Use of Flavin-Containing Monooxygenases for Conversion of Trimethylamine in Salmon Protein Hydrolysates

Abstract

Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. Despite this, their bitter taste and "fish" odor block implementation in food products and limit their economic potential. Trimethylamine (TMA) is a known contributor to malodor in fish. Current strategies to mask or remove the odor either are not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert TMA into the odorless trimethylamine N-oxide (TMAO) using TMA monooxygenases (Tmms). We identified a diverse set of bacterial Tmms using a sequence similarity network. Purified, recombinant enzymes were assessed for their biocatalytic capacity by monitoring NADPH consumption and TMAO generation. Selected Tmms were subjected to biochemical characterization and investigated for their ability to oxidize TMA in an industry-relevant substrate. From the 45 bacterial Tmm candidates investigated, eight enzymes from four different taxa were selected for their high activity toward TMA. The three most active enzymes were shown to vary in temperature optimum, with the highest being 45°C. Enzymatic activity dropped at high temperatures, likely due to structural unfolding. The enzymes were all active from pH 6.0 to 8.5, with functional stability being lowest around the optimal pH. All three Tmms, given sufficient NADPH cofactor, were found to generate TMAO in the TMA-rich salmon protein hydrolysate. The Tmms serve as unique starting points for engineering and should be useful for guiding process development for marine biorefineries.IMPORTANCE Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of "fish" caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries.

Keywords: enzyme discovery; fish protein hydrolysate; flavin-containing monooxygenases; malodor; oxidoreductases; trimethylamine; trimethylamine N-oxide; trimethylamine monooxygenases.