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

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.

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)

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).

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.

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.

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).

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).

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.

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.

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