New combined absorption/1H NMR method for qualitative and quantitative analysis of PET degradation products.

Poly(ethylene terephthalate) (PET) is a very valuable and beneficial material for industrial purposes, with various different applications. Due to the high annual production volume of over 50 million tons worldwide and the indiscriminate disposal by consumers, the polymers accumulate in the environment, causing negative effects on various ecosystems. Biodegradation via suitable enzymes represents a promising approach to combat the plastic waste issue so validated methods are required to measure the efficiency and efficacy of these enzymes. PETase and MHETase from Ideonella sakaiensis are suitable enzymes needed in combination to completely degrade PET into its environmentally friendly monomers. In this project, we compare and combine a previously described bulk absorbance measurement method with a newly established 1H NMR analysis method of the PET degradation products mono(2-hydroxyethyl) terephthalic acid, bis(2-hydroxyethyl) terephthalic acid and terephthalic acid. Both were optimized regarding different solvents, pH values and drying processes. The accuracy of the measurements can be confirmed with sensitivity limits of 2.5-5 µM for the absorption method and 5-10 µM for the 1H NMR analysis. The combination of the described methods therefore allows a quantitative analysis by using bulk absorption coupled with a qualitative analysis through 1H NMR. The methods established in our work can potentially contribute to the development of suitable recycling strategies of PET using recombinant enzymes.

Sequence diversity and functional characterization of the 5'-regulatory region of human CYP2C19.

Cytochrome p4502C19 (CYP2C19) plays an important role in drug biotransformation and has been shown to be genetically polymorphic. While polymorphisms in the coding region that have large effects on activity are well described, until recently, a lack of knowledge of the promoter sequence has hindered efforts to study it. Genetic variants in the promoter region have not been described and factors that influence its gene expression via promotor regulation remain largely undefined. We have cloned and sequenced 1.8 kb of the human CYP2C19 promoter. This promoter contains a number of putative transcription factor sites, including HepG2-specific factor 1, glucocorticoid response element, estrogen receptor element, constitutive androstane receptor and peroxisome proliferator-activated receptor. Sequencing of DNA obtained from 67 individuals identified eight single nucleotide polymorphisms within this region. No sequence of a known human pregnane X receptor response element was found in this section of the CYP2C19 promoter, despite the known effect of rifampin on the expression of this gene. A plasmid containing the 1.8-kb CYP2C19 promoter coupled to a luciferase reporter gene has been constructed and demonstrated to be functional and sensitive to induction by omeprazole in HuH7 cells. Nested deletions of CYP2C19 promoter were generated and the ability of serial promoter deletion constructs to activate luciferase expression in the HepG2 cell line was analysed. These data make possible future studies to elucidate the molecular mechanisms by which CYP2C19 can be induced in clinical settings and the consequences of genetic variability in its promoter.