Enzyme discovery for brown seaweed fucoidan modification

Abstract

The objective of this PhD study was to discover enzymes (fucoidanases and sulfatases) that are able to modify fucoidan from the brown seaweed Sargassum mcclurei. In this work, the discovery and characterization is reported of novel enzymes of marine bacteria isolated from sea cucumber gut. A collection of marine bacteria from sea cucumber gut in the Vietnam Sea was created and used for screening for ability to modify fucoidan from Sargassum mcclurei and Turbinaria ornata. Strains with fucoidan modifying potential were subsequently identified by 16S RNA analyses. The marine bacterial strains Pseudoalteromonas MB47 and Cobetia MB87 were selected for further study and their genome sequences were analyzed. The Pseudoalteromonas MB47 genome contained six potential sulfatases. One of these sulfatases, Ps1595, was found to be active on synthetic substrates (p-nitro catechol sulfate and p-nitro phenyl) and interestingly, for the first time, on fucoidan from S. mcclurei. The properties of the Ps1595 sulfatase were characterized on p-NCS substrate. The optimal reaction conditions for Ps1595 sulfatase were pH 6.5, 68oC, 10mM CaCl2. The Km and Vmax were 0.95 ± 0.15 and 26.6 ± 1.2 U, respectively, for the reaction without NaCl. With 125mM NaCl in reaction mixture, the Km did not change but Vmax decreased by 25%. No fucoidanase could be determined from the sequence data of Pseudoalteromonas MB47 and Cobetia MB87, therefore we used online databases to select fucoidanases that might be able to degrade fucoidan S. mcclurei. We selected five microbial fucoidan degrading enzymes, including three endo-fucoidanases FcnA2, Fda1 and Fda2 (EC3.2.1.-, GH 107) and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The five fucoidanases were expressed  heterologously in E. coli and purified. These enzymes were used to treat various fucoidan substrates and the resulting oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis (C-PAGE) and size exclusion chromatography. For the first time we show that all five fucoidanases in this study can degrade S. mcclurei galactofucan-fucoidan, but the recombinant FcnA2, Fda1 and Fda2 enzymes were unstable to different degrees. However, active and more stable enzymes were obtained by truncation of the C-terminal end (by removal of up to 47% of the protein in Fda1). In conclusion, the data obtained have implications for use of these enzymes, including the stabilized versions, in fucoidan processing. Hopefully, this will result in manufacturing of homogenous bioactive fucoidan oligomer products for use in the medicines industry.