Cloning Of HIS1 Gene And Its Function In Degrading Polypropylene Plastic

In recent decades,polypropylene(PP)plastics have been widely used in industrial,commercial and agricultural fields due to their unique physical and chemical properties.However,due to improper disposal of waste plastics,large amounts of plastic waste have accumulated in landfills or natural environments for long periods of time,posing a serious ecological challenge.Although it may take hundreds of years for plastics to be completely degraded,under the action of physical and biological factors,plastics will break down into micro/nanoscale plastic particles at a relatively fast rate,which in turn poses a serious threat to human health.Therefore,in this study,an Fe(II)/2-oxoglutarate(20G)-dependent oxygenase(HIS1)was screened by sequence-based macrogenomic screening and expressed by prokaryotic expression technique.The degradation of PP by HIS1 was successfully demonstrated by in vitro validation,and the interaction mechanism between HIS1 and PP molecules was investigated.This provides a promising pathway to achieve enzymatic degradation of PP plastics for recycling,and the main findings are as follows:(1)Screening and clonal expression of plastic degrading enzymes:The candidate plastic degrading enzyme HIS1,a member of the Fe(II)/20G-dependent oxygenase family with an open reading frame(ORF)length of 1056 bp,encoding a protein with a white molecular weight of 40.1 kDa,was screened from the macrogenomic database of plant dioxygenase class,and the theoretical isoelectric point(pI)and The total mean values of theoretical isoelectric point(pI)and hydrophilicity(GRAVY)of the protein were 5.04 and 0.806,respectively,which belonged to hydrophobic protein.The results of nucleic acid gel electrophoresis and SDS-PAGE showed that HIS1 was successfully expressed and purified at 10 mg/mL.(2)Characterization of the degradation effect of HIS 1 on PP plastics:scanning electron microscopy(SEM)observed many severe cracks,deep holes and fragmentation on the surface of HI S1-treated PP films and microplastics;atomic force microscopy(AFM)results showed that the roughness and thickness level of HIS1treated PP films became higher;the Two additional FTIR spectral absorption peaks were observed in the HIS 1-treated PP films,which were carbon-oxygen double bond(C=O-)and hydroxyl group(-OH);gel permeation chromatography(GPC)analysis showed that the molecular weight distribution(MWD)of the treated PP films showed a declustering trend;gas chromatography coupled with mass spectrometry(GC-MS)results indicated that the main degradation products were oxygenated compounds and short-chain olefins and alkyl substances.The experimental results fully demonstrated the degradation ability of HIS1 on PP plastics.the optimal conditions for HIS1 catalysis were Fe2+concentration at 0.2 mM-2 mM;optimal pH=7;optimal temperature at 30℃;the degradation rate(weight loss rate)was as high as 13.55%in the recovery of three PP plastics,while the degradation rates of the remaining materials were all around 5%,which might be due to the difference of plastic crystallinity.The degradation rate was different.(3)Interaction mechanism between HIS1 and PP molecules:During the kinetic simulation,HIS1 binds well to PP with the mean root mean square deviation(RMSD)of 0.235±0.023 nm and the mean radius of gyration(Rg)of 2.013±0.006 nm.The results indicate that the entire HIS1-PP complex is very stable throughout the simulation,and the average number of hydrogen bonds in the system is 5.37,which is favorable for the interaction between protein and PP.The root mean square fluctuation(RMSF)of amino acid residues showed that the flexibility of the entire protein amino acid residues was small except for the N-terminal and C-terminal,indicating that the binding of PP did not cause significant changes in the structure of some amino acids in the protein;the hydrophobic cavity of HIS1 tightly bound PP through hydrophobic interaction,and the binding free energy of HIS1-PP was-88.583 kJ/mol.The free energy of HIS1-PP is-88.583 kJ/mol,and the reaction process produces a deformation of PP that tends to be close to Fe2+.In summary,the discovery of HIS 1 provides an effective candidate enzyme for the biodegradation of PP plastics and an important treatment strategy for the green treatment and recycling of PP plastic waste.