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 are either not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert it 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 towards 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 6-8.5 with functional stability being lowest around 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 is limiting 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.

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

“…To investigate whether the mFMO variants had increased thermal stability compared to the native enzyme, we conducted protein melting studies using circular dichroism. The melting temperature ( T m ) of native mFMO was measured to be 46.2 °C (Table 3), which is in line with previous results (Goris et al, 2020). All mFMO variants demonstrated increased temperature stability compared to the wildtype enzyme, as reflected by their T m values, which ranged from 50.9 °C for mFMO_28 to 55.2 °C for mFMO_20 (Table 3).…”

“…All 7 mFMO mutant variants were expressed with a C-terminal hexa-histidine tag, at levels comparable to that of native mFMO, purified (Figure S2), and verified by mass spectrometry. When expressing mFMO in Escherichia coli , the culture medium turns blue due to the enzymatic conversion of endogenous indole to indigo (Choi et al, 2003; Fabara and Fraaije, 2020; Goris et al, 2020). The fact that the culture media of all mFMO variants turned blue following overnight expression suggested that the expressed mFMO variants were functional.…”

“…mFMO_20 reduces the TMA level by 95% in salmon protein hydrolysate at 65 °C Since mFMO_20 demonstrated the most prominent increase in thermal stability, we compared its ability to convert TMA to TMAO in a salmon protein hydrolysate to that of native mFMO. The cofactor NADPH was supplemented, as the hydrolysate did not contain sufficient amounts to drive the enzymatic reaction (Goris et al, 2020). Heatcalibrated enzymes and 0.5 mM NADPH were added to salmon protein hydrolysates (pH 6.1) and incubated for 1 hour at 30 °C, 50 °C and 65 °C, followed by measurements of TMA and TMAO concentrations (Figure 2).…”

Characterization of the Key Aroma Constituents in Fried Tilapia through the Sensorics Concept

“…As a precursor of trimethylamine (TMA), trimethylamine oxide (TMAO) widely exists in aquatic products [ 52 ]. In the process of frying tilapia, the oxide is decomposed and produces the main odor substance trimethylamine under the action of enzymes [ 53 ], forming the characteristic flavor and slightly fishy odor of fried tilapia.…”

Enzyme adaptation to habitat thermal legacy shapes the thermal plasticity of marine microbiomes

“…For determination, [total protein]: 5 μg ml −1 ; [substrates]: 25 mM; [pyridoxal phosphate]: 1 mM; reaction volume: 200 μL; T: 4-85 °C; and pH: 8.0 (50 mM Tris-HCl buffer). Aldo-keto reductase activity was determined using acetophenone as a substrate and NADPH as a cofactor, by following the consumption of NADPH at 340 nm (extinction coefficient, 6220 M −1 cm −1 ), as described 76 . For determination, [total protein]: 5 μg ml −1 ; [substrate]: 1 mM; [cofactor]: 1 mM; reaction volume: 200 μL; T: 4–85 °C; and pH: 8.0 (50 mM Tris-HCl buffer).…”