Rational design of disulfide bonds to increase thermostability of Rhodococcus opacus catechol 1,2 dioxygenase.

Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T ₅₀ , a 725% increase in half-life, a 5.5°C increase in T _m , and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase.
(© 2024 National Research Council Canada and The Authors. Biotechnology and Bioengineering by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)