リグノセルロース系バイオマスの有効活用に向けたフェルラ酸エステラーゼの構造と機能に関する研究

京都大学新制・課程博士博士(エネルギー科学)甲第25398号エネ博第477号京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻(主査)教授 片平 正人, 准教授 中田 栄司, 教授 菅瀬 謙治学位規則第4条第1項該当Doctor of Energy ScienceKyoto UniversityDFA

Inverse Correlation of Superoxide Dismutase and Catalase with Type 2 Diabetes among Rural Thais

Oxidative stress contributes to defective antioxidant defenses, which may lead to type 2 diabetes (T2D). This study aimed to elucidate the T2D risks and antioxidant defenses by investigating the superoxide dismutase (SOD), catalase (CAT), vitamin A, and vitamin E status. We observed 102 participants aged 35–66 years from Sung Neon, Nakhon Ratchasima, Thailand. The blood samples were collected to measure the SOD, CAT, vitamin A, and vitamin E concentrations. The SOD and CAT activities were inversely associated with T2D risk. When compared with participants in the highest quartile of SOD and CAT, those in the lowest quartile for T2D risk obtained multivariable-adjusted odds ratios of 4.77 (SOD: 95% confident interval CI, 1.01–22.40; p = 0.047) and 4.22 (CAT: 95% CI, 1.07–16.60; p = 0.039). The possible influencing factors (e.g., physical activity, total cholesterol, and triglyceride) might mediate the association of SOD and CAT with T2D risk. Meanwhile, the relationship between vitamin A and vitamin E concentrations and T2D risk was insignificant. In conclusion, lower concentrations of antioxidant enzyme activity (SOD and CAT) may be an additional risk factor for T2D

Overexpression of LAS21 in Cellulase-Displaying <i>Saccharomyces cerevisiae</i> for High-Yield Ethanol Production from Pretreated Sugarcane Bagasse

The valorization of lignocellulosic feedstocks into biofuels and biochemicals has received much attention due to its environmental friendliness and sustainability. However, engineering an ideal microorganism that can both produce sufficient cellulases and ferment ethanol is highly challenging. In this study, we have tested seven different genes that are involved in glycosylphosphatidylinositol (GPI) biosynthesis and remodeling for the improvement of cellulase activity tethered on the S. cerevisiae cell surface. It was found that the overexpression of LAS21 can improve β-glucosidase activity by 48.8% compared to the original strain. Then, the three cellulase genes (cellobiohydrolase, endoglucanase, and β-glucosidase) and the LAS21 gene were co-introduced into a diploid thermotolerant S. cerevisiae strain by a multiple-round transformation approach, resulting in the cellulolytic ECBLCCE5 strain. Further optimization of the bioprocess parameters was found to enhance the ethanol yield of the ECBLCCE5 strain. Scaling up the valorization of pretreated sugarcane bagasses in a 1 L bioreactor resulted in a maximum ethanol concentration of 28.0 g/L (86.5% of theoretical yield). Our study provides a promising way to improve the economic viability of second-generation ethanol production. Moreover, the engineering of genes involved in GPI biosynthesis and remodeling can be applied to other yeast cell surface display applications

Structure-Based Characterization and Improvement of an Enzymatic Activity of <i>Acremonium alcalophilum</i> Feruloyl Esterase

Bacteria and fungi utilize carbohydrate-active enzymes, such as feruloyl esterases (FAEs), to degrade lignocellulosic biomass. FAEs in subfamily 5 (SF5) of carbohydrate esterase family 1 target larger substrates, making them particularly interesting. However, their mechanisms are not well understood due to limited structural information. This study presents the first structure of the catalytic domain (CD) of an SF5 FAE from Acremonium alcalophilum (AaFaeD), both free and in a complex with ferulic acid (FA). FA binds within a hydrophobic cleft formed by two hydrophobic walls facing each other. Structure-based functional mutagenesis of key residues in these walls clarified their roles in catalysis. Notably, the F120Y mutant of the AaFaeD catalytic domain (AaFaeD-CD) showed a 1.5-fold increase in catalytic activity toward methyl ferulate compared with the wild type. Structural comparisons with SF2 and SF3 FAEs revealed a more open substrate-binding site in SF5. High-performance liquid chromatography and gas chromatography–mass spectrometry analysis of destarched wheat bran hydrolysis by AaFaeD-CD showed that SF5 FAEs can process both monomeric and dimeric phenolic substrates, like 5,5′-dehydrodiferulate, unlike SF2 and SF3 FAEs, which prefer monomeric substrates

Structure-Based Characterization and Improvement of an Enzymatic Activity of <i>Acremonium alcalophilum</i> Feruloyl Esterase

Bacteria and fungi utilize carbohydrate-active enzymes, such as feruloyl esterases (FAEs), to degrade lignocellulosic biomass. FAEs in subfamily 5 (SF5) of carbohydrate esterase family 1 target larger substrates, making them particularly interesting. However, their mechanisms are not well understood due to limited structural information. This study presents the first structure of the catalytic domain (CD) of an SF5 FAE from Acremonium alcalophilum (AaFaeD), both free and in a complex with ferulic acid (FA). FA binds within a hydrophobic cleft formed by two hydrophobic walls facing each other. Structure-based functional mutagenesis of key residues in these walls clarified their roles in catalysis. Notably, the F120Y mutant of the AaFaeD catalytic domain (AaFaeD-CD) showed a 1.5-fold increase in catalytic activity toward methyl ferulate compared with the wild type. Structural comparisons with SF2 and SF3 FAEs revealed a more open substrate-binding site in SF5. High-performance liquid chromatography and gas chromatography–mass spectrometry analysis of destarched wheat bran hydrolysis by AaFaeD-CD showed that SF5 FAEs can process both monomeric and dimeric phenolic substrates, like 5,5′-dehydrodiferulate, unlike SF2 and SF3 FAEs, which prefer monomeric substrates