| 研究実績の概要 |
Our aim of the current project is to develop an efficient method to depolymerize natural kelps available in the Hokkaido ocean by a combination of several enzyme functions, including laminarin-degrading enzymes and alginate-degrading enzymes. A set of kelp spp. were obtained and ready to analytically evaluate their chemical compositions for the next fiscal year. Regarding a laminarin-degrading enzyme, we had picked up a glycoside hydrolase 55 enzyme (GH55) from a highly cellulolytic insect symbiont bacterium (Bianchetti and Takasuka et al., 2015, Journal of Biological Chemistry). 7 polysaccharide lyases 18 enzymes (PL18s), which function in alginate degradation, were selected from 7 different bacterial species, and four PL18s had been recombinantly expressed and subjected to the initial enzyme screening by using pure alginic acid substrate. Moreover, two polysaccharide lyase 17 enzymes (PL17s), were gene synthesized and cloned into the bacterial expression vector. From the previous studies, PL18 is shown to be the endo-type polysaccharide lyase, which produces oligoalginic acids (>3 degrees of polymerization (DP)), while PL17 should catalyze an exo-type reaction. In the FY2021, collected kelps will be tested to be hydrolyzed by at least three different enzymes, GH55, PL17, and PL18, and end products will be analyzed.
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| 今後の研究の推進方策 |
We will prepare all 7 PL18 enzymes and 2 PL17 enzymes using recombinant protein expression methods, then purified by affinity tag purification. All enzymes will be individually tested for their specific activity, optimum reaction temperature, and optimum reaction pH for purified alginate hydrolysis by using a reported reducing end detection assay (DNS assay). The end products will be determined by an high-performance liquid chromatography. After determining the reaction optima of each enzyme, a combination of two enzyme classes (PL17 and PL18) will be tested under the optimized reaction condition in the presence of pure alginate to ask whether their functions are additive or synergistic. From these reactions, we hope a combination of PL17 and PL18 will produce monomeric alginic acid, but if not, we will search for another polysaccharide lyase that boosts up monomer end-product formation. Once the best combination is decided, they will be used to hydrolyze kelp spp. collected from the FY2020 in the presence of GH55 enzyme under optimum reaction temperature and PH. The reaction will be evaluated by a DNS assay, then end products will be assessed by HPLC and GC-MS. If possible, we would like to fractionate end products, analyzed their chemical compositions, and evaluate for their health beneficial functions.
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