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.

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.