| dc.description.abstract | Byproducts from the seafood industry are rich in nutrients, such as peptides, which might be utilized to a greater extent in the food market. Peptides can be extracted from fish byproducts by enzymatic hydrolysis, but the resulting product contain the smelly compound, trimethylamine (TMA). The flavin-dependent monooxygenase from Methylophaga aminisulfidivorans (mFMO), can oxygenate TMA, converting it to the odorless trimethylamine N’ oxide (TMAO). Adding it to the enzymatic hydrolyzation process might remove TMA from the protein hydrolysate.
mFMO is a versatile enzyme and can oxygenate a range of substrates with soft nucleophilic properties. mFMO uses the primary cofactor; falvin adeninine dinucleotide (FAD), and a secondary cofactor; nicotinamide adenine dinucleotide phosphate (NADPH). FAD is oxygen dependent, and other flavin-dependent monooxygenases (FMOs) have been shown to undergo futile oxidation of its secondary cofactor, to produce hydrogen peroxide (H2O2) through a mechanism known as uncoupling, rather than oxygenating the target substrate.
NADPH is a costly cofactor, and engineering mFMO to be active with the cheaper NADH, could increase industrial cost efficiency. However, mutant screening can be time consuming and would benefit from high throughput screening methods.
In this thesis, mFMO and a thermostable mutant, mFMO_20 were tested against a range of substrates. Two novel substrates were discovered: Triethylamine (TEA) and dimethylamine (DEA). And revealed a substrate preference for tri-substituted amines. Uncoupling was measured using the Xylenol orange assay, and both enzymes showed some uncoupling in absence of substrate, which dissipated when substrate was available. Finally, an NADH auxotrophic bacterial strain was used as a screening system in an attempt to identify NADH oxidizing activity in the mFMO and mFMO_20. However, neither of the enzymes successfully rescued the auxotrophic strain. Which might indicate low NADH oxidative capacity of the enzymes, or the substrate, TMA, might be too volatile, and unsuited for the screening system. | |
