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Waste is a big global issue that has a substantial influence on the environment and human health. One type of waste that is notoriously difficult to decompose and requires a considerable amount of time to degrade is polyethylene terephthalate (PET) plastic. Due to its remarkably persistent properties, the accumulation of PET waste poses a major threat to the ecosystem. Several attempts have been made to address the problem of PET plastic waste, one of which is to use an enzymatic approach to accelerate the decomposition of PET. PET hydrolase (PETase), found in Ideonella sakaiensis, is a potential agent for the biodegradation of PET. In this study, we used a synthetic biological approach (synthetic DNA) to develop a PETase biocatalyst, in which the PETase gene was chemically synthesized, cloned into a pET-22b vector, and then expressed in E. coli BL21 (DE3) with IPTG induction. During the construction process, the mutated N-terminal pelB signal sequence was included with the expectation that the end-product PETase could be either secreted into periplasmic or excreted to the culture medium. Several different extraction methods were utilized, including intracellular (cytoplasmic), periplasmic, and extracellular extraction. In addition, Western Blot Analysis was carried out to confirm that the target protein had been expressed. The pET22b(+)-petase was verified using restriction enzymes, and the result showed that the gene was properly cloned. The SDS-PAGE and western blot result showed that the pET22b-IsPETase has been successfully expressed and secreted, but the amount is still low so that further optimization needs to be done. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate (PET) is widely used as raw material for clear, strong, and lightweight plastics that have been exploited in the packaging market. Although the use of plastics makes human's life easier, their waste has become a major environmental problem. Recycling plastics-based waste is still expensive and highly energy consuming. Recently two steps synergic degradation of PET has been discovered in Ideonella sakaiensis in which PET is converted into mono-(2-hydroxyethyl) terephthalic acid (MHET) catalyzed by PETase, and MHET is further hydrolyzed into terephthalic acid and ethylene glycol catalyzed by MHETase. This discovery offers a promising and eco-friendlier solution to the global environmental crisis of plastic pollution. In this work, we have cloned the MHETase gene synthetically designed based on IsMHETase. The synthetic gene was codon optimized for protein expression in Escherichia coli. Aiming to obtain extracellular MHETase, the recombinant enzyme was produced in E. coli BL21 (DE3) and its secretion was mediated by a mutated pelB signal sequence. Expression of MHETase was induced by IPTG at 37°C for 5 h. Intracellular, periplasmic, and extracellular fractions were extracted using different methods. Intracellular and periplasmic fraction was obtained from a lysate of sonicated and osmotic shocked cells, respectively, while extracellular fraction was yielded from concentrated cell free culture. Those three fractions were further analyzed by SDS-PAGE and Western Blot using anti-6X HisTag antibodies. We found that the recombinant MHETase was majority expressed as inclusion bodies and distinctively observed after purification of solubilized protein. Western Blot analysis of solubilized fraction showed a stronger signal at ~62 kDa than soluble intracellular protein fraction. Active MHETase were observed in refolded protein fraction and generated 0.4015 U.mL-1 of esterase activity. [ABSTRACT FROM AUTHOR]