
environmental limitations (Sudakaran etal., 2015; Berasategui
etal., 2016). ey are more functional than degradative enzymes
produced by microorganisms in the free-living state, and have the
potential of industrial applications (Jang and Kikuchi, 2020).
Previous studies have highlighted that microorganisms extracted
from insect intestines can becultured to break down plastics such
as polyethene (PE) and polystyrene (PS; Yang etal., 2014, 2018).
ese microorganisms isolated from insects are a promising
source of end-of-life solutions for plastics and have the advantage
of not causing secondary pollution from plastic waste (Lee
etal., 2020).
In this study, the potential PU-degrading bacterium, Serratia
sp. strain HY-72, was isolated from the intestine of the Asian
mantis (Hierodula patellifera). PU-biodegrading potential of the
strain HY-72 was evaluated by comparing weight loss, scanning
electron micrographs, and chemical composition. In addition,
family I.3 lipases with PU-degrading activity were identied and
evaluated from the strain HY-72.
2. Materials and methods
2.1. Materials
e commercial polyester urethane dispersion agent, Impranil
DLN (Impranil; Bayer Materials Science, PA, UnitedStates), was
purchased to evaluate PU biodegradation by the isolated strain.
e two types of PU foams (polyester- and polyether-) were
purchased from KPX Chemical Co. (Seoul, Republic of Korea).
Each PU foam sample was cut into small pieces (polyester PU, 20
by 30 by 15 mm; polyether PU, 8 by 30 by 15 mm) and weighed
(polyester PU, 250 mg; polyether PU, 150 mg). ree replicates of
each type were soaked in 70% ethanol for 1 h and washed with
sterile distilled water. Subsequently, the pieces were place in an
oven at 50°C until the surface moisture was removed.
2.2. Isolation and identification of
PU-degrading bacteria
Adult Asian mantis (H. patellifera) were collected from
Bomoon Mountain (Daejeon, Republic of Korea) and transported
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to the laboratory. Insects were dissected to isolate bacterial strains
using the method described by Heo etal. (2006) with minor
modications. e insects were immersed in 70% (v/v) ethanol
for 1–2 min to remove the surface contaminants and then washed
twice with sterile distilled water. Next, the digestive tracts were
removed, and the intestinal contents were carefully recovered. e
intestinal contents were diluted in phosphate-buered saline (PBS;
0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, 0.024% KH2PO4, pH
7.4) and spread onto one-h strength of Reasoner’s 2A (R2A)
agar (KisanBio, Seoul, Republic of Korea) containing 1% Impranil
to isolate the PU-degrading gut bacteria. Aer incubation at 30°C
for 2 days, a HY-72 bacterial strain, which only formed a
translucent halo around areas of bacterial growth, was chosen for
further experiments. e bacterial strain was isolated and stored
at −70°C in R2A broth with 25% sterilized glycerol. Genomic
DNA was extracted from the strain HY-72 and 16S rRNA was
amplied by polymerase chain reaction (PCR) for its identication.
e universal primers 27F (5′-AGA GTT TGA TCM TGG CTC
A-3′) and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT T-3′)
were used. e sequence of the 16S rRNA gene was compared
with those of type strains available in the EzBioCloud database
(ChunLab Inc., Seoul, Republic of Korea) to nd closely related
species. Molecular phylogeny of 16S rRNA was inferred by the
neighbor-joining method in MEGA X soware (Kumar
etal., 2018).
2.3. Extracellular enzymatic activity
e strain HY-72 was assessed for protease and lipase
production using an agar plate assay as described by Bhagobaty
and Joshi (2012), with minor modications. e revived bacterial
culture was streaked onto R2A agar containing a specic substrate
and incubated at 30°C for 3 days. e ability to produce protease
was qualitatively conrmed by observing transparent halo zones
surrounding the colonies on the R2A agar containing 2% (w/v)
skim milk. e extracellular lipase activity was qualitatively
determined using R2A agar with 1% (w/v) Tween 80 and 0.01%
(w/v) CaCl2 and conrmed by the formation of crimson dots
around the colonies, which were further measured.
2.4. Identification and characterization of
PU-degradable enzymes
For the recombinant production in Escherichia coli BL21
(DE3) of mature lipase having PU-degrading capability, the
encoding gene was amplied using specic primers, SLPoly-F
containing an NdeI restriction site at the 5′-end
(5′-CGCCATATGATGGGAATCTTTAAT-3′) and SLPoly-R
containing a HindIII restriction site at the 5′-end
(5′-GGCAAGCTTTCAGGCCAGTAC-3′), using strain HY-72
genomic DNA as a template. PCR was performed using a thermal
cycler (Takara, Kyoto, Japan), and the initial template denaturation
was performed for 2 min at 94°C, followed by 35 cycles of 10 s at
98°C, 30 s at 57°C, and 1 min at 68°C. e PCR-amplied
fragment was inserted into the pET-28a (+) vector (Novagen,
Darmstadt, Germany) by a ligation reaction. Aer transformation
of the ligation mixture into E. coli BL21 (DE3), the E. coli strain
containing the recombinant plasmid was grown at 37°C in LB
broth (BD Difco, Franklin Lakes, NJ, UnitedStates) supplemented
with 50 μg ml−1 kanamycin. To overproduce lipase proteins,
recombinant E. coli BL21 (DE3) cells harboring pET-28a (+)/
lipase were cultivated in a 5-l baed ask with 1 l of LB broth and
50 μg ml
−1
of kanamycin in a rotary shaker (150 rpm) for 18 h at
30°C. Overexpression of the target gene was induced by adding
1 mM isopropyl β-D-1-thiogalactopyranoside when the optical
density of the culture at 600 nm reached approximately 0.6.
Following cultivation, lipase-expressing cells were harvested by
centrifugation (10,000 × g) for 20 min at 4°C. e cells were
thoroughly washed twice, re-suspended with PBS, and disrupted
via sonication. Lysates were harvested by centrifugation
(13,000 × g) for 10 min at 4°C. e recombinant protein in the
lysate was puried using a Ni-NTA Fast Start Kit (QUIAGEN,
Valencia, CA, United States) according to the manufacturer’s
instructions.
e cleavage site for the protein’s signal peptide was predicted
using the SignalIP5.0 server.1 Conserved domain searches and
protein family recognition were conducted using the Pfam server.
2
e sequence of lipase from HY-72 was retrieved from the
GenBank database by searching BLASTp.3 Multiple alignments of
lipase amino acid sequences were achieved using Clustal W in
MEGA X soware. Phylogenetic analysis of lipase was conducted
using MEGA X soware with the neighbor-joining method. e
reliability of the phylogenetic tree was checked by bootstrap
analysis based on 1,000 replications.
e relative molecular mass of the puried lipase was analyzed
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE; Mini-PROTEAN system, Bio-Rad, Hercules, CA,
UnitedStates). Aer electrophoresis, the proteins separated by
SDS-PAGE were visualized by staining the gel with Coomassie
Brilliant Blue R 250, and zymopraphy was performed on the other
side of the gel, according to Vandooren etal. (2013), with minor
modications. For zymograms, a gel containing puried
recombinant lipase was washed with 50 mM Tris–HCl buer (pH
8.0) for 15 min and subsequently placed on top of the zymogram
gel with 1% Impranil as substrate. e zymogram was incubated
overnight at 28°C and analyzed for clearing the following day.
Quantitative assays of protein concentrations were conducted
using the Qubit 4 Fluorometer (Invitrogen, ermo Fisher
Scientic, Waltham, United States) and associated kit (Qubit
Protein Assay Kit, Invitrogen, ermo Fischer Scientic,
Massachusetts, United States) according to the manufacturer’s
1 http://www.cbs.dtu.dk/services/SignalP/
2 http://pfam.sanger.ac.uk/
3 http://www.ncbi.nlm.nih.gov/BLAST/
Kim et al. 10.3389/fmicb.2022.1005415
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instructions. e lipolytic activity of the puried enzymes was
measured using a spectrophotometric method with p-nitrophenyl
acetate (pNPA) as the substrate (Shin etal., 2021). e puried
enzyme (0.1 ml) was added to 0.5 ml of 3 mM pNPA and 0.9 ml of
20 mM Tris-sulfate buer (pH 7.5), and the biocatalytic reaction
proceeded at 25°C for 5 min. Aer the reaction, the absorbance
was measured using a UV/Vis spectrophotometer (DU 730® Life
Science UV/Vis spectrophotometer; Beckman Coulter, Brea, CA,
UnitedStates) at 405 nm. One unit of lipase was dened as the
amount of enzyme that hydrolyzed 1 μmol p-nitrophenol
ml−1min−1.
2.5. Impranil degradation assay
PS-PU Impranil biodegradation was quantitatively determined
using cell-free supernatants of the strain HY-72 as described by
Hung et al. (2016) with minor modications. e isolate was
cultured in a baed Erlenmeyer ask containing 10 ml R2A medium
on a rotary shaker at 30°C for 2 days. Aer incubation, the
supernatants were separated by centrifugation at 10,000 × g and 4°C
for 15 min, and subsequently ltered using a 0.22-μm lter
(Millipore, UnitedStates). e cell-free ltrates were concentrated
by acetone precipitation (1:4 v/v), and the precipitates were
re-dissolved in 100 μl of PBS. An Impranil suspension (100 g l
−1
) in
PBS was used as the stock solution for this assay. Dilutions of the
Impranil suspension with distilled water were measured to construct
a standard curve for converting the absorbance to % clearance. Cell-
free precipitates (100 μl) and puried lipase (50 U) were added to
5 ml of the Impranil suspension and incubated at 30°C. e clearance
of the Impranil dispersion was measured using a spectrophotometer
(DU 730® Life Science UV/Vis spectrophotometer; Beckman
Coulter, Brea, CA, UnitedStates) at 600 nm for 72 h at 24 h intervals.
Fourier-transform infrared (FTIR) spectroscopy (VERTEX
80v with HYPERION 2000, Bruker, Hardt, Germany) was
conducted to determine the changes in the polymer bond
formation of PU. Aer 72 h of incubation, 1 ml of the Impranil-
containing supernatant was transferred into Eppendorf tubes and
subsequently lyophilized using a freeze dryer (Alpha 1–4 LD plus,
Martin Christ, Osterode, Germany). e spectra of freeze-dried
samples were recorded at 4 cm−1 resolution and wavenumbers
from 500 to 4,000 cm−1 at room temperature, including
non-treated samples as the negative control. All experiments were
performed in triplicates under the same conditions.
2.6. Degradation of PU foams
Pre-weighed pieces of PS-PU and PE-PU foams (as described
in Section 2.1) were added to baed Erlenmeyer asks containing
100 ml of R2A medium. e bacterial suspensions of the mid-log
phase were inoculated into each ask and incubated on a rotary
shaker (180 rpm) at 30°C for 14 days. A negative control without
bacterial inoculation was also maintained under the same
conditions. Aer incubation, the weight loss of each foam sample
was calculated. e PS-PU and PE-PU foams were thoroughly
washed with distilled water ve times to remove the colonized
bacteria on the surface and air-dried at 50°C until completely dry.
e weight loss percentage was calculated as weight loss
(%) = (initial weight - nal weight)/initial weight × 100. e
experiments were performed in triplicate under the same conditions.
e changes in the surface morphology of PU foams were
observed using scanning electron microscopy. e PS-PU and
PE-PU foams were thoroughly washed with distilled water to remove
impurities on the surface and air-dried at 50°C until completely dry.
e samples were then gold-coated with a sputter coater (Q15ORS,
Quorum, East Sussex, UnitedKingdom) and observed using an FEI
Quanta 250 FEG (FEI, Hillsboro, OR, UnitedStates). Non inoculated
PU foams were examined as negative controls.
2.7. Statistical analyses
One-way ANOVA was performed using SPSS soware
(version 24, SPSS, Inc., Chicago, IL, UnitedStates). e mean
values was compared using Scheé’s method, and p values <0.05
were considered statistically signicant.
3. Results
3.1. Isolation and identification of
Impranil-degrading strain
e Impranil-degrading bacterium with a high degree of
transparent halo zones on R2A agar containing 1% (v/v) Impranil
was isolated from the intestines of the adult Asian mantis
(H. patellifera). e isolate was selected as a promising candidate
for the degradation of PU and was stored for further
biodegradation studies. For phylogenetic proling, the 16S rRNA
gene sequence of the isolate (2,000 bp) was compared with those
of type strains available in the EzBioCloud database. e isolated
strain was most closely related to Serratia liquefaciens strain ATCC
27592T (NR121703), with 99.93% 16S rRNA nucleotide sequence
similarity (query coverage of 92%; Figure1A). e HY-72 strain
was evaluated for the production of extracellular enzymes
associated with hydrolysis of PU components (Figure1B) e
results of agar plate assay indicated that strain HY-72 has the
ability to produce extracellular protease (the formation of
transparent halo zone) and lipase (the formation of calcium
complex); therefore, the isolate has the potential to degrade PU.
3.2. Identification and characterization of
PU-degrading enzymes
e 1,848 bp lipase gene encoding an extracellular lipase from
HY-72 was identied (Figure2). e nucleotide sequence of the
Kim et al. 10.3389/fmicb.2022.1005415
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lipase was predicted to express a premature protein of 615 amino
acids with a molecular weight of approximately 65 kDa. e active
site of the extracellular lipase (serine hydrolase motif ),
G-X-S-X-G, was found at Gly 205, and the secretion signal,
G-G-X-G-X-D-X-X-X, was found at Gly 382. A protein BLAST
survey indicated that the primary sequence of premature lipase
was similar to that of a S. liquefaciens S33 DB-1 lipase (ABP04234)
with 99.35% identity and S. liquefaciens polyurethanase
(WP_048762691) with 95.77% identity. Phylogenetic analysis
showed that the primary sequence of the lipase from HY-72
shared a close evolutionary relationship with that of family I.3
extracellular lipases (Figure3). e puried recombinant lipase
from HY-72 was observed by SDS-PAGE, and their PU
degradation activity was conrmed by zymogram analysis. e
puried recombinant lipase had a relative molecular mass of
approximately 65 kDa, and the gel used to obtain a zymogram
revealed PU-clear regions (Figure4). e specic lipolytic activity
of the recombinant PU-degrading lipase for pNPA was
2,883 U mg−1 (Table1).
3.3. Biodegradation of Impranil
e strain HY-72 and lipase were quantitatively evaluated by
turbidimetric analysis to conrm the biodegradation of Impranil.
Optical absorbance at 600 nm was used as a direct measurement
of clearance by the bacterial ltrates and puried lipase. FTIR
analysis was performed to conrm Impranil biodegradation.
Visual observation of the Impranil suspension treated with
ltrates of HY-72 and puried lipase showed that the turbid
suspension became transparent and the value of OD600 notably
decreased in a time-dependent manner (Figure5A). Aer 72 h
incubation, the degradation rate of the bacterial ltrates and the
puried lipase was 85.24 and 70.37%, respectively, while the
negative control not treated with HY 72 did not show detectable
clearance of the suspension (Figure5B). FTIR spectra of Impranil
degradation by the strain HY-72 and its lipase revealed changes in
functional groups, accompanied by more subtle changes at other
wave numbers (Figure6). e ltrates and puried lipase from
HY-72 demonstrated a signicant decrease in the signal intensity
of the peak at 1,735 cm−1, indicating that the ester functional
group was aected by the hydrolysis and subsequent catabolism
of urethanes. e peak at 1,524 cm−1 corresponds to the N-H
bending, with a notably increase in the C-N stretch. e
characteristic peaks from the urethane group at 1,140 cm−1,
representing C-O stretching, also increased in intensity. e peak
at 704 cm−1 (aliphatic chain signals) decreased aer 72 h of
incubation. e results correlated with the biodegradative
enzymatic activities detected in the HY-72 ltrates, as indicated by
the degradation assays and FTIR analysis.
A B
FIGURE1
Characterization of HY-72 strain isolated from the intestine of Asian mantis (Hierodula patellifera). (A) Phylogenetic relationship of the strain HY-72
based on 16S rRNA gene sequence. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences and closely related species
constructed using MEGA X software. Numbers at each branches indicate the bootstrap percentage of 1,000 replications. (B) Extracellular
enzymatic activities of HY-72 strain.
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3.4. Biodegradation of PS and PE-PU
foam
e degradation eciency of the strain HY-72 for two types
of PU foams (PS-PU and PE-PU) was determined by weight loss
measurement and analyzing scanning electron micrographs.
PS-PU and PE-PU foams showed approximately 20.34 and 5.13%
weight loss, respectively (Figure7A). e dry weight of untreated
foams did not change aer 2 weeks of incubation. e PU foams
incubated with the bacterial isolate showed morphological
FIGURE2
Amino acid sequences of lipase from HY-72 strain. HY-72 lipase, Serratia liquefaciens L135 polyurethanase (Sll135; WP_048762691), and S.
liquefaciens S33 DB-1 lipase (DB-1; ABP04234) were compared using CLUSTALW program. Orange = catalytic domain; red; nucleophilic elbow,
GXSXG; blue = domain of interaction with calcium; green = glycine-rich repeats, GGXGXDXXX, interacting with calcium and type Isecretion system;
purple closed circles = amino acid residues that interact with calcium; black closed circles = catalytic triad.
Kim et al. 10.3389/fmicb.2022.1005415
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changes caused by bacterial degradation. In scanning electron
micrographs, PS-PU foam treated with the strain HY-72 showed
holes and loss of integrity of their reticulated cell structure in
panoramic views (70 × magnication); the development of bends
was detected in a closer view (1,500 × magnication). Similarly,
the PE-PU foam structure was disrupted, and the development of
cracks, roughness, and surface roughening was evident, while the
non-treated control foam maintained its structure and a smooth,
intact and clear surface (Figure7B).
4. Discussion
Insects exhibit remarkable adaptations to various
environmental factors. ese adaptation are closely related to the
changes in the prole of their gut microorganisms as well as
associated functions (Dillon and Dillon, 2004). Insect-associated
microbes produce various bioactive molecules and eective
digestive enzymes, which have great potential for industrial
applications (Jang and Kikuchi, 2020). Insect-associated gut
microbes have been studied for their bioremediation ability using
contaminants, pollutants, and toxins as nutritional sources
(Loredo-Treviño etal., 2012; Banerjee etal., 2021). Insects and
their gut microbes have gained attention as promising resources
for plastic biodegradation (Bilal etal., 2021). Recent studies have
reported that bacterial strains from Tenebrio molitor (Yang etal.,
2015) and Triboluim castaneum (Wang et al., 2020) eciently
degrade polystyrene. Bacteria isolated from Plodia initerpunctella
(Yang etal., 2014) and T. molitor (Yin etal., 2020) have been
reported to degrade PE. is study evaluated insect symbiotic
microbes and their secreted lipases for PU biodegradation.
Serratia sp. strain HY-72 was isolated from the intestine of the
FIGURE3
Phylogenetic analysis of HY-72 family I.3 lipase and its closely related functional homologs. Multiple alignments of the amino acid sequences were
achieved using Clustal W in the MEGA X software. The protein sequence data used for phylogenetic analysis were retrieved from the GenBank
database.
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adult Asian mantis (H. patellifera), and its PU-degradation activity
was investigated. is is the rst report demonstrating PU
degradation by a Serratia sp. and its exogenous lipases isolated
from the intestine of the insects.
Various bacteria utilize exogenous enzymes to degrade
polymers, and use them as nutritional sources for their
assimilation and metabolism. Several extracellular enzymes
responsible for PU biodegradation have been studied (Binger
et al., 2015). e enzymes that degrade PU by hydrolysis
include protease, lipase, esterase, and urease, and the
synergistic action of these enzymes could improve the
biodegradation of PU through their co-metabolic interactions
(Howard, 2002; shah etal., 2016). Based on the results, it was
hypothesized that the biodegradation of PU by HY-72 strain
mainly occurs via extracellular enzymatic activities. e genus
Serratia produces exogenous enzymes, such as proteases and
esterases (Gupta etal., 2004). e 65 kDa puried recombinant
lipolytic enzyme from HY-72 showed lipase activity and
degraded PU. e results were consistent with those of a
previous study in which 65 kDa lipase from S. liquefaciens L135
from cow’s milk had lipolytic activity, and the modeled and
validated structure of this polyurethanase was able to bind
urethane by molecular docking (Salgado et al., 2021).
Comparing the puried recombinant lipase from HY-72 with
polyurethanase from S. liquefaciens L135, there were 26 amino
acids, of which 6 were dierent in the catalytic domain and
10in the interaction domain with calcium. In addition, r 385
of the secession signal in the interaction domain with calcium
was dierent. e specic activity of lipase from HY-72 was
2,883 U mg
−1
, and polyurethanase from S. liquefaciens L135 was
2,793 U mg−1, using pNPA as a substrate (Salgado etal., 2021).
e present study conrms family I.3 lipase has a
PU-degrading ability.
Impranil has been widely used as a substrate for detecting
the PS-PU hydrolyzing activity of bacterial strains (Schmidt
etal., 2017); it is an aqueous aliphatic polymer composed of
spheres 200 nm or less that can remain suspended in aqueous
media (Binger etal., 2015). e HY-72 ltrates and puried
recombinant lipase showed PU degradation rates of 85.24 and
70.37%, respectively. Various types of protease are known to
hydrolyze urethane bonds, leading to release of carbon dioxide
and formation of amines and alcohols (Mahajan and Gupta,
2015). A previous study suggested that all tested strains with PU
degradative capabilities were closely related in their protease
activity (Loredo-Treviño etal., 2012). In this study, the higher
PU degradation rate achieved by cell ltrates is believed to
bedue to a synergistic eect between the protease and lipase of
the HY-72 strain. In comparison, Pseudomonas putida A12 from
soil showed that Impranil was degraded by 45% aer 2 days
(Peng et al., 2014). Lasiodiplodia sp. strain E2611A and
Cladosporium pseudocladosporioides T1.PL.1 showed 85 and
FIGURE4
SDS-PAGE and zymography of lipase from HY-72 strain after
purification. Lane M, standard marker proteins; lane 1, the total
cell lysate of lipase-expressing Escherichia coli BL21 after IPTG
induction; lane 2, purified lipase; lane 3, corresponding
zymogram.
TABLE1 Enzyme characteristics of lipase for polyurethane degradation.
Strain Mr (kDa) Optimal pH Optimal Temp.
(°C)
Specific activity
(U mg−1)
Reference
Serratia sp. HY-72 65 8.0 30 2,883ais study
Serratia liquefaciens L135 65 8.0 30 2,793bSalgado etal., 2021
Acinetobacter gerneri P7 66 8.0 25 37.58cHoward etal., 2012
Pseudomonas chlororaphis 63 8.4 - 8.5aRuiz etal., 1999
One unit (U) of lipolytic activity is dened as the amount of protein that hydrolyzed 1 μmol p-nitrophenol ml−1min−1. All assays were performed in triplicates.
aSpecic activity toward p-nitrophenyl acetate.
bSpecic activity toward p-nitrophenyl palmitate.
cSpecic activity toward p-nitrophenyl propanate.
Kim et al. 10.3389/fmicb.2022.1005415
Frontiers in Microbiology 09 frontiersin.org
87% Impranil degradation rate aer 14 days (Russell etal., 2011;
Álvarez-Barragán et al., 2016). e FTIR spectrum analysis
demonstrated Impranil breakdown due to the attack on
functional groups, in accordance with those previously reported
by Peng etal. (2014) and Álvarez-Barragán etal. (2016). Based
on the Impranil degradation assay and FTIR analysis, the strain
AB
FIGURE5
Time course of degradation of Impranil (100 g l−1) by the cell-free filtrates and purified lipase of HY-72 strain. (A) Photograph of Impranil
suspensions according to incubation with the cell-free precipitates and purified lipase of HY-72 strain compared to the non-treated control.
(B) Quantitative analysis of Impranil degradation. Percent clearance was determined by a spectrophotometer at 600 nm. All data were normalized
to the negative control. Values followed data are presented as means ± SD (n = 3). Dierent letters above error bars indicate a significant dierence
by Scheé’s test (p < 0.05).
FIGURE6
Comparison of FTIR spectra of Impranil degradation after 3 days of incubation with the cell-free precipitates and purified lipase of HY-72 strain and
non-treated control. Arrows represent the position of peaks for the functional groups decreased or increased in intensity.
Kim et al. 10.3389/fmicb.2022.1005415
Frontiers in Microbiology 10 frontiersin.org
A
B
FIGURE7
Weight loss (A) and scanning electron micrographs (B) of PU foam by HY-72 strain. PS-PU foam and PE-PU foam were incubated with the HY-72
strain for 14 days. Each pair of foam pieces presents the control foam (left) compared to the HY-72 treated foam (right). Values followed data are
presented as means ± SD (n = 3). Statistical significance between compared groups is indicated as * p < 0.05.
HY-72 and family I.3 lipase could bea potential resource for the
biodegradation of colloidal PS-PU. e quantitative and visual
analyses of PS- and PE- PU foam degraded by the strain HY-72
indicated that PS-PU (with weight loss of 20.34%) had a higher
degradation rate than PE-PU aer 2 weeks of incubation. ese
results indicate that the strain HY-72 and its exogenous enzyme
could also participate in the biodegradation of foam type-PU,
which has urethane and ester groups. In the scanning electron
micrographs, the PS- and PE-PU foams showed disruption and
loss of integrity of their reticulated cell structure and changes in
the surface morphology, which is consistent with previous
reports (Gautam etal., 2007; Spontón etal., 2013; Gómez etal.,
2014; Pellizzi etal., 2014; Álvarez-Barragán etal., 2016; Pérez-
Lara etal., 2016; Khan etal., 2020). erefore, the strain HY-72
has the potential to biodegrade PU. However, practical
biodegradation on a large scale and information on the
bioconversion of PU by microbial strains are limited and remains
a challenging subject for managing plastic waste. erefore,
further studies are needed to verify the metabolic pathways and
the mechanism of bioconversion, also its degradation
system optimization.
A PU-degrading bacterium, Serratia sp. strain HY-72, was
isolated from the intestine of the Asian mantis (H. patellifera) and
evaluated its PU-degrading activity. e results conrmed that
this strain can degrade aqueous aliphatic PS-PU polymer,
Impranil, and two types of foam (PS- and PE-PU; Figure8). e
65 kDa lipase of I.3 lipase family isolated from this strain was
identied as a PU-degrading enzyme with high specic lipolytic
activity. Further studies on the degradation mechanism and its
optimization as well as on the practical applications of the HY-72
strain for real-world PU waste management is required and
should employ multi-omics (e.g., genomics, proteomics,
metabolomics), synthetic microbial communities (e.g., cell–cell
interaction, microbial consortium, molecular interaction), and
Kim et al. 10.3389/fmicb.2022.1005415
Frontiers in Microbiology 11 frontiersin.org
gene editing tools (CRISPR, TALEN, and ZFN; Jaiswal etal.,
2020; Liu etal., 2021). e present study suggests that the insect-
associated bacteria and their exogenous enzymes have potential
applications in plastic biodegradation, and can oer a promising
approach for plastic waste management.
Data availability statement
e datasets presented in this study can befound in online
repositories. e names of the repository/repositories and accession
number(s) can befound in the article/supplementary material.
Author contributions
J-HK, K-HS, and H-YP participated in acquiring the data and
the study design, draed the manuscript, and revised the nal
manuscript. SHC performed the experiments and analyzed the
data. MGP and DHP participated in study protocol design and
supplied samples. All authors contributed to the article and
approved the submitted version.
Funding
is research was supported by the Korea Research Institute
of Bioscience and Biotechnology (KRIBB) Research Initiative
Program (KGM5492221).
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their aliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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