Increased thermostability of an engineered flavin-containing monooxygenase to remediate trimethylamine in fish protein hydrolysates

Abstract

Protein hydrolysates made from marine by-products are very nutritious, but frequently contain trimethylamine (TMA) which has an unattractive fish-like smell. Bacterial trimethylamine monooxygenases can oxidize TMA into the odorless trimethylamine N-oxide (TMAO) and have been shown to reduce TMA-levels in a salmon protein hydrolysate. To make the Methylophaga aminisulfidivorans trimethylamine monooxygenase, mFMO, more suitable for industrial application, we engineered it using the Protein Repair One-Stop Shop (PROSS) algorithm. All seven mutant variants, containing 8-28 mutations, displayed increases in melting temperature between 4.7 °C and 9.0 °C. The crystal structure of the most thermostable variant, mFMO_20, revealed the presence of four new stabilizing interhelical salt bridges, each involving a mutated residue. Finally, mFMO_20 significantly outperformed native mFMO in its ability to reduce TMA levels in a salmon protein hydrolysate at industrially relevant temperatures.