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

Abstract

AbstractCatechol 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 T50, a 725% increase in half‐life, a 5.5°C increase in Tm, 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.