posted on 2024-03-13, 16:06authored byYang Zhao, Kun Chen, Haixia Yang, Yongtao Wang, Xiaojun Liao
In this study, the phenomenon of
the stability-activity trade-off,
which is increasingly recognized in enzyme engineering, was explored.
Typically, enhanced stability in enzymes correlates with diminished
activity. Utilizing Rosa roxburghii copper–zinc superoxide dismutase (RrCuZnSOD) as a model, single-site mutations were introduced based on a semirational
design derived from consensus sequences. The initial set of mutants
was selected based on activity, followed by combinatorial mutation.
This approach yielded two double-site mutants, D25/A115T (18,688 ±
206 U/mg) and A115T/S135P (18,095 ± 1556 U/mg), exhibiting superior
enzymatic properties due to additive and synergistic effects. These
mutants demonstrated increased half-lives (T1/2) at 80 °C by 1.2- and 1.6-fold, respectively, and
their melting temperatures (Tm) rose by
3.4 and 2.5 °C, respectively, without any loss in activity relative
to the wild type. Via an integration of structural analysis and molecular
dynamics simulations, we elucidated the underlying mechanism facilitating
the concurrent enhancement of both thermostability and enzymatic activity.