4.
Conclusion
In summary, we report here the development of a sensitive, simple, and
efficient fluorogenic assay that is suitable for high-throughput
screening of ADHs. We demonstrated
the potential of SDFA by performing directed evolution on PfODH through
screening a combinatorial library. The selected enzyme variant exhibited
a substantially higher kcat/km value
toward (S )-2-octanol compared to its wild-type. In addition, the
secretion-based nature of SDFA exempts the need for cell lysis, thus
bypassing its disadvantages including the release of interfering
cytosolic components, the additional cost of lysis reagent, the
potential inconsistency of lysis efficiency, the extra effort of
preserving intact culture for genome recovery. From this perspective,
SDFA not only provides a sensitive and reliable way for determining ADHs
activity, but also simplifies the screening
procedure.
To the best of our knowledge, SDFA is the first secretion-based assay
that can easily enable normalizing the catalytic activity of ADHs for
high throughput screening. SDFA is
in theory also applicable to other NAD(P)(H)-dependent enzymes besides
ADHs and is suitable for high-throughput screening methods such as
96/384 well plate format. Although the method we have developed so far
only works with the oxidative direction of the ADH-catalyzed reaction,
SDFA should still be attractive to many protein engineers, because there
are a large number of useful oxidative reactions catalyzed ADHs.