iGEM 2024 — Team xju-china

SAFETY | XJU-China
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Experimental Samples
Experimental Material Safety
Experimental Process Safety
Laboratory Safety

# Experimental Samples

Source of experimental samples

![](https://static.igem.wiki/teams/5268/notebook-page/table-1.webp)

# Strains and carriers used in the experiment

Strains and plasmids used

![](https://static.igem.wiki/teams/5268/notebook-page/table-2.webp)

# Main chemical reagents in the experiment

Main chemical reagents in the experiment

![](https://static.igem.wiki/teams/5268/notebook-page/table-3.webp)

# Reagent formulations

(1) Luria-Bertani ((LB) medium: peptone 10g / L, yeast extract 5g / L, NaCl / 10g / L, pH 7.0121 ℃, 20 min),
solid LB
medium plus 2% (Wamp V) Agar.

(2) Inorganic salt culture medium (MSM) with polyethylene as the sole carbon source: KH2PO4 0.7g shock L,
K2HPO4 0.7g
shock L, MgSO4 ·7H2O 0.7g K2HPO4 1.0g shock L, FeSO4 ·7H2O 0.002 g shock L, ZnSO4 ·7H2O 0.002 g max L, MnSO4
·H2O 0.01g
max L. pH 7.0 After sterilization at 121℃for 20 min, polyethylene (PE) film with 3 × 3 cm and PE microsphere
with a
diameter of 30 μ m were added for 10 mg respectively. Pretreatment of the film and polyethylene microspheres:
90%
ethanol (1 h) and 75% ethanol (1 h) treated the film (3 cm × 3 cm) and 30 μ m microsphere particles and dried
before use
(40 °C, 30 min). The treated polyethylene samples were cultured in LB (pH 7. 0) for 24 h to confirm that they
were fully
sterilized.

(3)50 × TAE buffer: weighing 242gTris-base,37.2 g Na2EDTA ·2H2O, then adding 800 mL distilled water, fully
stirring and
dissolving, then adding 57.1 mL acetic acid, fully mixing, finally distilled water fixed volume to 1 L, room
temperature. Dilute it to 1×TAE when in use (for example, if you need to use 1 × TAE of 1000 mL, then take 50
× TAE,980
mL of deionized water of 20 mL, mix and rest for a while before use).

(4) IPTG storage solution: weigh 2 g
isopropylthiogalactoside solid, dissolve it in 8 mL distilled water, configure it into 50 mg/mL storage
solution, use
0.22 μ m filter membrane to remove bacteria in a 2 mL centrifuge tube and store it in a refrigerator at-20 ℃
for use.

(5) preparation of Kana solution: 0.5g kanamycin sulfate solid was dissolved in 9 ml distilled water and
configured as
50 mg/mL storage solution. 0.22 um filter membrane was used to remove bacteria in 2 mL centrifuge tube in
super clean
table, and stored in refrigerator at-20 ℃. Its working concentration was 50 μ g/mL.

(6) Coomassie brilliant blue dye: 1
g Coomassie brilliant blue R250 was dissolved in 400 mL ddH2O, 450 mL methanol, 100 mL glacial acetic acid was
added,
and finally ddH2O was added to fix the volume to 1 L.

(7) SDS-PAGE decolorizing solution: methanol 100mL, glacial acetic acid 100mL, ddH2O volume 1L.

(8) SDS-PAGE protein loading buffer (5 × SDS-PAGE Protein Loading Buffer): 1.25 mL 0.5 mol/L Tris-HCl pH6.8,
glycerol 2
mL, 10%SDS 2 mL β-mercaptoethanol 1 mL, 0.1% bromophenol blue 0.5 mL, dissolved in distilled water to 10 mL,
then packed
the prepared solution into 1.5 mL EP tubes, 1 mL per tube, stored in a refrigerator at-20 ℃.

# Bacterial colony PCR

PCR Reaction system

![](https://static.igem.wiki/teams/5268/notebook-page/table-4.webp)

PCR Amplification reaction conditions

![](https://static.igem.wiki/teams/5268/notebook-page/table-5.webp)

![](https://static.igem.wiki/teams/4835/wiki/avater/avatar.png)

![](https://static.igem.wiki/teams/4835/wiki/avater/walking.gif)

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Engineering | XJU-China
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Overview
1. Looking for the intended sequences
2. Target gene heterologous expression
3. Confirming the activity of the enzyme that breaks down polyethylene

# OVERVIEW

Microplastics (MPs) are made up of plastics that are smaller than 5 mm and are released into the environment
every year.
These polymers undergo a variety of processes including photo-oxidation, weathering, mechanical abrasion,
water
movement, and microbial degradation.

When microplastics enter the body through the previously mentioned exposure
pathways, however, they may have a range of harmful effects on mammals.At the same time, research is being
done to
identify convenient, affordable, and environmentally friendly ways to process polyethylene, as it makes up
around 25% of
all plastics produced worldwide.

The use of bio-enzymes to break down polyethylene has progressively gained popularity
in recent years due to their ability to convert polyethylene wastes into carbon dioxide (CO2) and monomer
substances.
These methods are safer and more environmentally friendly than recycling techniques like landfilling and
incineration.

Currently, the multi-omics linkage approach is mostly used to mine the degradative enzymes from known
polyethylene-degrading bacteria.

However, a significant barrier to mining PE degradative enzymes is the low culturable
fraction of natural microorganisms—less than 2%. In order to test the genes encoding PE-degrading enzymes, the
target
sequences that were retrieved using macrogenome mining were subsequently directly bioinformatically examined,
heterologously produced, and functionally confirmed.

The limitation that traditional microbial isolation and culture
techniques are unable to excavate PE degradative enzyme-related genes of non-culturable microorganisms is
broken by this
method, which avoids the isolation and laboratory culture of microorganisms and excavates PE degradative
enzymes in a
faster and more thorough manner.

![](https://static.igem.wiki/teams/5268/engineering-page/project-structure-replace-1.webp)

Project Struture

# 1. Looking for the intended sequences

Look up in public databases the DNA and amino acid sequences (probe sequences) of known PE-degrading enzymes.
The target
DNA and amino acid sequences were obtained by comparing the probe sequence with those in the macro genome
library.

Next,
the Alphafold was used to predict the tertiary structure of the probe sequence. Subsequently, the tertiary
structures of
the target and probe amino acid sequences were further constructed, and the software was used to compare the
target and
probe gene sequences.

Following a comparison of the tertiary structures of the target and probe amino acid sequences,
the molecular docking software was utilized to molecularly dock the target amino acid with a small molecule
(16 alkyls)
that were retrieved from the Pubchem database. High-quality target sequences were picked out after the three
stages
mentioned above were examined.

# 2. Target gene heterologous expression

Once the high-quality target sequence from screening has been codon optimized, construct the expression
vector. Then,
transfer the recombinant plasmid into E. coli to create the engineering bacteria, verify the sequencing
correctly,
extract the recombinant plasmid containing the target gene from the engineering bacteria, and express the
target
gene.

The modified bacteria into which the recombinant plasmid was injected produced the recombinant plasmid
carrying the
target gene, which was then extracted and confirmed by PCR. The prokaryotic expression vector carrying the
target gene
has been effectively generated if the fragment size matches the size of the target gene. Once the prokaryotic
expression
vector encoding the target gene was driven to express itself by IPTG, the bacteria was crushed, and the
resulting
crushed crude enzyme solution was collected.

# 3．Confirming the activity of the enzyme that breaks down polyethylene

The crude enzyme solution after cell crushing was mixed with PE film and PE microspheres for 30 days, with
three
parallels in each group; after that, the PE film and microspheres were put into an ultrasonic cleaner
(RH-CX1000W) and
washed by adding sodium dodecyl sulphate for 30 min, and the PE film was completely immersed in 75% (v/v)
alcohol for 30
min, and then rinsed in sterile water, and dried naturally overnight.

Then the film was rinsed with sterile water and
dried naturally overnight. The weight loss rate of PE film, surface morphology of PE film and microspheres,
and changes
in surface functional groups were examined using an analytical balance, scanning electron microscope (SEM,
SU800), and
Fourier Transform Infrared Spectrometer (FTIR, VERTEX 70), respectively, and the results were used to
determine whether
the recombinant enzyme had PE degradation activity.

At the same time, only the microspheres after 30 days of mixing were
subjected to scanning electron microscopy (SEM) and Fourier infrared spectroscopy (FTIR) to investigate the
degradation
characteristics of the microspheres by the recombinant enzyme.

![](https://static.igem.wiki/teams/4835/wiki/avater/avatar.png)

![](https://static.igem.wiki/teams/4835/wiki/avater/walking.gif)

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Experiments | XJU-China
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Validation of plasmid construction by enzymatic digestion experiments
Preparation of Escherichia coli receptor cells
Transformation of plasmids
Screening of positive clones
Expression of target genes
Protein detection
E coli. Dh5α-pET-28a(+)-LS21sufB salt concentration shake flask test
Determination of salinity tolerance of LS21sufB engineering bacteria

# Validation of plasmid construction by enzymatic digestion experiments

## experimental step：

Reaction system:
total volume 20 μL
NcoⅠ:1μL
HindⅢ:1μL
10×H Buffer:2μL
Plasmid DNA: 10μL
ddH2O :6μL

## Reaction conditions:

Temperature 37°C, enzyme digestion for 3 hours.
1. Agarose gel electrophoresis to detect the results of enzyme digestion
Prepare 1% agarose gel (containing 0.5μg/mL nucleic acid dye), electrophoresis at 90V for 30 min.
2. UV transilluminator to observe the results (lane 1: original plasmid; lane 2: plasmid digested by restriction endonuclease.

# Preparation of Escherichia coli receptor cells

## Experimental Steps:

### Preparation:

Configuration and sterilization of 0.05mol/L CaCl2 solution, 0.05mol/L CaCl2 solution containing 15% glycerol; E. coli BL21, E. coli S17-1 plate delineation to ensure single colonies
1、Pick single colony from the plate and incubate it in 5ml LB medium overnight.
2, Take 100ul of bacterial solution and add it into 10mL LB medium. Shake at 220 rpm for about 3-5 hours to make OD600 between 0.4~0.5.
3、Sub-transfer the bacterial solution into 1.5ml centrifuge tubes (one tube of 10ml bacterial solution can be divided into about 6 tubes of 1.5ml centrifuge tubes), place on ice for 10 minutes, and centrifuge at 3000g for 10 minutes at 4℃.
4. Discard the supernatant, gently suspend the cells with 200 μL of pre-cooled 0.05 mol/L CaCl2 solution, and place on ice for 10 min.
The cells were suspended in 200 μL of pre-cooled 0.05 mol/L CaCl2 solution, placed on ice for 30 min, and then centrifuged at 3000g for 10 min at 4℃.
5. Discard the supernatant, add 200 μL of pre-cooled 0.05 mol/L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for a few minutes to form a sensory cell suspension. The cells were kept on ice for a few minutes to form a sensory cell suspension. Plasmid transformation

# Transformation of plasmids

## Experimental steps:

1, 10μL of recombinant plasmid was mixed with 50μL of susceptible E. coli BL21
2、Mix the recombinant plasmid with the receptor cells in an ice bath for 30min.
3、Hot excitation at 42℃ for 90s, and then ice bath for 2-3min.
4、Add 500μl of LB medium, 37℃, 220rpm, incubate for 1-2h.
5、Take 200μL and spread it evenly on LB solid medium containing 100μg/ml Kana, incubate at 37℃ inverted overnight.

# Screening of positive clones

## Experimental steps:

1 Pick the overnight cultured colonies (2 parallels per plate) into the LB liquid medium containing 100μg/ml Kana, 37℃, 150rpm, overnight culture, prepare the seed solution, and then carry out bacterial liquid PCR for verification.
2 Bacterial liquid PCR: take 2μL of bacterial liquid on the ultra-clean bench, add 100μL of ddH2O, mix well, and boil in a 100℃ water bath for 15min.

3 20μL system
Bacterial liquid: 4μL
Upstream primer: 1μL
Downstream primer: 1μL
Master mix:6μL
ddH2O:8μL
(Upstream primer: ATGGCAAGCGAAGAAATGGAAC)
Downstream primer: TTAACCCACTGCGCCTTCAAG)
PCR Parameters.
95°C pre-denaturation:5min➔main cycle 94°C:30s➔55°C:30s➔72°C:2min cycle 30 times➔72°C:10min➔4°C:50min

# Expression of target genes

The activated engineering bacteria were pre-cultured 37°C overnight. Growth at 1% inoculum reached 0.6 ≤ OD600 ≤ 0.8 at 37°C. 0.1 mM IPTG was added (no IPTG was added to the control group) and growth was carried out at 30°C for 2 hrs. The experimental group with OD600 ±1.0, diluted the above bacterial solution with LB at 0.01% inoculum. The growth was observed every 12h by inoculating into 8% NaCl, pH=8 LB plates at an inoculum of 50μl.

# Protein detection

Take 30ml of the induced bacterial solution,at 4℃,,centrifuge for 5min (5000g). Discard the supernatant and add 1% lysozyme containing 1mg/ml. Then freeze and thaw three times: at 37℃ for 5min, and then at -80℃ for 5min. ultrasonic cell crusher was used to sonicate for 3s, stop for 3s, and work for 20min (power: 40%, 360W), and the obtained solution was centrifuged at room temperature for 10min, and the supernatant was taken as the upper sample solution, and electrophoresis was performed.

# Salt concentration shake flask test

Procedure: inoculate the induced bacterial solution at 1% inoculum into 250 ml conical flasks. (100 ml sample volume)

Control group: A wild-type Dh5α (WT) medium without kana, B empty (EV) medium with kana.

Experimental group: E coli. Dh5α-pET-28a(+)-LS21sufB(LSD) medium with kana.

Note: The addition of the antibiotic kana to the medium of the empty and experimental groups serves to prevent contamination by stray bacteria.

# Determination of salinity tolerance of engineering bacteria

Engineering bacteria: E coli.BL21(DE3)-pET-28a(+)-LS21sufB .

Saline plate medium pH 8.5, diluted to 10-2 after induction, 30μL per compartment.

![](https://static.igem.wiki/teams/4835/wiki/avater/avatar.png)

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Results | XJU-China
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[Dry Lab](#section1)
Wet Lab

# Dry Lab

After Dry Lab, we obtained the protein primary structure, tertiary structure comparison and molecular docking
results of
the target and probe sequences.

Primary and tertiary structure comparison and molecular docking results

![](https://static.igem.wiki/teams/5268/result-page/table-1.webp)

# Wet Lab

## Measure One- Scanning Electron Microscope (SEM)

### PE film

PE films were mixed with recombinant enzymes and controls for 30 days and then subjected to SEM assay. The
results
showed that the surface of PE films treated with laccase (Lac-NJ-1, Lac-S102-1, Lac-S12-4), alkane
monooxygenase
(AlkB-S102-2, AlkB-NJ-3, AlkB-SKL-1) were all smoother and flatter (Figure 1). Laccase Lac-S11-1, Lac-PM2-2,
Lac-PM3-3,
Lac-PM2-1, Lac-S12-2, Lac-S12-3, Lac-SKL-1, Lac-PM2-3, Lac-PM3-1, Lac-NJ2-1, Lac-S12-1, Lac-PM3-2, Lac-S100
-1,
hydrolases (Hyd-S11-1, Hyd-S12-1), alkane monooxygenases AlkB-NJ1-1, AlkB-NJ1-2, etc. produced different
morphological
changes on the PE surface, such as cracks, holes, folds and dents. Lac-S12-2, Lac-SKL-1, Lac-PM2-3, Lac-S12-3,
Hyd-S12-1; Lac-S11-1; Hyd-S11-1 produced cracks, holes, and dents on the surface of the PE film and did not
produce any
other morphological changes on the surface of the PE film, respectively. Lac-PM3-2, Lac-S12-2, Lac-PM2-1,
AlkB-NJ1-2,
Lac-PM3-3, Lac-PM2-2, the surface of the polyethylene surface appeared wrinkles, dents, cracks, and other
morphological
changes. Lac-PM3-1, Lac-NJ2-1, Lac-S100-1, resulting in the surface of the PE film is more serious damage,
respectively,
a large number of PE fragments and larger holes (Figure 1c), whereas AlkB-NJ1-1 caused less surface breakage
of PE films
and only produced smaller holes on its surface.

In order to show more visually the difference in the degree of surface breakage caused by different
recombinant enzymes
on the surface of PE films, the percentage of surface breakage was calculated in this study. Compared to the
control
group. The percentage of surface breakage of PE films treated with laccase Lac-NJ-1, Lac-S102-1, Lac-S12-4,
alkane
monooxygenase AlkB-S102-2 AlkB-NJ-3 AlkB-SKL-1 were 4.84%, 5.98%, 8.95%, 1.92%, 2.39%, 1.92%, and 2.39%,
respectively,
which were not significantly higher compared to control. significantly higher than that of the control group.
While
laccase Lac-S12-1, Lac-PM2-1, Lac-PM2-3, Lac-PM3-3, Lac-S12-2, Lac-S11-1, Lac-S12-3, Lac-PM3-1, Lac-NJ2-1,
Lac-SKL-1,
Lac-PM3-2, Lac-PM2-2, Lac-S100-1, hydrolase (Lac-NJ2), Lac-S100-1, and Lac-S100-1, hydrolysin (Lac-NJ2) were
not
significantly higher than the control. S100-1, hydrolase (Hyd-S11-1, Hyd-S12-1), alkane monooxygenase
AlkB-NJ1-1, and
AlkB-NJ1-2 treated PE film surfaces were broken at 20.02%, 20.02%, 34.63%, 24.36%, 26.21%, 23.63%, 20.13%,
20.13%,
20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, 20.13%, and 20.13%,
respectively. 36.89%, 30.51%, 28.63%, 28.53%, 24.29%, 24.76%, 26.34%, 32.89%, 23.82%, 23.05% were
significantly higher
compared to the control group (Fig. 2) However, based on the changes in polyethylene surface morphology alone,
it was
not possible to determine whether the potential polyethylene-degrading enzyme has polyethylene-degrading
function, so it
is necessary to combine the Fourier infrared spectroscopy results for analysis.

![](https://static.igem.wiki/teams/5268/result-page/figure-1.webp)

SEM results of films after reaction with recombinase (a, SEM results of the first part of PE
films after reaction with
recombinase; b, SEM results of the second part of PE films after reaction with recombinase; c, SEM results of
the third
part of PE films after reaction with recombinase; and d, SEM results of PE films after reaction with alkane
monooxygenase)

![](https://static.igem.wiki/teams/5268/result-page/figure-2.webp)

Proportion of PE film surfaces broken (a, proportion of first part PE films broken after reaction
with recombinase; b,
proportion of second part PE films broken after reaction with recombinase; c, proportion of third part PE
films broken
after reaction with recombinase; and d, proportion of PE films broken after reaction with alkane
monooxygenase)

### Surface microscopic characterisation of PE microspheres

Laccase Lac-S12-1, Lac-PM2-1, Lac-S12-2, Lac-S11-1, Lac-S12-3, Lac-PM3-1, Lac-NJ2-1, Lac-SKL-1, Lac-PM3-2,
Lac-PM3-3,
Lac-PM2-2, Lac-PM2-3, Lac-S100 -1, hydrolases (Hyd-S11-1, Hyd-S12-1), alkane monooxygenase AlkB-NJ1-1,
AlkB-NJ1-2, etc.
caused different degrees of changes in the surface morphology of PE microspheres. Among them, Hyd-S12-1,
Lac-NJ-2-1,
Lac-PM3-2, Lac-S100-1, Lac-S12-3, Lac-PM3-1, etc. led to the change of PE microspheres from spherical to
oblate with
cracks on the surface, and Lac-PM3-1 led to the degradation of PE microspheres into smaller irregular
particles (Fig.
3a).Lac-PM3 -3, AlkB-NJ1-1, AlkB-NJ1-2, Hyd-S11-1, and Lac-PM2-3 caused less damage to the PE microspheres
with only
small pits and holes on the surface (Figure 3b).Lac-PM2-1, Lac-S12-1, Lac-S11-1, Lac-SKL-1, Lac-PM2-2, and
Lac-S12-2
caused almost the same degree of damage to the polyethylene surface, all of which resulted in craters on the
surface of
the polyethylene microspheres, while Lac-S12-1, Lac-S12-2, and Lac-S11-1 caused craters on the surface of the
polyethylene microspheres and also adsorbed incompletely degraded microsphere debris on the surface(Fig. 3c).

![](https://static.igem.wiki/teams/5268/result-page/figure-3.webp)

Microscopic characterizations of the surface of PE microspheres

Note: SEM images of polyethylene microspheres with similar degree of surface morphology damage and
morphological changes
were combined and numbered with a, b, and c according to polyethylene microspheres subjected to recombinant
enzyme
action.

## Measure Two- Fourier Transform Infrared Spectroscopy

### Changes in functional groups on the surface of polyethylene films

Polyethylene in the presence of degrading enzymes usually leads to the formation of new functional groups and
FTIR
spectroscopy was used to detect the chemical changes produced in the polymers by the reaction of enzymes with
PE films.
As shown in Figure 4a after the reaction of PE films with laccase Lac-S12-1, Lac-PM2-1, Lac-NJ-1, Lac-S102-1,
new
absorption peaks appeared on the surface of PE films treated with laccase Lac-S12-1 near 1650 cm-1, 1747 cm-1,
etc., as
compared to the control group (CK), and the absorption at 1650 cm-1 The absorption peak at 1650 cm-1 indicates
the
presence of C=C in PE, and 1747 cm-1 corresponds to C=O. According to the characteristics of the absorption
peaks in IR
absorption spectra, it can be seen that the absorption peaks between 1720 cm-1 and 1770 cm-1 are generated by
the
stretching and vibration of C=O in the ester bond.A new absorption peak was generated in the Lac-PM2-1 treated
film at
1727 cm-1, and a broader absorption peak was generated in the range of 1150-1087 cm-1. The new absorption peak
at 1727
cm-1 corresponds to the expansion and contraction vibration of C=O, a newly generated oxidation functional
group in the
PE long chain, and 1150-1087 cm-1 represents the expansion and contraction vibration of C-O in the PE long
chain. And
Lac-NJ-1, Lac-S102-1 did not cause the PE film to generate new oxidation functional groups, indicating that
Lac-NJ-1,
Lac-S102-1 did not cause oxidative decomposition of PE film.

The second part of the recombinant enzymes mixed with PE films after 30 days of action, except for laccase
(Lac-S12-4)
both hydrolases (Hyd-S11-1, Hyd-S12-1) and laccases (Lac-S12-2, Lac-S11-1, Lac-S12-3) caused new absorption
peaks on the
surface of the PE films (Figures 4b, 4c). After 30 days of action of hydrolase Hyd-S11-1, new absorption peaks
were
generated at 1018 cm-1, 1747 cm-1, and 1643 cm-1, which corresponded to the expansion and contraction
vibrations of the
oxidation functional groups such as C-O, C=O, and C=C, respectively, and those appeared near 1010 cm-1, 1087
cm-1, and
1087 cm-1, respectively, for the PE films under the action of laccase Lac-S12-3. The new peak near 1087 cm-1
corresponds
to the generation of C-O, and the absorption peak at 1727 cm-1 is generated by the C=O stretching vibration in
the ester
bond. Compared to the control group, the PE films treated with Lac-S12-2 produced a new absorption peak at
1654 cm-1
corresponding to C=C. After 30 days of Hyd-S12-1, the PE films produced a new chemical group, C=O, at 1724
cm-1. After
30 days of incubation with Lac-S11-1, new absorption peaks were formed on the surface of the PE films at 1757
cm-1, 1639
cm-1 and 3286 cm-3. The new absorption peaks at 1757 cm-1, 1639 cm-1 and 3286 cm-1-3200 cm-1 on the surface of
PE film
after 30 days of incubation with Lac-S11-1, 1639 cm-1 corresponds to C=C. Since multiple hydroxyl groups in
carboxylic
acid form hydrogen bonds with each other which results in a wider absorption peak at 3300 cm-1-2500 cm-1, and
the
absorption peaks of C=O in carboxylic acid are usually generated near 1760 cm-1, Lac-S11-1 The absorption
peaks at 3286
cm-1-3200 cm-1 and 1757 cm-1 are generated in the PE film, so the PE hydrocarbon chain may be oxidised to
generate fatty
acids under the action of Lac-S11-1.

![](https://static.igem.wiki/teams/5268/result-page/figure-4.webp)

Results of changes in surface functional groups of the first and second batches of PE films after
reaction with
recombinant enzymes (a, FITR test results from the first degradation experiments; b, c, FITR observations from
the
second degradation experiments)

Fourier infrared spectroscopy results from the third part of the recombinant enzyme polyethylene degradation
validation
experiment showed that the reaction of PE with the laccases Lac-PM3-1, Lac-NJ2-1, Lac-SKL-1, Lac-PM3-2,
Lac-PM3-3,
Lac-PM2-2, Lac-PM2-3, Lac-S100-1, the alkane monooxygenase AlkB- NJ1-1, AlkB-NJ1-2, Lac-PM3-1, Lac-S100-1,
Alkane
monooxygenase AlkB-1, and AlkB-NJ1-2 all resulted in new characteristic peaks on the surface of the
polyethylene after
30 days of reaction, and Lac-PM3-1 resulted in broader absorption peaks in the range of 3643-3266 cm-1 on the
surface of
the polyethylene, and blunter absorption peaks in the range of 3201 cm-1, which corresponded to the stretching
vibrations of the intermolecular hydrogen bonds formed between the multiple -OHs. In view of the production of
-OH, the
strong absorption peaks at 1056 cm-1, 1160 cm-1, and 1056 cm-1 correspond to the C-O in the alcohol molecule,
and in
addition to -OH, the C=O, C=C, and C=C stretching vibration peaks are also produced at 1751 cm-1 and 1670
cm-1,
respectively. stretching vibration peaks. In contrast, Lac-NJ2-1 did not cause polyethylene to produce -OH
absorption
peaks, so the broader absorption peaks at 1184-1010 cm-1 may be C-O-C. Lac-SKL-1 caused the PE film to produce
absorption peaks at 3489-3200 cm-1 consisting of multiple -OH, while the absorption peaks at 3656 cm-1, 3606
cm-1
represent the generation of free -OH in the hydrocarbon chain, given the generation of the characteristic
peaks of
alcohols and the generation of the C=O stretching vibration peak at 1743 cm-1, 1068- 975 cm-1 may be a mixture
of the
C-O stretching vibration peaks in the alcohol molecule and the -C-H- out-of-plane bending vibration peaks of
Cα in the
aldehyde group, which also suggests that after 30 days of mixing with Lac-SKL-1 the sub-methyl and methyl
groups of the
hydrocarbon chain were oxidised to produce free -OH, respectively. methyl and methyl groups were oxidised to
produce
hydroxyl and aldehyde groups respectively (Figure 5a).

The oxidative degradation of the PE film after 30 days of Lac-PM2-2 resulted in the expansion and contraction
vibration
peaks of oxidised groups such as -OH (3293 cm-1, 3614 cm-1), C=C (1646 cm-1) and C-O (1257 cm-1), but there
was no
production of C=O, so that 1183-983 cm-1 was not a good indication of oxidative degradation. So the absorption
peaks
generated at 1183-983 cm-1 may be the mixed absorption peaks of C-O and the -C-H-plane bending vibration of Cα
in the
carbon-carbon double bond.Lac-PM3-3-3 caused the strong absorption peaks generated on the PE surface at 3266
cm-1, the
1654 cm-1 produces strong absorption peaks corresponding to the free -OH, C=C stretching vibration peaks.
Since the PE
surface at 3266 cm-1 produces stretching vibration peaks of intermolecular hydrogen bonds formed by multiple
-OH.
Therefore, the strong absorption peaks at 1249 cm-1 and 1091 cm-1 correspond to the C-O in primary and
secondary
alcohols, respectively, which also indicates that some of the methyl and methylene groups in the PE
hydrocarbon chain
oxidised to form -OH, and the two adjacent methylene groups can be oxidised to form -OH by further catalytic
oxidation
to form β-diketone, which can be further oxidised to form β-diketone. The oxidation of -OH generated by the
oxidation of
two neighbouring methylene groups can be further catalyzed to produce β-diketone, and normally the C=O in
β-diketone
will generate a telescopic vibration peak at 1640-1540 cm-1 , so the absorption peak at 1546 cm-1 corresponds
to the
β-diketone.The Lac-PM3-2 only caused broader absorption peaks on the surface of PE at 1646-1595 cm-1 and
1122-1018 cm-1
, and the -OH was not generated on the surface of PE film. film surface did not produce -OH, so 1122-1018 cm-1
corresponds to C-O-C. The absorption peaks generated at 1646-1595 cm-1 may be the superposition peaks of C=C
and C=O
stretching vibration peaks (Fig. 5b).The absorption peaks generated by the PE film at Lac-PM2 -3 and
Lac-S100-1 after 30
days of action, new absorption peaks were generated at 1589 cm-1, 1146 cm-1, 1045 cm-1 and 1646 cm-1, 1122
cm-1, 1064
cm-1, respectively. The four absorption peaks generated at 1146 cm-1, 1045 cm-1 and 1122 cm-1, 1064 cm-1, etc.
were
C-O-C stretching vibration peaks.1589 cm-1 and 1646 cm--1 were C=O, C=C stretching vibration peaks,
respectively (Fig.
5c).The PE films were treated with the Alkane monooxygenase AlkB-NJ1-2 for 30 days produced new absorption
peaks at
3409-3320 cm-1, 1751 cm-1, 1639 cm-1, 1164 cm-1 and 1076 cm-1 for the stretching vibration of intermolecular
hydrogen
bonds formed by -OH, C=O, C=C, C=C, C -O, C=C, C-O in secondary alcohols, etc. (Fig. 5d).The absorption peaks
at 3606
cm-1, 3398 cm-1, and 1650 cm-1 of PE films after the action of AlkB-NJ1-1 correspond to the free -OH and
multiple -OH,
C=C and other oxidation functional groups (Figure 5e). The alkane monooxygenases AlkB-S102-2, AlkB-NJ-3, and
AlkB-SKL-1
did not cause the generation of new functional groups on the PE surface compared to the control (Figure 5f),
indicating
that the three alkane monooxygenases did not produce oxidative degradation of PE films.

![](https://static.igem.wiki/teams/5268/result-page/figure-5.webp)

Results of changes in surface functional groups of the third batch of PE films after reaction
with recombinant enzymes
(a, b, c, changes in surface functional groups of PE films after 30 days of reaction with laccase; d, e, f,
changes in
surface functional groups of PE films after 30 days of incubation with alkane monooxygenase)

In addition to the production of oxidation groups such as C=O, C=C, and C-O on the surface of PE films under
the action
of recombinant enzymes the intensity of the peaks at 2923 cm-1 (-C-H- asymmetric stretching vibration in
methyl group)
and 2850 cm-1 (-C-H- symmetric stretching vibration in methyl group) decreased. -C-H- symmetric stretching
vibration) at
2923 cm-1 (-C-H- asymmetric stretching vibration in methyl group) the intensity of the peaks decreased. The
intensity of
the absorption peaks at 2923 cm-1 and 2850 cm-1, etc., decreased in PE films treated with laccase Lac-PM3-1,
Lac-SKL-1,
Lac-PM2-2, and Lac-PM3-3 (Fig. 5a, b). A decrease in peak intensity at 2923 cm-1 and 2850 cm-1 also occurred
after
treatment with alkane monooxygenase AlkB-NJ1-1, AlkB-NJ1-2, while the PE treated with AlkB-NJ1-2 was
accompanied by a
slight shift of the absorption peak to the right. The decrease of peak intensity and the change of absorption
peak
position indicated that the conformation and arrangement of hydrocarbon chains were changed due to the
breakage of PE
long chains during the enzyme-catalysed degradation process (Figure 5d). For other recombinant enzymes that
can catalyse
the production of oxidative functional groups on the surface of PE films, the absence of absorption peak
shifts or the
decrease in absorption peak intensities at 2923 cm-1 and 2850 cm-1 after the interaction of PE films with them
may be
due to the uneven degradation of the surface of the PE films and the random cutting of the PE films at the
time of
delivery of the samples, which may have resulted in the non-detection of the peaks at 2923 cm-1 and 2850 cm-1.
2850 cm-1
, which may have led to the non-detection of the changes in peak intensity and peak position at 2923 cm-1 and
2850 cm-1
.

The oxidation of PE films catalyzed by recombinant enzymes produced various oxidation groups including
carbonyl,
hydroxyl, and carbon-carbon double bonds, but the FTIR spectroscopy alone could not determine the degree of
oxidation of
PE after different recombinant enzymes, and the carbonyl index could be a more intuitive demonstration of the
degree of
oxidation of PE. As shown in Figure 6a, the carbonyl indices of PE films treated with laccase Lac-S12-1 and
Lac-PM2-1
were 3.16% and 2.09%, respectively, which were significantly higher than those of the control group (CK)
(0.26%),
whereas the carbonyl indices of PE films treated with laccase (Lac-NJ-1 and Lac-S102-1) were 0.26% and 0.18%,
respectively, which were not significantly higher than those of the control group (CK). ) did not
significantly
increase; the carbonyl index of PE films after laccase (Lac-S12-2, Lac-S11-1, Lac-S12-3) and hydrolase
(Hyd-S12-1,
Hyd-S11-1) were 2.73%, 2.60%, 2.27%, 2.32%, 2.22% higher than that of control, and the carbonyl index of PE
films after
the reaction of Lac-S12-4 was 0.26% and 0.18% respectively compared to that of control (CK), and the carbonyl
index of
PE films after the reaction of Lac-S12-1, Lac-S102-1 was 0.26% and 0.18% respectively compared to that of
control (CK).
-4 after the reaction, there was no significant difference in the carbonyl index of the PE film surface
compared to the
control (Figure 6b).

The third part of the validation results as shown, the surface carbonyl index of PE films mixed with laccase
Lac-PM3-1,
Lac-NJ2-1, Lac-SKL-1, Lac-PM3-2, Lac-PM3-3, Lac-PM2-2, Lac-PM2-3, Lac-S100-1 for 30 days were 3.46%, 3.34%,
3.66%,
3.77%, and 3.34%, respectively. 3.66%, 3.77%, 3.16%, 2.98%, 3.99%, and 3.63% were all significantly higher
than the
control (Figure 6c). The carbonyl indices on the film surface after the action of alkane monooxygenases
AlkB-NJ1-1,
AlkB-NJ1-2, AlkB-S102-2, AlkB-NJ-3, and AlkB-SKL-1 were 3.88%, 4.08%, 1.08%, 1.50%, and 1.48%,
respectively.The carbonyl
indices of AlkB-NJ1-1, AlkB-NJ1-2 were significantly higher than the control, and the carbonyl indices of
AlkB-SKL-1
were significantly higher than the control. indices were significantly higher than the control, and the
carbonyl indices
of AlkB-S102-2, AlkB-NJ-3, and AlkB-SKL-1-treated PE surfaces were not significantly higher compared to the
control
(Figure 6d).

![](https://static.igem.wiki/teams/5268/result-page/figure-6.webp)

Carbonyl index changes on the surface of PE films (a, carbonyl index changes after reaction of
the first batch of PE
films with recombinant enzyme; b, carbonyl index changes after reaction of the second batch of PE films with
recombinant
enzyme; c, carbonyl index changes after reaction of the third batch of PE films with recombinant enzyme; and
d, carbonyl
index changes of carbonyl index changes of the PE films after reaction of the films with alkane monooxygenase)

### Changes of functional groups on the surface of PE microspheres

After the recombinant enzyme action of PE film weight loss rate calculation, Fourier infrared spectroscopy,
surface
microanalysis found that there are 17 recombinant enzymes can produce degradation of PE film, in order to
explore the PE
microplastics in the role of these 17 recombinant enzymes in PE surface property changes. Fourier transform
infrared
(FTIR) spectroscopy of PE microspheres mixed with these 17 recombinant enzymes for 30 days revealed that a new
absorption peak near 1747 cm-1 appeared on the surface of PE microspheres treated with laccase Lac-S12-1,
which was
generated by the C=O stretching vibration in the ester bond. The characteristic peaks also appeared on the
surface of PE
films treated with Lac-S12-1, and the new absorption peaks at 1724 cm-1 and 1662-1600 cm-1 of
Lac-PM2-1-treated PE
microspheres corresponded to the expansion and contraction vibration of C=O, C=C, and C=C, respectively, which
are the
newly generated oxidation functional groups in the PE long chains, and the expansion and contraction vibration
absorption peaks of C -O stretching vibration absorption peaks at 1150-1087 cm-1 in Lac-S12-1 and Lac-PM2-1
treated PE
microspheres (Figure 7a).

The absorption peaks produced by PE microspheres after 30 days of interaction with hydrolase Hyd-S11-1,
laccase(Lac-S11-1) at 1115-1060 cm-1, 1115-1052 cm-1, etc., respectively, corresponded to the C-O-C stretching
vibration. Also
laccase (Lac-S11-1) with the action of PE microspheres produced C=C stretching vibration peaks at 1631-1600
cm-1 (Figure
7b). Laccase Lac-S12-3, Lac-S12-2, and Hyd-S12-1 produced new absorption peaks at 1735 cm-1, 1127 cm-1;
3405-3263 cm-1,
1653 cm-1, 1095-1018 cm-1; and 1345-1122 cm-1 after 30 days of action corresponding to C=O, C -O-C; stretching
vibration
peaks of intermolecular hydrogen bonds formed by multiple -OHs; and stretching vibration peaks of oxidation
functional
groups such as C=C, C-O; and C-O-C, respectively (Figure 7c). The stretching vibration peaks of C=O, C=C,
C-O-C, and
C-O-C oxidation groups at 1747 cm-1, 1646 cm-1, and 1153-1076 cm-1 were produced by PE microspheres under the
action of
Lac-NJ2-1. PE microspheres under the action of Lac-SKL-1 showed the stretching vibration peaks of C=O, C=C,
and C-O-C
oxidation functionalities at 3610 cm-1, 3282 cm-1, 1753-1076 cm-1, 3610 cm-1, 3282 cm-1, 1753-176 cm-1, and
3610 cm-1.
and 3282 cm-1, 1743 cm-1, 1658 cm-1, 1531 cm-1, 1248 cm-1, 1078 cm-1, and 948-869 cm-1, respectively, where
3610 cm-1
and 3282 cm-1 are the peaks of free -OH and multiple -OHs, respectively. OH formation of intermolecular
hydrogen bonds;
the absorption peaks at 1658 cm-1, 1531 cm-1 correspond to C=O and C=C; since C=C is generated in the long
chain of PE,
the absorption peaks at 948-869 cm-1 are -C-H-plane bending vibration peaks; since the ester bond is generated
at 1750
cm-1, the absorption peaks at 1750 cm-1 are -C-H-plane bending vibration peaks. peak; since the C=O stretching
vibration
absorption peak occurs at 1750-1735 cm' for the ester bond, and two C-O stretching vibration absorption peaks
are
generated simultaneously at 1300-1150 cm-1 and 1140-1030 cm-1, the absorption peaks at 1743 cm-1, 1248 cm-1
and 1078
cm-1 are generated respectively for the ester bond at 1750-1735 cm-1 with C=O stretching vibration absorption
peaks and
two C-O stretching vibration absorption peaks. The resulting absorption peaks at 3610 cm-1, 1900 cm-1, 1751
cm-1, 1658
cm-1, 1172-1114 cm-1, 948-869 cm-1 under the action of Lac-PM3-1 correspond to -OH, C=O (1900 cm-1 and 1751
cm-1), C=C,
C-O , C=C, C-O-C stretching vibrational peaks, and -C-H- in-plane bending vibrational peaks (Figure 7d).

From Figure 7e, it can be seen that compared to the control group, the PE microspheres after the action of
laccase
Lac-PM3-2, Lac-PM3-3, and Lac-PM2-2 were found to be in the range of 3286 cm-1, 1654 cm-1, 1234 cm-1,
1116-1072 cm-1;
3606 cm-1, 3286 cm-1, 1889 cm-1, 1627 cm-1, 1535 cm-1, 971-929 cm-1; 3610 cm-1, 3363 cm-1-3270 cm-1, 1881
cm-1, 1643
cm-1, etc. New absorption peaks were generated. Among them, 3286 cm-1, 3363 cm-1-3270 cm-1 are the stretching
vibration
peaks of multiple -OHs forming intermolecular hydrogen bonds; 3606 cm-1, 3610 cm-1 are the stretching
vibration
absorption peaks of free -OH; 1881 cm-1, 1889 cm-1, 1535 cm-1 correspond to the C=O stretching vibration; 1654
cm-1,
1643 cm-1, 1627 cm-1 are the C=C stretching vibration absorption peaks; 1234 cm-1, 1116-, 1072 cm-1 correspond
to the
C=C stretching vibration absorption peaks, because PE microspheres generate multiple -OH at 3286 cm-1 after
Lac-PM3-2
action to form the stretching vibration peaks of intermolecular hydrogen bonding; 1234 cm-1, 1116-, 1072 cm-1
correspond
to the C=O stretching vibration absorption peaks. 1072 cm-1 correspond to the C-O stretching vibration peaks;
971-929
cm-1 correspond to the -C-H- out-of-plane bending vibration peaks.

The PE microspheres treated with laccase Lac-PM2-3, Lac-S100-1 were found at 3614 cm-1, 3341-3270 cm-1, 1893
cm-1, 1745
cm-1, 1645 cm-1, 1118-1076 cm-1, 906-860 cm-1; 1893 cm-1, 1638 cm-1 1307 cm-1, 1018-1000 cm-1, etc. New
absorption peaks
were produced. The absorption peaks at 3614 cm-1, 3341-3270 cm-1 are the stretching vibration peaks of free
-OH and the
stretching vibration absorption peaks of intermolecular hydrogen bonds formed between multiple hydroxyl
groups,
respectively.The absorption peaks at 1893 cm-1 correspond to the C=O stretching vibration, 1645 cm-1, the C=C
stretching
vibration absorption peak, 1745 cm-1. The absorption peaks at 1118-1076 cm-1 were the C=O in aldehyde group
and C-O in
hydroxyl group, respectively; since the PE microspheres treated with Lac-S100-1 did not produce -OH vibration
absorption
peaks, and C=C vibration peaks at 1638 cm-1, the absorption peaks near 1018-1000 cm-1 corresponded to C=O
vibration. The
absorption peaks near 1000 cm-1 correspond to C-O-C. The absorption peaks at 906-860 cm-1 and 1307 cm-1
correspond to
the out-of-plane and in-plane bending of -C-H- in olefins, respectively. vibration peaks (7f).PE microspheres
treated
with alkane monooxygenase AlkB-NJ1-1 produced new absorption peaks at 3610 cm-1, 1893 cm-1, 1747 cm-1, 1311
cm-1,
1180-1112 cm-1, and 952-917 cm-1, respectively, which corresponded to free -OH. C=O (1893 cm-1), C=O in
aldehydes (1747
cm-1), C-O stretching vibration, -OH in-plane bending vibration, and -C-H- out-of-plane bending vibration in
olefins.
And the new absorption peaks produced by the microspheres after 30 days of AlkB-NJ1-2 treatment were the same
as those
produced by the polyethylene microspheres after AlkB-NJ1-1 treatment, which were at 3610 cm-1, 1893 cm-1, 1747
cm-1,
1307 cm-1, and 1172 cm-1, respectively, and were produced to correspond to the -OH, C =O (1893 cm-1, 1747
cm-1) and C-O
(1307 cm-1, 1172 cm-1) stretching vibrational peaks, respectively, and -C-H- (914 cm-1) out-of-plane bending
vibrational
peaks in the olefins (7 g).

![](https://static.igem.wiki/teams/5268/result-page/figure-7.webp)

Changes in functional groups on the surface of PE microspheres

Note: Due to the Fourier infrared spectroscopy of microspheres treated with recombinant enzyme there are more
results
detected so they are plotted separately and numbered with a, b, c, d, e, f, g. Among them, 7g shows the change
of
functional groups on the surface of PE microspheres after the action of alkane monooxygenase.

The surface of PE microspheres under the action of recombinase in addition to the production of oxidised
groups such as
C=O, C=C, C-O at 2923 cm-1 (-C-H- asymmetric stretching vibration in methyl group) and 2850 cm-1 (-C-H-
symmetric
stretching vibration in methyl group) showed a decrease in peak intensity. -C-H- symmetric stretching
vibration) the
intensity of the peak at 2923 cm-1 (-C-H- asymmetric stretching vibration in methyl) decreased. The intensity
of the
absorption peaks of PE microspheres treated with laccase Lac-S12-1, Lac-PM3-1, Lac-SKL-1, Lac-PM2-2,
Lac-PM3-3,
Lac-PM3-2, Lac-PM2-3, AlkB-NJ1-1, AlkB-NJ1-2, etc. was decreased at 2923 cm-1 and 2850 cm-1, etc. . At the
same time, PE
microspheres treated with alkane monooxygenase AlkB-NJ1-2, AlkB-NJ1-1, laccase Lac-PM3-2, Lac-PM3-1,
Lac-PM2-3,
Lac-PM3-1, Lac-SKL-1 showed slight shifts to the right in addition to a decrease in the intensity of the
absorption
peaks at 2923 cm-1 and 2850 cm-1. The decrease in peak intensity and the shift of the absorption peaks
indicate that the
conformation and arrangement of the hydrocarbon chains were changed due to the breakage of the PE long chains
during the
enzyme-catalysed degradation process. For other recombinant enzymes that can catalyse the production of
oxidative
functional groups on the surface of PE microspheres, the lack of a slight shift of the absorption peaks to the
right and
the decrease in absorption intensity at 2923 cm-1 and 2850 cm-1 may be attributed to the fact that some of the
highly
oxidised microspheres were not collected during the collection of the samples because of the smaller particle
size of
the PE microspheres or to the catalytic ability of the rest of the samples, both of which may have contributed
to the
lack of oxidation. oxidative capacity of the rest of the samples, which could be the reason for the
non-detection of the
changes in peak intensity and peak position at 2923 cm-1 and 2850 cm-1.

The PE microspheres produced a variety of oxidative groups including carbonyl group under the catalysis of
recombinant
enzymes, in order to show the catalytic oxidation degree on the surface of PE microspheres more intuitively,
the
carbonyl index of PE microspheres after recombinant enzyme treatment was calculated, and the results were
shown in
Figure 1 that carbonyl index of the surface of PE microspheres was significantly elevated after the action of
the 17
recombinant enzymes for 30 days as shown in Figure 1, and the carbonyl index of the surface of the PE
microspheres was
significantly higher for the laccases Lac-S12-1, Lac PM2-1, Lac-S12-2, Lac-S11-1, Lac-S12-3, Lac-PM3-1,
Lac-NJ2-1,
Lac-SKL-1Lac-PM3-2, Lac-PM3-3, Lac-PM2-2, Lac-PM2-3, Lac-S100-1; the hydrolases (Hyd-S11-1. Hyd-S12-1); alkane
monooxygenase AlkB-NJ1-1, AlkB-NJ1-2 treated PE microspheres with carbonyl indices of 3.65%, 3.10%, 2.90%,
3.50%, 3.33%,
2.90%, 3.50%, 5.35%, 4.01%, 15.63%, 15.60%, 6.53%; 7.13%, 6.08%; 13.17%, 15.65%, were higher than the carbonyl
index of
the polyethylene film after 30 days of reaction with the polyethylene film, which may be due to the fact that
the
polyethylene powder has a larger surface area compared to the PE film, so the rate of oxidation under the
action of
recombinant enzymes may be faster compared to the PE film, resulting in a higher carbonyl index of the surface
of the PE
microspheres in the same period of time.

![](https://static.igem.wiki/teams/5268/result-page/figure-8.webp)

Significance analysis of carbonyl index on the surface of PE microspheres (a, changes in surface
functional groups of PE
microspheres after 30 days of laccase and hydrolase action; b, changes in surface functional groups of PE
microspheres
after 30 days of alkane monooxygenase action)

## Measure-Three-Weight Loss Rate

The mass of PE films was measured and the weight loss rate was calculated after 30 days of interaction with
crude enzyme
solution or recombinant engineered bacteria at 30°C and 150 rpm, and a one-way analysis of variance (ANOVA)
was
performed on the weight loss results to determine whether the weight loss of PE films was related to the
action of
potentially functional polyethylene-degrading enzymes.

The degradation rates of laccase Lac-S12-1, Lac-PM2-1 were
0.876%, 0.451% significantly higher than that of the control (CK), while laccase (Lac-NJ-1, Lac-S102-1) did
not result
in loss of film mass (Fig. 9a); laccase (Lac-S12-2, Lac-S11-1, Lac-S12-3. Lac-S12-4) hydrolase (Hyd-S12-1,
Hyd-S11-1)
caused 0.929%, 0.648%, 0.857%, 0.630%, 0.423%, and 0.238% mass loss of polyethylene films, respectively (Fig.
9b);
however, the results of the analysis of variance (ANOVA) indicated that the hydrolase (Hyd-S11-1) and laccase
(Lac S12-4) were not significantly different (P < 0.05) compared to the control; the laccase Lac-PM3-1,
Lac-NJ2-1, Lac-SKL-1, Lac-PM3-2, Lac-PM3-3, Lac-PM2-2, Lac-PM2-3, and Lac-S100-1 caused PE films to lose
1.062%, 1.814%, 0.814%, and 0.814% of mass, respectively.

0.814%, 0.833%, 0.822%, 0.729%, 0.792%, 0.822%, 0.760%,
respectively (Fig. 9c); alkane monooxygenases AlkB-NJ1-1, AlkB-NJ1-2, AlkB-S102-2 AlkB-NJ-3 AlkB-SKL-1 and
others caused film weight loss 0.838%, 0.746%, 0.746% and 0.760%, respectively. 0.746%, 0.087%, 0.113%,
and
0.09%, respectively, and the results of ANOVA indicated that the weight loss of polyethylene films caused
by
AlkB-NJ1-1, AlkB-NJ1-2 was significantly higher than that of the control group except for the three alkane
monooxygenases, AlkB-S102-2, AlkB-NJ-3, and AlkB-SKL-1, which caused significantly higher weight loss of
polyethylene films than that of the control group after treating the PE films for 30 days (Fig. 9d). While
laccase (Lac-NJ-1, Lac-S102-1), hydrolase (Hyd-S11-1) and laccase (Lac-S12-4). Alkane monooxygenases
(AlkB-S102-2, AlkB-NJ-3, AlkB-SKL-1) did not cause significant weight loss to occur in PE films.

![](https://static.igem.wiki/teams/5268/result-page/figure-9.webp)

Polyethylene film weight loss (0.01 < P ＜ 0.05 labelled \*, 0.001 < P ＜0.01 labelled \*\*, P ＜
0.001 labelled \*\*\*, P> 0.05
indicates no statistical significance labelled ns)

In order to demonstrate more visually the weight loss of PE films in the presence of enzymes, the rate
constant (k) for
the PE degradation reaction catalysed by degradative enzymes and the half-life, i.e., the time required
for the PE film
to decompose and reduce to half of its initial amount, were determined by using first-order kinetic
formulas according
to Sanjeevani et al. Half-life calculations were not performed for laccase (Lac-NJ-1, Lac-S102-1),
hydrolase (Hyd-S11-1)
and laccase (Lac-S12-4), and alkane monooxygenase (AlkB-S102-2, AlkB-NJ-3, AlkB-SKL-1), as they did not
result in a
significant loss of weight in the PE film. Its half-life is 100-200 years.

After 30 days of recombinant enzyme action,
the degradation cycle of PE film under the action of laccase Lac-PM3-1 and other enzymes can be reduced to
5.3-9.0 years
(Table 2), causing significant weight loss of PE film polyethylene degradation enzyme, which indicates
that it can
significantly shorten the half-life of PE plastic degradation and accelerate the rate of PE degradation.

Half-life of PE degradation in the presence of different recombinant enzymes

![](https://static.igem.wiki/teams/5268/result-page/table-2.webp)

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