Protein engineering for polyethylene terephthalate degradation

Plastics play an important part in nowadays life since it became the material of choice used in countless application (e.g. packaging, automobiles or furniture). Among all plastics, polyethylene terephthalate (PET) is globally one of the most abundant and in 2014 approximately 40 million tons of PET were used for the production of plastic bottles, synthetic fibers, and foils. In order to meet requirements of modern life style, most of the commonly sport clothing are made of highly hydrophobic PET polymer making the clothes waterproofed and antimicrobial if linked to silver. Washing of synthetic clothes causes microplastic PET accumulation into freshwater systems through household sewage discharge. Additionally, low PET biodegradability causes its accumulation in terrestrial and aqueous environment in form of either by products during PET production or accumulated PET after final usage. Chemical or mechanical PET degradation is usually performed under harsh conditions (high acidic, high basic, high pressure) in a time consuming manner resulting in incomplete PET degradation. Alternatively, enzyme based PET degradation showed to have an application potential to completely degrade PET into monomers and offers environmentally friendly method for removal of accumulated plastic particles. Up to date no successful enzyme based PET degradation was achieved since principle and underlying molecular basis of hydrolysis of PET is still not elucidated. In previous studies it was shown that potential enzymes with an ability to catalyse PET degradation are hydrolases (i.e. esterases and cutinases) with still weak activities towards PET. In order to be applied in biodegradation processes (e.g. waste water treatment, laundry detergents) the enzyme candidates need to be further optimized regarding its specific activity towards PET, and resistance towards high temperatures. The enzyme optimization was since 1970s done using directed evolution and rational design. A main bottleneck of every directed evolution experiment is the design of a screening system which mimics application conditions as close as possible. In order to evolve hydrolases having high specific activity towards PET the complex aromatic model substrate, phenol benzoate was used to develop and optimize microtiter plate (MTP) based screening system. The screening system is based on indirect monitoring of esterolytic activity through detection of phenols with 1,5-dimethyl-1-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2 phenyl-1,2-dihydropyrazol-3-one. The product formation is followed continuously as an increase of absorbance at 509 nm. Validation of novel screening system was done by screening error-prone PCR (epPCR) esterase mutant library for improved esterase activity at elevated temperature. The identified esterase variant T3 (Ser378Pro) showed a 4.7 fold improved residual activity after thermal treatment. MTP screening formats offer screening throughput of 10^4-10^5 variants which is insufficient to cover sequence space in generated gene diversity library (10^8-10^9). In order to increase screening throughput and increase coverage of sequence space a novel ultra-high throughput screening system was optimized as a part of a universal screening toolbox for hydrolases. The screening is based on coupled enzyme reaction using glucose derivatives as substrates which upon hydrolysis forms a fluorescent hydrogel layer on the surface of the E. coli cells. Applying a developed screening platform on epPCR esterase library resulted in a variant E1 (Glu256Gly, Gly401Val) with a 7.1-fold higher kcat and 2-fold reduced KM. Additionally, in order to generate first hypothesis on PET degradation mechanism, an in depth analysis of esterase lid like loop (18 amino acids) in close proximity of the active site was done. Shortening and complete deletion of the esterase loop was performed and its influence on PET degradation activity was followed. The activity decrease correlates with the decrease in loop length and revealed in an inactive esterase variant in case the complete lid like loop was deleted. This confirmed that the lid like loop has a key importance for the esterase activity.