Enhancing the organic solvent resistance of transaminase from
Aspergillus terreus by regional random mutation
Abstract
Biocatalysis in high-concentration organic solvents has been applied to
produce various industrial products with many advantages. However, using
enzymes in organic solvents often suffers from inactivation or decreased
catalytic activity and stability. So, improving the tolerance of enzymes
in organic solvents is essential. Herein, the method of regional random
mutation combined with combinatorial mutation was used to improve the
resistance of transaminase from Aspergillus terreus (AtATA) in organic
solvents, and the best mutant T23I/T200K/P260S (M3) was acquired. In
different concentrations of dimethyl sulfoxide (DMSO), the catalytic
efficiency toward 1-acetylnaphthalene and the stability were higher than
the wild-type (WT) of AtATA. M3 also showed enhanced stability against
six organic solvents with different oil-water partition coefficients
(log P values). The results of decreased Root Mean Square Fluctuation
(RMSF) values via 20-ns molecular dynamics simulations under different
concentration DMSO revealed that mutant M3 had lower flexibility,
acquiring a more stable protein structure and contributing to its
organic solvents stability than WT. Intra- and intermolecular
interaction analysis indicated that the increased hydrogen bonds and
hydrophobic interactions within monomers or at the interface of two
monomers also strengthened the stability of the overall structure
against organic solvents. Furthermore, M3 was applied to convert
1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine
((R)-NEA), which was and an intermediate of Cinacalcet Hydrochloride.
Moreover, 3~10 mM 1-acetylnaphthalene can be converted
to (R)-NEA with 94.2~38.9% yield and a strict
R-stereoselectivity within 10 h under 25% DMSO, which was higher than
WT and expected to be a potential biocatalyst for industrial
application.