論文の公開元へIn vitro synthesis and chemical modification of biorenewable unbranched polymer of a(1→3) linked glucose residues
概要
Chapter 1: Introduction
This research was initiated from the driving force to find new bio-based materials replacing petroleum- based plastics. Polysaccharides are the most abundant one and mostly produced naturally. An attempt to create non-natural polymers has been done and then applied in this research. GtfJ (Fig. 1), glucosyltransferase J, from Streptococcus salivarius ATCC 25975 was used as a biocatalyst to synthesize unbranched α-1,3-glucan (Fig. 2). The in vitro synthesis methods were designed and factors affecting the synthesis were explored in greater details in order to optimize and develop the production of α-1,3-glucan. According to the availability of hydroxyl groups in α-1,3-glucan, the in vitro synthesized material was then esterified followed by the characterization with an aim to develop this new bio-based polymer as thermoplastics.
Chapter 2: The in vitro synthesis of α-1,3-glucan
The isolated GtfJ enzyme from recombinant E. Coli can effectively catalyze the one-pot water-based enzymatic polymerization of linear α-1,3-glucan without branches based on NMR results (Fig. 3). The optimum pH for the catalysis of this enzyme is 5.3-5.8. The synthesis method is environmentally friendly with a reaction in water medium, without organic solvent, and also convenient - only contacting sucrose solution with GtfJ enzyme and letting the reaction undergo at designated temperature (Fig. 4). The raw material, sucrose as a source of monomer, is also from renewable resources and commercially available. Reaction temperature has an effect on Mw and yield of a-1,3-glucan, i.e., lower temperature leads to higher average molecular weight and Mw increases approximately 3.5 times with a reduction of reaction temperature from 30 °C to 15 °C. The reactions at higher temperature result in an increase in the yield of a-1,3-glucan. In addition, average molecular weight also decreases following an increase in reaction time. Besides, enzyme (Fig. 6) and sucrose concentration are directly proportional to yield, but inversely proportional to average molecular weight of α-1,3-glucan (Fig. 5). As a result, the molecular weight and yield of a-1,3-glucan can be tailored by these factors.
Chapter 3: Effect of additives on the production of α-1,3-glucan
Methods to improve molecular weight and yield of α-1,3-glucan during the in vitro enzymatic polymerization were investigated. The first attempt was tried by using surfactants; however, the surfactants do not increase average molecular weight and yield. During the in vitro enzymatic polymerization by GtfJ, leucrose is be found as a by-product (Fig. 2), formed by the chemical binding between glucose and fructose released from the hydrolysis of sucrose. The addition of borate-containing compounds, such as tetraborate and boric acid, in the sucrose solution results in an increase in the yield of α-1,3-glucan. Borate ions enhance the ongoing of reaction by capturing fructose molecules released from the hydrolysis. The maximum yield is found when the concentration of boric acid or tetraborate is at 250 mM or 50 mM, respectively. Therefore, using borate-containing compounds results in a decrease in the formation of leucrose by-product (Fig. 7).
The reaction medium is another influential factor on average molecular weight and yield of α-1,3-glucan. The addition of water-miscible organic solvent in the sucrose solution leads to a decrease in average molecular weight of α-1,3-glucan. Solvents having lower relative polarity result in α-1,3-glucan with lower average molecular weight (Fig. 8). In addition, aprotic organic solvents have higher impact on the reduction of average molecular weight. Furthermore, the presence of water-miscible solvent in the reaction also causes a reduction of yield, except in the case of ethylene glycol. Consequently, the addition of water-miscible solvent in reaction medium is another approach to control the average molecular weight of α-1,3-glucan, increasing an opportunity to provide more molecular weight options of the product.
Chapter 4: Synthesis and properties of α-1,3-glucan homo esters
Esterification is a strategy to improve the thermoplasticity of α-1,3-glucan and it can be conducted via the heterogeneous or homogeneous method. TFAA and carboxylic acid can be utilized in the heterogeneous method, whereas acid anhydride, pyridine and solvent system LiCl/DMAc can be used in the homogeneous method. According to DS results, fully substituted (DS=3) α-1,3-glucan esters can be synthesized from both methods. Average molecular weight of α-1,3-glucan esters synthesized via the homogeneous reaction is higher than that of via the heterogeneous reaction. Owing to the chain degradation occurring in heterogeneous reaction, the obtained molecular weight of α-1,3-glucan esters is reduced in comparison with native α-1,3-glucan. However, the homogeneous method requires longer reaction time than the heterogeneous method does to complete the synthesis.
Thermal degradation temperature is improved after the esterification, from 237°C to ca. 350°C. A presence of crystalline structure in α-1,3-glucan esters can be observed by DSC curves. Tm is detectable in all α- 1,3-glucan esters except for octanoate, and the value is inversely proportional to carbon chain length of acyl group (Fig. 9). In addition, an increase in acyl chain length also results in a decrease in Tg. Furthermore, α-1,3-glucan ester films change from hard to soft behavior with increasing side chain acyl length (Fig. 10).
Chapter 5: Synthesis and properties of α-1,3-glucan mixed esters
The properties of α-1,3-glucan ester can be adjusted and varied by the preparation of mixed esters. Type of mixed esters, side chain length and DS of each ester component in mixed esters do not significantly differentiate the degradation pattern of α-1,3-glucan mixed esters. Tm and Tg of synthesized products are varied between the value of two homo esters. For example, Tm’s and Tg’s of α-1,3-glucan mixed acetate hexanoate range around 172 - 339 °C and 48 - 177 °C, respectively (Fig. 11). Therefore, Tm and Tg of mixed esters can be tailored and predicted by considering the initial values of two ester components and together with DS of each component. Furthermore, hexanoate, as a long chain component, plays an important role in reducing both Tm and Tg in a similar way to octanoate. The crystalline structure of α-1,3-glucan mixed esters still exists, as revealed by the presence of endothermic melting peaks, after the variation of hexanoate content between DS of 0 and 3.0. On the contrary, once hexanoate is replaced by octanoate, the crystalline structure seems to disappear after DS of octanoate reaches near and beyond 2.0 in the case of α-1,3-glucan mixed acetate (or propionate) octanoate. Therefore, a longer chain component, octanoate, has a higher influential effect on the transformation of crystalline structure of α-1,3-glucan mixed esters into amorphous structure.
Tensile strength and elongation at break are varied by changing the content of each ester component. Similar to each other, between mixed hexanoate and mixed octanoate esters, the higher DS of hexanoate or octanoate component α-1,3-glucan mixed esters have, the higher elongation at break but lower tensile strength they exhibit (Fig. 12). Therefore, both hexanoate and octanoate components contribute soft characteristic and chain flexibility to the polymers, whereas acetate and propionate provide a hard characteristic. In conclusion, tensile strength and elongation at break of α-1,3-glucan mixed esters can be adjusted and optimized by changing DS of each component. However, a trade-off behavior - longer elongation in exchange with lower tensile strength, is a common consequence.
Chapter 6: Conclusions
Engineered α-1,3-glucan can be handily and copiously produced from the biorenewable feedstock, having advantages such as the low-cost materials and the organic reaction without organic solvents. The yield and average molecular weight of α-1,3-glucan can be altered by changing the reaction conditions including temperature, time, sucrose concentration and enzyme concentration. The addition of the additive such as borate- containing compounds and water-miscible solvents also influences yield and average molecular weight of α-1,3- glucan. Esterification is an effective approach to handle with the limited thermoplasticity of native α-1,3-glucan. The crystalline α-1,3-glucan esters with outstanding thermal properties - high thermal stability and melting temperature - are of interest for developing new thermoplastic materials in the future. In addition, both thermal and mechanical properties of α-1,3-glucan esters are tunable by the introduction of mixed esters. In conclusion, the in vitro enzymatic polymerization is the advancement of the new synthesis option to provide tailor-made polysaccharide materials with the unique structural polymer. The further development and commercialization of this process will open the gate to numerous industrially important applications.
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