Glucose Isomerase Research Articles

Advances in the biosynthesis of D-allulose.

D-allulose is a rare monosaccharide and a C-3 epimer of D-fructose. It has physiological functions, such as antihyperglycemic, obesity-preventing, neuroprotective, and reactive oxygen species (ROS) scavenging effects, making it an ideal sugar substitute. The synthesis methods for D-allulose include chemical synthesis and biosynthesis. Chemical synthesis requires strict reaction conditions and tends to produce byproducts. Biosynthesis is mainly an enzymatic process. Enzymatic catalysis for the conversion of starch or glycerol to D-allulose is performed mainly by enzymes such as isoamylase (IA), glucose isomerase (GI), D-allulose 3-epimerase (DPE), D-allulose-6-phosphate 3-epimerase (A6PE), D-allulose 6-phosphate phosphatase (A6PP), ribitol 2-dehydrogenase (RDH), glycerophosphate kinase (GK), glycerophosphate oxidase (GPO), and dihydroxyacetone phosphate (DHAP)-dependent aldolase. Biosynthesis is a more energy-efficient process, producing fewer harmful by-products and pollutants, and significantly reducing negative environmental impacts. Furthermore, the specific catalytic activity of enzymes facilitates the production of compounds of higher purity, thereby facilitating the isolation and purification of the products. It has thus become the main method for producing D-allulose. This article reviews the progress in research on the biosynthetic production of D-allulose, focusing on the enzymes involved and their enzymatic properties, and discusses the production prospects for D-allulose.

Xylooligosaccharide interferes with the cell cycle and reduces the antibiotic tolerance of avian pathogenic Escherichia coli by associating with its potential antimetabolic actions

This study aimed to probe if xylooligosaccharide (XOS) could act as an antimetabolite to impact the cell cycle and antibiotic tolerance of avian pathogenic Escherichia coli (APEC). We firstly measured the bacteriostasis of XOS against APEC O78 and its effect on the growth of APEC O78 growing on different medium. Afterwards, the effects of XOS on xylose operon activation along with the cell cycle and antibiotic tolerance of APEC O78 were analyzed. The results showed that XOS caused no inhibitory circle against APEC O78 and did not affect (P > 0.05) the growth of APEC O78 growing on LB medium. Besides, APEC O78 was unable to grow on M9 medium (carbon-free) added with XOS. However, XOS exerted a similar role as xylose in increasing (P < 0.05) the expression of certain xylose operon genes including xylose isomerase (XylA)-encoding gene (xylA) and xylose-binding periplasmic protein (XylF)-encoding gene (xylF) in APEC O78. The molecular docking simulation revealed that the major monomer components (xylobiose, xylotriose and xylotetraose) of XOS had stable binding potentials to both XylA and XylF proteins of E. coli, as supported by the low binding free energy and the formation of considerable hydrogen bonds between them. The subsequent analysis showed that XOS altered certain cell cycle-related genes expression, especially elevated (P < 0.05) nrdB expression and decreased ihfB expression to a degree. Moreover, XOS played a similar role as 2-deoxy-glucose (a glucose analogue serving as a typical antimetabolite) in lowering (P < 0.05) the number of ampicillin-tolerant APEC O78. Collectively, XOS had no direct bacteriostasis against APEC and could not be metabolized/utilized by APEC O78. However, it might become an analogue of xylose and then activate xylose transport- and metabolism-related proteins in APEC O78, thus functioning as a potential antimetabolite and exerting anti-metabolic actions. This could at least partially interpret the observed roles of XOS in interfering with the cell cycle and diminishing the antibiotic tolerance of APEC O78. The above findings expand the knowledges about the functions of XOS and provide a basis for exploring novel strategies to reduce the antibiotic tolerance of APEC.

Lactose hydrolysis in sheep and cow milk with varying fat levels to evaluate enzymatic production of lactulose

AbstractThis study aimed to perform lactose hydrolysis using free and immobilised β-galactosidase in cow and sheep milk with different fat levels, followed by lactulose synthesis through the isomerisation of glucose to fructose using immobilised glucose isomerase. There was no significant difference (P &gt; 0.05) in lactose hydrolysis (&gt;95%) using the free enzyme in the different types of milk. In contrast, significant differences were observed for the hydrolysis of whole cow milk (WCM) and skimmed cow milk (SCM) with the immobilised enzyme (IE). WCM required a longer hydrolysis time (43 h). Thus, the fat content may have affected the lactose hydrolysis of bovine milk with immobilised β-galactosidase. No difference was observed for whole sheep milk and skimmed sheep milk hydrolysed by IE, possibly due to the presence of smaller fat globules, which did not affect the lactose hydrolysis. No significant differences were observed for lactulose synthesis in whole milk from the different species evaluated. The lactulose synthesis in cow and sheep milk by the enzymatic route may be a promising alternative, though further studies are required to optimise the production of this prebiotic in situ.

Keratin-reinforced encapsulation of whole cells expressing glucose isomerase: Development of robust and reusable biocatalyst microbeads.

Glucose isomerase (GI) is crucial in high-fructose corn syrup production. This study introduces a novel approach to enhance GI stability and reusability through whole-cell encapsulation of Streptomyces olivochromogenes PTCC 1457 in hybrid microbeads, utilizing keratin as a multifunctional stabilizer and cross-linker. Optimal bead formation was achieved using 2 % alginate, 2-3 % CaCl2, and 2.5 % keratin at pH 7.0 and 37-40 °C. Keratin played a vital role in forming a robust and flexible matrix. Immobilization in keratin-alginate-biomass beads maintained GI activity (655 GIU·g-1) comparable to free enzyme (650 GIU·g-1), while silicate incorporation reduced activity to 234 GIU·g-1. The immobilized enzyme exhibited enhanced stability over a wider pH (6-9) and temperature (4-60 °C) range compared to the free enzyme. Importantly, the immobilized GI maintained 80 % of its initial activity after 20 reaction cycles. Thermogravimetric analysis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and tensile testing confirmed the formation of hybrid beads with improved thermal and mechanical stability. This novel immobilization strategy, leveraging keratin's unique properties, offers a promising approach for enhancing GI stability, reusability, and storage longevity, potentially improving its industrial applicability in high-fructose corn syrup production.

Functionalized mesoporous biosilica for immobilizing D-allulose 3-epimerase: A multienzyme cascade strategy for calorie reduction in fruit tea drinks

D-allulose, a low-calorie rare sugar, can be biosynthesized by D-allulose 3-epimerase (DAE). Here, DAE was covalently immobilized on polyethyleneimine-modified diatomite using two methods: a biopolymer coating approach (DAE@PEI-GA@PDP) and a direct attachment method (DAE-GA@PEI@PDP). Both immobilized DAE enzymes exhibited enhanced thermal stability and broader pH tolerance compared to the free DAE. They also exhibited excellent reusability and maintained more than 70% of their activity after 10 cycles. Additionally, a multienzyme cascade system was developed to produce D-allulose from low-cost D-glucose using immobilized glucose isomerase (GI) and immobilized DAE enzymes. This system obtained 76 g/L D-allulose from 500 g/L D-glucose. Notably, the application was extended to fruit tea drinks, successfully transforming high-calorie sugars (D-glucose and D-fructose) into D-allulose. This research offers a promising immobilization strategy for DAE on silica-based materials and provides robust biocatalysts for creating functional fruit tea drinks aimed at diabetics and individuals with specific dietary needs.

Engineering the xylose metabolism of Saccharomyces cerevisiae for ethanol and single cell protein bioconversion

Xylose isomerase (XI) pathway has been widely employed to enable Saccharomyces cerevisiae to convert xylose and glucose into commercially feasible lignocellulosic ethanol products. Nevertheless, studies about the effect of different promoters to the expression of xylA are lacking. Therefore, five strains with ADH1, GAPDH, PDC1, PGK, TEF1 promoters were constructed. Among them, S. cerevisiae INVSc1/pHM368-PADH1-xylA generated with xylA driven by ADH1 promoter displayed the highest xylose utilization rate (approximately 19.98 %) using xylose as the only carbon source. With 4 g/L glucose and 10 g/L xylose as the carbon sources, the xylose utilization rate was 60.04 %. Moreover, the utilization rate increased to 64.04 % with fermentation temperature elevated from 28 °C to 30 °C and reached 83.09 % with peptone and yeast extract as the nitrogen sources. The ethanol titer reached 1.74 g/L with a yield of 0.38 g/g sugar under this condition. In Comparison with direct fermentation, the single cell protein (SCP) was 1.27-fold higher during aerobic fed-batch fermentation. Furthermore, INVSc1/pHM368-PADH1-xylA attains high ethanol productivities and yields by converting glucose and xylose from non-detoxified bagasse hydrolysates as carbon sources. The results extend our understanding of the xylose metabolism of S. cerevisiae and provide a platform for biomass conversion to ethanol and SCP, hence paving the way for the development of a more economical and sustainable approach to co-fermentation performance and capabilities for future engineering.

One-pot synthesis of cellobiose from sucrose using sucrose phosphorylase and cellobiose phosphorylase co-displaying Pichia pastoris as a reusable whole-cell biocatalyst

Cellobiose has received increasing attention in various industrial sectors, ranging from food and feed to cosmetics. The development of large-scale cellobiose applications requires a cost-effective production technology as currently used methods based on cellulose hydrolysis are costly. Here, a one-pot synthesis of cellobiose from sucrose was conducted using a recombinant Pichia pastoris strain as a reusable whole-cell biocatalyst. Thermophilic sucrose phosphorylase from Bifidobacterium longum (BlSP) and cellobiose phosphorylase from Clostridium stercorarium (CsCBP) were co-displayed on the cell surface of P. pastoris via a glycosylphosphatidylinositol-anchoring system. Cells of the BlSP and CsCBP co-displaying P. pastoris strain were used as whole-cell biocatalysts to convert sucrose to cellobiose with commercial thermophilic xylose isomerase. Cellobiose productivity significantly improved with yeast cells grown on glycerol compared to glucose-grown cells. In one-pot bioconversion using glycerol-grown yeast cells, approximately 81.2 g/L of cellobiose was produced from 100 g/L of sucrose, corresponding to 81.2% of the theoretical maximum yield, within 24 h at 60 °C. Moreover, recombinant yeast cells maintained a cellobiose titer > 80 g/L, even after three consecutive cell-recycling one-pot bioconversion cycles. These results indicated that one-pot bioconversion using yeast cells displaying two phosphorylases as whole-cell catalysts is a promising approach for cost-effective cellobiose production.

Reaction and Kinetics in Immobilized Glucose Isomerase of Packed-Bed Reactors Using Akbari-Ganji’s Method

Reaction and Kinetics in Immobilized Glucose Isomerase of Packed-Bed Reactors Using Akbari-Ganji’s Method

Benchmarking predictive methods for small-angle X-ray scattering from atomic coordinates of proteins using maximum likelihood consensus data.

Stimulated by informal conversations at the XVII International Small Angle Scattering (SAS) conference (Traverse City, 2017), an international team of experts undertook a round-robin exercise to produce a large dataset from proteins under standard solution conditions. These data were used to generate consensus SAS profiles for xylose isomerase, urate oxidase, xylanase, lysozyme and ribonuclease A. Here, we apply a new protocol using maximum likelihood with a larger number of the contributed datasets to generate improved consensus profiles. We investigate the fits of these profiles to predicted profiles from atomic coordinates that incorporate different models to account for the contribution to the scattering of water molecules of hydration surrounding proteins in solution. Programs using an implicit, shell-type hydration layer generally optimize fits to experimental data with the aid of two parameters that adjust the volume of the bulk solvent excluded by the protein and the contrast of the hydration layer. For these models, we found the error-weighted residual differences between the model and the experiment generally reflected the subsidiary maxima and minima in the consensus profiles that are determined by the size of the protein plus the hydration layer. By comparison, all-atom solute and solvent molecular dynamics (MD) simulations are without the benefit of adjustable parameters and, nonetheless, they yielded at least equally good fits with residual differences that are less reflective of the structure in the consensus profile. Further, where MD simulations accounted for the precise solvent composition of the experiment, specifically the inclusion of ions, the modelled radius of gyration values were significantly closer to the experiment. The power of adjustable parameters to mask real differences between a model and the structure present in solution is demonstrated by the results for the conformationally dynamic ribonuclease A and calculations with pseudo-experimental data. This study shows that, while methods invoking an implicit hydration layer have the unequivocal advantage of speed, care is needed to understand the influence of the adjustable parameters. All-atom solute and solvent MD simulations are slower but are less susceptible to false positives, and can account for thermal fluctuations in atomic positions, and more accurately represent the water molecules of hydration that contribute to the scattering profile.

Structural Analysis of Xylose Isomerase from Streptomyces avermitilis

Xylose isomerase (XI, also known as glucose isomerase) is an oxidoreductase that interconverts aldoses and ketoses. XI catalyzes the reversible isomerization of D-glucose and D-xylose into D-fructose and D-xylulose, respectively. The molecular function of XI is widely applied in producing high-fructose corn syrup (HFCS) in the food industry and bioethanol from hemicellulose in the biofuel industry. The structural information of XI from diverse strains is important for understanding molecular properties that can provide insights into protein engineering to improve enzyme efficiency. To extend the knowledge of the structural information on XI, the crystal structure of XI from Streptomyces avermitilis (SavXI) was determined at a 2.81 Å resolution. SavXI containing TIM barrel and extended α-helix domains formed the tetrameric assembly. The two metal-binding sites and their coordinating residues showed diverse conformations, providing the structural flexibility of the active site of SavXI. The structural comparison of SavXI and XI homologs exhibited unique metal-binding sites and conformations of the C-terminal α-helix domain. These structural results extend our knowledge of the molecular flexibility and mechanism of the XI family.

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Highly Selective Isomerization of Glucose to Fructose through a Biphasic and Recyclable Mimetic Enzyme Catalyst

AbstractDeveloping a biphasic catalyst is of great significance for improving reaction efficiency and its industrial application. Herein a novel biphasic catalyst containing three functional groups of chlorophenol, amino and carboxyl was synthesized and used as mimic glucose isomerase to catalyze the isomerization. The mimic enzyme exhibited excellent catalytic performance for isomerization of glucose to fructose under mild conditions. Temperature and pH of solution showed significant influence on the conversion of glucose and the selectivity of fructose. The yield and selectivity of fructose reached 32.4 % and 91.0 % after reacting 2 h, respectively, at pH 9.5 and 90 °C. The solubility of the catalyst exhibited dual sensitivity to both pH and temperature. The catalyst was in a soluble state during the reaction process and could be precipitated by reducing the pH and temperature of the reaction system. The catalyst could be reused and still maintained good catalytic activity after five consecutive cycles.

Solution-mediated phase transition of protein crystals

A snapshot of the evidence of solution-mediated phase transition of glucose isomerase (GI) crystals more than 7 months after the start of an experiment was successfully captured. The transition to [Formula: see text] crystals started about 2 months after the first I222 crystals nucleated. To conduct such long-term experiments, we developed novel observation cells with liquid paraffin layers to prevent aqueous GI solutions from evaporation of water. Changes in the weights of cells were measured, and much less evaporation was successfully achieved by continuously adding liquid paraffin as a liquid sealant.

Synthetically-primed adaptation of Pseudomonas putida to a non-native substrate D-xylose

To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.

Construction and investigation of multi-enzyme immobilized matrix for the production of HFCS.

Enzymes are biological molecules that act as catalysts and speed up the biochemical reactions. The world's biotechnological ventures are development of enzyme productiveness, and advancement of novel techniques for thriving their shelf existence. Nowadays, the most burning questions in enzyme technology are how to improve the enzyme productivity and reuse them. The immobilization of enzymes provides an excellent scope to reuse the enzymes several times to increase productivity. The main aim of the present study is the establishment of an immobilized multi-enzyme bio-system engineering process for the production of High-fructose corn syrup (HFCS) with an industrial focus. In this study, multi-enzyme such as α-amylase, glucoamylase and glucose isomerase were immobilized in various support matrices like sodium alginate, sawdust, sugarcane bagasse, rice bran and combination of alginate with cellulosic materials. The activities of the immobilized multi-enzyme system for the production of HFCS from the starch solution were determined. The multi-enzyme immobilized in sodium alginate shows better fructose conversion than free enzyme. Among the support matrices, multi-enzyme immobilized in sawdust produced total 80.74 mg/mL of fructose from starch solution and it was able to be used in several production cycles. On the other hand, multi-enzyme immobilized in combination of sodium alginate and sawdust produced the maximum amount of fructose (total 84.82 mg/mL). The free enzyme produced 17.25 mg/mL of fructose from the starch solution in only a single cycle. In this study a new fixed bed immobilized multi-enzyme bioreactor system was developed for the production of HFCS directly from starch. This finding will create a new opportunity for the application of immobilized multi-enzyme systems in many sectors of industrial biotechnology.

Method for Glucose and Fructose Quantitation in Beverages Using an Off-the-Shelf Glucose Test Strip.

A method was developed for quantifying both glucose and fructose in solutions and grape juice using commercially available glucose test strips connected to a mini-potentiostat. The first step of this sensing approach involved exposing the sample solution to an Accu Chek Aviva glucose test strip, which allowed for the direct quantitation of glucose. To quantify fructose, the solution was exposed to glucose isomerase, which led to the conversion of glucose to fructose and vice versa until an equilibrium was reached. Once equilibrium was reached, the solution was exposed to another glucose test strip; the signal obtained was shown to be related to the total amounts of glucose and fructose in solution. Finally, fructose was quantified by subtracting the glucose concentration (from the initial measurement) from the total concentration of glucose and fructose (from the second measurement after the reaction with glucose isomerase). The method yielded a limit of detection of 0.047 g L-1 for glucose and 0.49 g L-1 for fructose. Importantly, this method was shown to work well for analyzing glucose and fructose concentration in grape juice, which contains >60 g L-1 glucose and fructose. Since the ratio of glucose and fructose concentration in ripe grapes is close to 1, this method can be used to aid in the determination of grape ripeness to guide harvesting times.

Theoretical Improvements in Enzyme Efficiency Associated with Noisy Rate Constants and Increased Dissipation.

Previous studies have revealed the extraordinarily large catalytic efficiency of some enzymes. High catalytic proficiency is an essential accomplishment of biological evolution. Natural selection led to the increased turnover number, kcat, and enzyme efficiency, kcat/KM, of uni-uni enzymes, which convert a single substrate into a single product. We added or multiplied random noise with chosen rate constants to explore the correlation between dissipation and catalytic efficiency for ten enzymes: beta-galactosidase, glucose isomerase, β-lactamases from three bacterial strains, ketosteroid isomerase, triosephosphate isomerase, and carbonic anhydrase I, II, and T200H. Our results highlight the role of biological evolution in accelerating thermodynamic evolution. The catalytic performance of these enzymes is proportional to overall entropy production-the main parameter from irreversible thermodynamics. That parameter is also proportional to the evolutionary distance of β-lactamases PC1, RTEM, and Lac-1 when natural or artificial evolution produces the optimal or maximal possible catalytic efficiency. De novo enzyme design and attempts to speed up the rate-limiting catalytic steps may profit from the described connection between kinetics and thermodynamics.

Enhanced High-Fructose Corn Syrup Production: Immobilizing Serratia marcescens Glucose Isomerase on MOF (Co)-525 Reduces Co2+ Dependency in Glucose Isomerization to Fructose.

The escalating demand for processed foods has led to the widespread industrial use of glucose isomerase (GI) for high-fructose corn syrup (HFCS) production. This reliance on GIs necessitates continual Co2+ supplementation to sustain high catalytic activity across multiple reaction cycles. In this study, Serratia marcescens GI (SmGI) was immobilized onto surfaces of the metal-organic framework (MOF) material MOF (Co)-525 to generate MOF (Co)-525-GI for use in catalyzing glucose isomerization to generate fructose. Examination of MOF (Co)-525-GI structural features using scanning electron microscopy-energy dispersive spectroscopy, Fourier-transform infrared spectroscopy, and ultraviolet spectroscopy revealed no structural changes after SmGI immobilization and the addition of Co2+. Notably, MOF (Co)-525-GI exhibited optimal catalytic activity at pH 7.5 and 70 °C, with a maximum reaction rate (Vmax) of 37.24 ± 1.91 μM/min and Km value of 46.25 ± 3.03 mM observed. Remarkably, immobilized SmGI exhibited sustained high catalytic activity over multiple cycles without continuous Co2+ infusion, retaining its molecular structure and 96.38% of its initial activity after six reaction cycles. These results underscore the potential of MOF (Co)-525-GI to serve as a safer and more efficient immobilized enzyme technology compared to traditional GI-based food-processing technologies.

Engineered Saccharomyces cerevisiae harbors xylose isomerase and xylose transporter improves co-fermentation of xylose and glucose for ethanol production

Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INVSc-XI and INVSc-XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INVSc-XI/XT was 2.3-fold higher than that of INVSc-XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INVSc-XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase (ACS), alcohol dehydrogenase (ADH), and transketolase (TKL) gene expression in INVSc-XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INVSc-XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INVSc-XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INVSc-XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization.

Reactor development for a one‐step hybrid catalytic conversion of D‐glucose to HMF

AbstractThe design of an “H”‐shaped reactor has been studied to satisfy the possibility of using a triphasic medium to transform d‐glucose into 5‐hydroxymethylfurfural (HMF) by a hybrid catalytic process using an immobilized glucose isomerase, allowing the isomerization of D‐glucose to d‐fructose, and a heterogeneous chemical catalyst to catalyze the dehydration of d‐fructose to HMF. The various parameters influencing the transport of d‐fructose within the reactor were studied, namely the difference in pH between the two aqueous phases, the agitation and the quantity of 3,4‐DCPBA/Aliquat336® used to extract and transport continuously the d‐fructose. The pH discrepancy, which must be between 5.0 and 5.5, turns out to be the real driving force behind the extraction of D‐fructose. The D‐Fru/3,4‐DCPBA molar ratio of 1/0.25 allowed the transport of more than 80 % of the D‐fructose initially present and shows that the same molecule of 3,4‐DCPBA is involved at least twice in the transport of D‐fructose and that the 3,4‐DCPBA/Aliquat336® couple performs a turnover within the organic phase. Finally, a HMF production yield of 30.9 % and an isomerization yield of 79.1 % were obtained, illustrating the shift in the isomerization equilibrium made possible by a D‐fructose extraction yield of 96.8 %.