Chemistry Student Seminar: Shaghayegh (Sherry) Dezvarei (PhD)
- Date: Fri, 8 Mar 2019, 12:00 pm - 1:00 pm
- Location: MacBeth Lecture Theatre - Badger Building
- Cost: N/A
- Contact: Dr Christopher Newton 8313 5308
- Email: firstname.lastname@example.org
Improving the oxidation activity of P450Bm3
Cytochrome P450 enzymes provide an enzymatic alternative route to the selective oxidation of unreactive carbon-hydrogen (C-H) bonds and epoxidation of alkenes.
P450Bm3 is one of the most applicable members of the P450 superfamily because of its solubility, self-sufficient nature and high activity towards fatty acids (C12-15).
However, the wild type (WT) enzyme has limited substrate range which restricts its application as a biocatalyst. Therefore, to expand the substrate range, a combination of decoy molecules and enzyme engineering was employed.
Decoy molecules are dummy substrates that have a similar structure as native substrates and place enzyme in the active state while leaving enough space for a non-native substrate. For P450Bm3 the decoy molecules are based on perfluorinated fatty acids.
The regio- and stereoselectivity of the RLYFIP, KT2, R19, RLYFAP and WT variants of Bm3 were explored for the oxidation of ethylbenzene, styrene, propylbenzene, trans-β-methylstyrene, 3-ethyltoluene, 2-methylstyrene, 1-bromo-2-ethylbenzene and isophorone in presence and absence of the decoy molecules. To explore how the size of substrate change the oxidation activity of Bm3 variant, cycloalkanes (C5-C10) were also studied with decoy molecules.
A single mutation of Thr268 to Glu converted Bm3 into H2O2-dependent variant (Bm3TE). The Bm3TE variant successfully oxidised styrene, ethylbenzene and methylthiobenzene in presence of H2O2. Addition of decoy molecules showed no improvement in the activity of Bm3TE but enhanced the holoenzyme monooxygenases productivity.
To increase the stability of Bm3TE and Bm3R19, their encapsulation in the metal-organic frameworks (ZIF-8 and ZIF-90 crystals) and biological compartments (encapsulins) was investigated.