The previously laboratory-evolved cytochrome P450 2B1
quadruple mutant V183L/F202L/L209A/S334P (QM),
which showed enhanced H2O2-mediated substrate oxidation,
has now been shown to exhibit a >3.0-fold decrease
in Km,HOOH for 7-ethoxy-4-trifluoromethylcoumarin
(7-EFC) O-deethylation compared with the parental
enzyme L209A. Subsequently, a streamlined random mutagenesis
and a high-throughput screening method were
developed using QM to screen and select mutants with
enhanced tolerance of catalytic activity to temperature and
dimethyl sulfoxide (DMSO). Upon screening >3000 colonies,
we identified QM/L295H and QM/K236I/D257N with
enhanced catalytic tolerance to temperature and DMSO.
QM/L295H exhibited higher activity than QM at a broad
range of temperatures (35C55C) and maintained 1.4-fold
higher activity than QM at 45C for 6 h. In addition,
QM/L295H showed a significant increase in Tm,app compared
with L209A. QM/L295H and QM/K236I/D257N exhibited
higher activity thanQMat a broad range ofDMSO concentrations
(2.5C15%). Furthermore, QM/K236I/D257N/
L295H was constructed by combining QM/K236I/D257N
with L295H using site-directed mutagenesis and exhibited
a >2-fold higher activity than QM at nearly the entire
range of DMSO concentrations. In conclusion, in addition
to engineering mammalian cytochromes P450 for enhanced
activity, directed evolution can also be used to optimize
catalytic tolerance to temperature and organic solvent.