Chloroperoxidase (CPO), secreted by marine fungus Caldariomyces fumago, is the most versatile catalyst among known heme enzymes. Chloroperoxidase can catalyze epoxidation reactions with high enantioselectivity and high yield, which makes CPO an attractive candidate for both industrial and medicinal chiral synthesis. Toward this end, we have constructed two CPO mutants, F103A and N74V. Chiral HPLC was used to evaluate the enantioselectivity and yield of CPO and the mutants toward the epoxidation of styrene and its derivatives. Both of the mutants show dramatically changed epoxidation profiles compared to the parent protein. This information provided fresh insight into the mechanism through which CPO achieves its enantioselectivity. Furthermore, effort was made to understand the biological function of CPO through characterization of CPO catalyzed oxidation of dimethylsulfoniopropionate (DMSP), a secondary metabolite of many marine algal species that plays a pivotal role in marine ecology and global climate.

In the last two decades, particular interest has been given to the cycle of dimethylsulfide (DMS), a climate active gas, and its precursors the dimethylsulfoniopropionate (DMSP) and the dimethylsulfoxide (DMSO). DMS is involved in the Earth’s radiation budget while the DMS(P,O) are produced by a wide variety of micro- and macroalgae, corals, bacteria, or angiosperms in response to diverse environmental stresses. Several functions have been suggested for these sulfur compounds such as antioxidants, cryoprotectants, overflow mechanisms, osmolytes, zooplankton deterrents or signalling compounds. This research aims at improving the knowledge about the antioxidant role of DMS(P,O) within three major phytoplankton groups: diatom (i.e. Skeletonema costatum), Prymnesiophyceae (i.e. Phaeocystis globosa) and dinoflagellate (i.e. Heterocapsa triquetra). The experimental results demonstrate that cellular DMS(P,O) have the ability to lower cellular reactive oxygen species concentrations produced during high-light and chemically-induced oxidative stresses; thus supporting the antioxidant function. However, the initial DMS(P,O) concentrations of each species are not informative of their ability to tolerate a further oxidative stress, and their concentrations do not increase in high-light grown cells. The DMS(P,O) may then act as antioxidants without being part of the antioxidant response of the cell. We recommend analysing more constituents of the antioxidant system (i.e. enzymes, carotenoids, redox buffer) along with DMS(P,O) by-products and DMSP-lyase activity to better understand the cellular function of DMSP. Field measurements in the North Sea, including the Belgian Coastal Zone (BCZ) and the Northern North Sea (NNS), bring additional information on the DMS(P,O) variations. While abiotic parameters (nutrients, temperature, and incident light) influence the Chlorophyll-a (Chl-a) concentrations in the BCZ, this impact is not reflected in the DMS(P,O) concentrations. The latter depend on the succession of low- and high-DMSP producing species (i.e. diatoms and Phaeocystis, respectively). In the NNS in August, no distinct pattern can be drawn for the DMS(P,O) evolution regarding the phytoplankton diversity or abiotic parameters. Investigated by correlations between DMS(P,O), photoprotective pigments and incident light, the antioxidant function is not observed for this short-term period of sampling in a temperate sea. Based on Chl-a linear regressions, DMS(P,O) concentrations are successfully estimated with two distinct relationships for diatoms and Prymnesiophyceae in the BCZ. However, this estimation lacks accuracy in the NNS due to the mixed phytoplankton community observed. Further work will provide a better understanding about the antioxidant function – especially on the field – and its association with the phytoplankton diversity in temperate regions such as the North Sea. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

The eutrophication of the Southern Bight of the North Sea has been benefitting to the prymnesiophyte Phaeocystis globosa (P. globosa). This species is a known high dimethylsulfoniopropionate (DMSP) producer whose bloom accounts for 95% of spring phytoplankton biomass. An increase in DMS(P) and its oxidation product dimethylsulfoxide (DMSO) cellular contents have been frequently observed in cellular stress conditions. To test this, we have first analysed the natural distribution of DMS(P,O) cellular contents in the North Sea. Secondly, we have measured DMS(P,O) cellular contents in monospecific cultures of several key species of the North Sea and their responses to salinity variations. Our main working hypothesis is that DMSP acts as an osmoregulator and/or as an antioxidant, depending on the species. The DMS(P,O) annual cycle in the Southern Bight of the North Sea revealed a seasonality linked to the spring phytoplankton communities succession: (1) colonial diatoms (reappearing in autumn), (2) Chaetoceros spp. (3) P. globosa, (4) large-size summer diatoms (mainly Guinardia spp.), and (5) dinoflagellates. Spatial gradients of DMS(P) were related to those of phytoplankton biomass, itself related to the inputs of nutrients from the Scheldt estuary. It also discharges suspended matter in which DMSO may have been produced by anaerobic oxidation of DMS. Laboratory measurements confirmed a large variability in DMSP cellular contents between the six studied diatoms (Nitzschia closterium, Skeletonema costatum, Thalassiosira rotula, Chaetoceros socialis, Chaetoceros debilis, and Guinardia delicatula), low producers in comparison with P. globosa and even more with Heterocapsa triquetra (Dinoflagellate). In particular, communities 2 and 4 have lower DMSP cellular contents than community 1 (N. closterium, S. costatum and T. rotula). Senescence induces a decrease in DMSP/DMSO suggesting an oxidative stress caused by nutrients and/or light limitation in DMSP producers. In S. costatum, DMSP seems to play an osmoregulatory role and is oxidised into DMSO in hyposaline conditions. In P. globosa and H. triquetra, an oxidative stress appears in hypo- and hypersaline conditions diverging from their salinity optimum. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished