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.
Highlights
Enzymatic processing of fish by-products for recovery of peptides is a promising technology to reach food grade ingredients of high nutritional quality
The known TMA monooxygenases (Tmms) belong to the flavin monooxygenase (FMO) family (InterPro accession number IPR000960), we expanded the search to the encompassing flavin-containing monooxygenases (FMOs)-like family (InterPro accession number IPR020946) to ensure that all relevant sequences were included
The sequence similarity network (SSN) analysis was performed on all 19,736 FMO-like family UniRef90 sequences, resulting in a representative node network of 6,740 nodes, where each node represents a collection of proteins with 40% or higher identity
Summary
Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. 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. There has been great progress in the development of enzyme-based processes by which fish by-products are converted into marine ingredients, such as peptides and oils Today, these ingredients are mainly used to manufacture animal and pet feed, but since the by-products contain high-quality proteins, they have a great potential for human consumption. Enzymatically derived fish protein hydrolysates may suffer from a distinct malodor [3], which is described as an unpleasant smell associated with rotting fish This currently limits their potential in the human consumption market. Whether the NADPϩ remains bound to the active site during catalysis has been debated [13,14,15]
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