Biotechnologically production of polyethylene terephthalate (PET) type plastic degrading enzyme petase in escherichia coli

Polyethylene terephthalate (PET) is one of the most used plastic types in the world, thus causing the most environmental pollution. Compared to physical and chemical methods used for recycling PET, biological recycling stands out as a low-cost and effective solution that does not increase the degradability of the plastic material and does not have toxic effects. Ideonella sakaiensis bacterium; degrades PET into its monomers with Polyethylene terephthalate hydrolase (PETase) and Monoethylene terephthalate hydrolase (MHETase) enzymes it secretes and uses these monomers as carbon source. The issue of recycling PET type plastic, which can be recycle by physical and chemical methods at the present time, through biotechnological methods has been studied intensively for the last decade, and PETase enzyme constitutes a strong alternative in this field. In this thesis, it is aimed to bioinformatically design PETase enzyme, which is produced by I. sakaiensis bacterium and hydrolyzes PET to MHET in an efficient and effective manner, and biotechnologically produce it in E. coli bacterium. For this goal; by using bioinformatics tools, (1) codon optimization of PETase gene, (2) addition of restriction enzyme cleavage sites to the gene for cloning into pET26b expression vector, (3) keeping the pelB signal sequence of plasmid that directs the recombinant protein to be produced to the periplasmic area within the boundaries of the restriction enzymes, (4) addition of the Histidine tag to the gene that needed for purification after the enzyme production, and cloning of the bioinformatically designed synthetic gene into expression vector by recombinant DNA technology; then, produce PETase enzyme with high activity in the cytoplasm of the cell with high activity, it was aimed to be transferred to E.coli SHuffle bacteria, which is capable of making disulfide bonds in the cytoplasm, thanks to DsbC protein in it produces. In this thesis, the original PETase gene of I. sakaiensis was bioinformatically redesigned in order to be able to produce actively and efficiently. In this context, with codon optimization of the designed synthetic PETase gene, the GC content was reduced from 68.73% to 56.78%, while the Codon Adaptation Index (CAI) was increased from 0.64 to 0.80. The NdeI at the 5' and HindIII restriction enzymes recognition sites at the 3' end and the nucleotide sequence of 6X Histidine tag were added to the 3' end of the gene so that the enzyme produced after gene expression can be purified from bacteria. The designed PETase gene was synthetically obtained in pTZ57R/T plasmid successfully transferred to E.coli by transformation with a transformation efficiency of 4.5x102 CFU / μg. The PETase gene was extracted from pTZ57R/T-PETase plasmid DNA that was amplified and isolated in E.coli DH5α with HindIII and NdeI restriction enzymes. The pET26b expression plasmid, which was previously amplified with transferred into E. coli DH5α by transformation (transformation efficiency: 3,08x102 CFU/g) and then isolated from bacteria, was also cut with the same restriction enzymes. PETase gene and pET26b plasmid backbone with expected size isolated from agarose gel and put into the ligation reaction with T4 DNA Ligase enzyme. Thus, the pET26b-PETase recombinant plasmid was obtained. The obtained pET26b-PETase recombinant plasmid was successfully transformed to E. coli DH5α bacteria for amplification, with a transformation efficiency: 1.62x102 CFU/μg. Then, the obtained recombinant plasmid was transferred by transformation to E. coli SHuffle bacterium, which is capable of making disulfide bonds in the cytoplasm, in order to produce PETase enzyme in cytoplasm which carries 2 disulfide bonds, transformation efficiency: 1.24x103 CFU/μg. By the colony PCR technique, 4 colonies determined to contain the recombinant plasmid were selected and gene expression was induced with 100 mM IPTG. After the induction, PETase enzyme was purified from 3 out of 4 clone cultures by magnetic purification system that enables isolation of polyhistidine tagged proteins. Enzyme purity was analyzed spectrophotometrically at 280 nm wavelength by absorbance measurement and Bradford Assay. Pure protein concentrations of these 3 samples, isolated from recombinant cells with high yield, were determined as 0.935, 0.56 and 0.59 mg/mL. The homogenate of fourth culture was used as a protein source. The total protein concentration in this sample was determined as 3.9544 mg/mL. It was determined that the lowest and highest pure protein concentrations obtained after protein isolation were 1.44 and 2.44 times higher, respectively, compared to the lowest and highest pure protein concentrations obtained from Chlamydomonas reinhardtii. Again, in this study, the total amount of protein obtained from bioinformatically redesigned and produced PETase gene were 29 and 60 times higher than the lowest and highest total protein amounts that are obtained from the medium of E. coli in an previous study, which MalE and LamB signal sequences were used for the production of PETase enzyme in the periplasm of E.coli. In this thesis, the ability to reproduce the PETase gene efficiently by cloning in the pET26b expression plasmid, and produce and purification of high concentration enzyme in E.coli SHuffle bacterium, which can make disulfide bonds in th cytoplasm reveals that; th codon optimisation based bioinformatics design of the PETase enzyme and the applied genetic engineering approaches have been successfully implemented. The findings obtained can be used for large scale production of PETase enzyme, which has an important potential in biotechnological recycling of PET type plastic.