Design of a thermostable bilirubin oxidase from Myrotheciumverrucaria.

Abstract

Bilirubin oxidase (BOD), identified as a multicopper oxidase produced by Myrothecium verrucaria, plays a critical role in the oxidation of bilirubin to biliverdin, which is pivotal in various biochemical processes. To construct a highly thermostable BOD, we have used three protein engineering methods: (i) stabilization of the main chain (proline substitution), (ii) design of salt bridges, and (iii) improvement of hydrophobic interactions. Significant enhancement of thermostability was achieved through stabilization of the main chain (L476P, A496P), introduction of a salt bridge (Q495R), and improvement of hydrophobic interactions (A264V). Furthermore, the combination of these point mutations, which contributed to structural stabilization, resulted in a novel thermostable mutant. Utilizing the cumulative effect of point mutations based on the three strategies, we were able to obtain a thermostable enzyme that exhibited approximately 3.9-fold higher residual activity than wild-type BOD (WT) even after incubation at 60 °C for 1 h and nearly 10 °C higher optimum temperature than that of WT. Importantly, these mutations did not affect its 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation activity. This approach provides a valuable strategy for improving the thermostability of multivalent copper oxidases and offers promising prospects for industrial applications.