We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

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Marine algae are not only a rich source of dietary fibre, proteins, vitamins, and minerals, but also contain a great variety of secondary metabolites with diverse biological activities. Marine macroalgae are a rich source of various natural antioxidants such as polyphenols, especially phlorotannins (made of polyphloroglucinol units) derived from brown algae, which play an important role in preventing lipid peroxidation. In recent years, a number of potent antioxidant compounds have been isolated and identified from different types of edible seaweeds. Extraction methods commonly used for the isolation of antioxidants are based on conventional water or organic solvent extractions. However, recent advances have shown that enzymatic hydrolysis can achieve higher yield of bioactive compounds from algae. Here we describe a method based on enzymatic hydrolysis which both increases yield and decreases cost associated with organic solvents. This method achieves cell wall disruption and breakdown of internal storage components for more effective release of intracellular bioactive compounds. In addition, hydrolysis of proteins produces peptides which may have antioxidant properties, thus enhancing the bioactivity of the algal extract. The method described can be used for production of extracts from red and brown macroalgal species.