Chemical Analysis of Uronic Acids in Macroalgae and Bioconversion of Macroalgae-Derived Uronic Acids

Abstract

Recently, macroalgae have gained attention as possible renewable sources for biofuel and biochemical production. However, there have been insufficient studies on the utilization of macroalgae-derived uronic acids in industrial aspects. The objective of this study is to elucidate the compositional characteristics of macroalgae and to investigate enzymatic reactions that can utilize uronic acids from macroalgae. The content of the work can be categorized into two large groups. In the first part, we investigate the possibility of using macroalgae as biomass feedstock based on chemical composition analysis. For this purpose, we analyzed the proximate composition and element, amino acid, and mineral content of U. pertusa, a model biomass containing uronic acid. Based on the combined results of the chemical composition analysis, the feasibility of using U. pertusa as feedstock for bioenergy and bioproducts was discussed. In addition, we examined the uronic acid composition of macroalgae and the equilibrium between uronic acid and its lactone. In our research, U. pertusa was found to contain 52.3% carbohydrate, 25.1% protein, 0.1% lipid, and 22.5% ash by proximate composition analysis. The high carbohydrate content in U. pertusa is expected to be advantageous for the production of carbon-based products, such as biofuels and bioplastics. U. pertusa’s degree of reductance was the lowest among the examined biomasses, including land crops and plants, due to its high D-glucuronic acid content (34.9% of total carbohydrate). The low degree of reductance of U. pertusa would be beneficial for producing value-added acidic chemicals, such as D-glucaric acid and succinic acid. In addition, we examined the uronic acid content in commercially available alginic acid. The ratio of L-guluronic acid to D-mannuronic acid in alginic acid from the brown macroalgae Macrocystis pyrifera was 32%:68%. The equilibrium between uronic acid and its lactone was also tested. In our experiment, uronic acid lactones were spontaneously converted into uronic acids in alkaline conditions. On the other hand, uronic acids tended to become uronic acid lactones in acidic conditions. Using this approach, we were able to make pure uronic acid or its lactone easily from their mixture. In the second part, we investigated the enzymes that can convert three major uronic acids (D-glucuronate, L-guluronate, and D-mannuronate) from seaweed to useful biochemicals. We investigated D-glucuronic acid dehydrogenase, which can convert D-glucuronic acid to D-glucaric acid, a possible building block of bioplastics. We not only characterized the enzyme, but also analyzed the three-dimensional structure and binding conformation of D-glucuronic acid dehydrogenase. We also proposed metabolic pathways of D-mannuronic acid and L-guluronic acid that can be utilized for the production of useful biochemicals. Moreover, we examined the activity of tartronate semialdehyde reductase (TSAR) from Candidatus P. ubique in order to better understand the metabolism of D-glucuronic acid-utilizing bacteria as carbon sources. We characterized D-glucuronic acid dehydrogenase from Chromohalobacter salexigens, including optimal temperature, pH of enzyme reaction, and kinetic parameters. Through a structural analysis of D-glucuronic acid dehydrogenase with a docking simulation, binding conformation and critical residues were investigated. As a part of the utilization of D-glucuronic acid, we investigated TSAR from Cand. P. ubique. We confirmed the oxidation and reduction reactions of TSAR, including TSAR from E. coli. By comparing this to the structure and sequence of TSAR from Salmonella typhimurium, we showed that Ser121, Gly122, Lys169, and Gln173 of SAR11_TSAR are important residues in substrate binding. By performing cloning, expression, and enzyme assays of four enzymes from Marine Bacterium A, we identified novel metabolic pathways of L-guluronate and D-mannuronate. In the L-guluronate pathway, L-guluronate is converted into D-gluconate by GulA, L-guluronate reductase, with a preference for NADPH. D-gluconate is phosphorylated to 6-phospho-D-gluconate by GulB, D-gluconate kinase. In the D-mannuronate pathway, D-mannuronate is converted into D-fructuronate by ManA, D-mannuronate isomerase. D-fructuronate is reduced to D-mannonate by ManB, D-fructuronate reductase. Then, ManC, D-mannonate dehydratase, dehydrates D-mannonate to produce 2-keto-3-deoxy-D-gluconate. From the results of the chemical analysis of macroalgae and the bioconversion of uronic acids, we concluded that macroalgae-derived uronic acids have potential as next-generation biomass feedstock. We expect that many novel bioprocesses for the conversion of macroalgae-uronic acids will be developed in the future based on the findings of our study.