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Identifier

000461904

Title

Towards more active and stable BVMOs via protein engineering

Alternative Title

Στοχεύοντας σε πιο ενεργές και σταθερές μονοοξυγενάσες μέσω πρωτεϊνικής μηχανικής

Author

Μιχαλοπούλου, Χριστίνα Δ.

Thesis advisor

Παυλίδης, Ιωάννης

Reviewer

Παυλίδης, Παύλος
Ελευθεριάδης, Νικόλαος

Abstract

Baeyer−Villiger monooxygenases (BVMOs, EC 1.14.13.X) offer a “green” route for lactone synthesis from ketones; however, they are quite unstable under oxidative and thermal stress. Furthermore, one of the challenges of BVMO processes is the generation of H₂O₂ instead of product formation, through a futile cycle of uncoupling reaction. This study focuses on stabilizing the 2-oxo-D3-4,5,5- trimethylcyclopentenylacetyl-CoA monooxygenase from Pseudomonas putida (OTEMO). Rational design was used as a first approach in our group, where the amino acids liable to oxidation were mutated; thus, a cysteine located in the active site of the OTEMO (Cys₄₄₄) was mutated to serine. This effort has improved thermal stability and increased the specific activity of the variant by 1.5-fold compared to the wild-type enzyme, at the price of decreased oxidative stability and higher uncoupling ratio. To overcome this limitation, this thesis focused on introducing mutations in OTEMO_C444S guided by directed evolution approach and specifically error-prone polymerase chain reaction (epPCR). The mutagenesis was performed with OTEMO_C444S as a stable scaffold is prone to evolution, leading to more functional mutants, proportionally. Several mutant libraries were created, and the best one was selected after the quality control. From this one, ~800 clones were screened for variants with enhanced activity, and stability towards H₂O₂. Variants with improved expression level (1.5 to 3.3-fold) and increased oxidative stability were identified. The variant OTEMO_C444S/K155E showed great improvement with respect to H2O2 stability, retaining ~20% more activity at 5-25 mM H₂O₂ compared to OTEMO_C444S. Nevertheless, the thermal stability of this variant is decreased, as it completely loses its activity after 24 h of incubation at 35 ℃. OTEMO_C444S/D375G, exhibited lower uncoupling rate by 3-fold compared to the template, however no favorable results in terms of oxidative stability. This observation suggests a lack of direct correlation between uncoupling rate and oxidative stability. Both positions are found away from the active site of the enzyme. These results confirmed that epPCR was an effective tool for predicting distal mutations for the development of robust industrially enzymes, which would not have been designed by rational design.

Language

English

Subject

Error-prone PCR

Monooxygenases

Stability

Thermal stability

Επιρρεπή-στα-λάθη αλυσιδωτή αντίδραση πολυεράσης

Θερμοσταθερότητα

Πρωτεϊνική μηχανική

Σταθερότητα

Issue date