Zhaohui Guo,Christian Marcus Pedersen,Honghong Chang,Yingxiong Wang,Yan Qiao
As a renewable source, fructose is of great significance in biomass conversion and food industries. Currently, most of the chemo-catalysts studied, with glucose as a substrate, are limited by the thermodynamic equilibrium of the reaction, resulting in the maximum yield of fructose of approximately 30–40%. The similar chemical structures and physical properties of monosaccharides bring another challenge for the separation of fructose from the reaction mixture. In this study, the combination of CaCl2 and Et3N was found to alter the thermodynamic equilibrium and promote fructose separation. The reaction parameters were optimized and up to 78% fructose yield could be obtained. The pathways of the isomerization reaction were found to involve both intramolecular hydride shift and an enediol intermediate mechanism, and the former was major. It could be speculated reasonably that Ca2+ played a key role in glucose conversion and interacted with glucose at its C1 and C2 positions, and Et3N interacted with Cl− while providing an alkaline environment. The formation of the α-fructofuranose’ complex, from Et3N, CaCl2 and α-fructofuranose, could shift the equilibrium of glucose isomerization towards fructose formation. According to the diffusion-ordered spectroscopy (DOSY) NMR results, the strong complexation slowed down the diffusion rate of the α-fructofuranose’ complex, allowing it to gradually precipitate, and hence drove the reaction equilibrium towards the desired fructose. The precipitate was filtered from the reaction mixture and processed further to obtain high-purity fructose in 45% isolated yield. Moreover, the filtrate containing solvent and reagents obtained in the separation process could be recycled.
Xinnan Ma,Wenrui Huang,Yongqing Song,Juan Han,Jiacong Wu,Lei Wang,Yun Wang
The development of a suitable immobilization strategy to improve the performance of immobilized glucose isomerase for the isomerization of glucose to fructose is crucial to promoting the industrial production of high-fructose syrup. In this work, a novel recyclable upper critical solution temperature (UCST)-type mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst (PVAA@GI) was prepared, and the immobilized glucose isomerase could capture the glucose substrate through the affinity of 4-vinylbenzeneboronic acid (4-VBA) and the glucose substrate, which led to the enhanced substrate affinity and catalytic efficiency of the PVAA@GI. The biocatalyst exhibited excellent stability in pH, thermal, storage, and recycling compared to the free enzyme. The mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst displayed reversibly soluble–insoluble characteristics with temperature change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the temperature after the reaction. The highest fructose production rate reached 62.79%, which would have potential application in the industrial production of high-fructose syrup.
Jianlin Chu,Yani Tian,Qian Li,Gaofei Liu,Qi Yu,Tianyue Jiang,Bingfang He
Levan-type fructooligosaccharides (FOS) exhibit enhanced health-promoting prebiotic effects on gut microbiota. The wild type (WT) of β-fructofuranosidase Fru6 could mainly yield 6-ketose. Semirational design and mutagenesis of Fru6 were exploited to promote the transfructosylating capacity for FOS. The promising variants not only improved the formation of 6-kestose but also newly produced tetrasaccharides of 6,6-nystose and 1,6-nystose (a new type of FOS), and combinatorial mutation boosted the production of 6-kestose and tetrasaccharides (39.9 g/L 6,6-nystose and 4.6 g/L 1,6-nystose). Molecular docking and molecular dynamics (MD) simulation confirmed that the mutated positions reshaped the pocket of Fru6 to accommodate bulky 6-kestose in a reactive conformation with better accessibility for tetrasaccharides formation. Using favored conditions, the variant S165A/H357A could yield 6-kestose up to 335 g/L, and tetrasaccharides (6,6-nystose and 1,6-nystose) reached a high level of 121.1 g/L (134.5 times of the mutant S423A). The β-(2,6)-linked FOS may show the potential application for the prebiotic ingredients.
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