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.
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