Production Of D-allulose 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.

Biosynthesis of nonnutritive monosaccharide d-allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose.

Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. d-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for d-allulose production from sucrose using Escherichia coli JM109 (DE3) as a chassis host. The genes cscA, cscB, cscK, alsE, and a6PP were co-expressed for the construction of the synthesis pathway. Then, the introduction of ptsG-F and knockout of ptsG, fruA, ptsI, and ptsH to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of pfkA and zwf, achieving an increase in d-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of d-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.

Comprehensive Analysis of Allulose Production: A Review and Update.

Advancements in D-allulose production have seen significant strides in recent years, focusing on enzymatic conversion methods. Key developments include traditional immobilization techniques, the discovery of novel enzymes, directed evolution studies, and biosynthesis through metabolic pathway modification. Enzymatic conversion, particularly utilizing D-allulose 3-epimerase, remains fundamental for industrial-scale production. Innovative immobilization strategies, such as functionalized nano-beads and magnetic MOF nanoparticles, have significantly enhanced enzyme stability and reusability. Directed evolution has led to improved enzyme thermostability and catalytic efficiency, while synthetic biology methods, including phosphorylation-driven and thermodynamics-driven pathways, have optimized production processes. High-throughput screening methods have been crucial in identifying and refining enzyme variants for industrial applications. Collectively, these advancements not only enhance production efficiency and cost-effectiveness but also adhere to sustainable and economically viable manufacturing practices. The past five years have witnessed critical developments with significant potential impact on the commercial viability and global demand for allulose.

Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor.

D-Allulose is one of the most well-known rare sugarswidely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60to 27.56h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

Enhanced Biosynthesis of d-Allulose from a d-Xylose-Methanol Mixture and Its Self-Inductive Detoxification by Using Antisense RNAs in Escherichia coli.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.

Immobilization of a novel d-allulose 3-epimerase from Bacillus cihuensis within metal-organic frameworks

As a functional low-calorie sugar, d-allulose demonstrates significant potential for applications in the food industry. Ketose 3-epimerases (KE) catalyzing the epimerization of d-fructose into d-allulose, play a critical role in the production of d-allulose. Majority of the reported KE exhibited maximum activity at high temperatures (60–70 °C) and pH (7.5–8.0) with metal ions (Mn2+ or Co2+), resulting in browning reaction and complex processes (adjusting pH of fructose solution, removing heavy metal ions, etc.) in allulose production. In this study, a novel d-allulose 3-epimerase from B. cihuensis (BcDAE) was identified and characterized. The BcDAE showed maximum activity at 40–50 °C and pH 7.0, alongside exhibiting board pH tolerance in weakly acidic environments. As a mesophilic enzyme, BcDAE could provide advantages by mitigating browning reactions of substrate and product, minimizing by-product formation, and reducing energy consumption during production. Robustness in acidic environments of BcDAE might renders the enzyme well-suited for fructose conversion without pH adjustments. Considering the poor thermostability and dependence on Mn2+ of BcDAE, the enzyme was captured in Mn-BTC MOF material using a straightforward co-precipitation method. The MOF-based enzyme was subsequently encapsulated to form micrometer-sized immobilized enzyme PDA@BcDAE@Mn-BTC using polydopamine as the cross-linker. Metal ions are not required in the reaction using the immobilized BcDAE. Significantly, after 10 repeated usage cycles, the immobilized BcDAE maintained a relative enzyme activity exceeding 70%, surpassing the performance of other reported immobilized DAE within MOF. Furthermore, comprehensive characterization of the immobilized BcDAE confirmed the success and feasibility of the immobilization strategy.

Enhancing the stability of a novel D-allulose 3-epimerase from Ruminococcus sp. CAG55 by interface interaction engineering and terminally attached a self-assembling peptide

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.

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.

Rational design improves both thermostability and activity of a new D-tagatose 3-epimerase from Kroppenstedtia eburnean to produce D-allulose

D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency. Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds, and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; Tm value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Enhancing enzyme immobilization: Fabrication of biosilica-based organic-inorganic composite carriers for efficient covalent binding of D-allulose 3-epimerase

D-allulose, an ideal low-calorie sweetener, is primarily produced through the isomerization of d-fructose using D-allulose 3-epimerase (DAE; EC 5.1.3.30). Addressing the gap in available immobilized DAE enzymes for scalable commercial D-allulose production, three core-shell structured organic-inorganic composite silica-based carriers were designed for efficient covalent immobilization of DAE. Natural inorganic diatomite was used as the core, while 3-aminopropyltriethoxysilane (APTES), polyethyleneimine (PEI), and chitosan organic layers were coated as the shells, respectively. These tailored carriers successfully formed robust covalent bonds with DAE enzyme conjugates, cross-linked via glutaraldehyde, and demonstrated enzyme activities of 372 U/g, 1198 U/g, and 381 U/g, respectively. These immobilized enzymes exhibited an expanded pH tolerance and improved thermal stability compared to free DAE. Particularly, the modified diatomite with PEI exhibited a higher density of binding sites than the other carriers and the PEI-coated immobilized DAE enzyme retained 70.4 % of its relative enzyme activity after ten cycles of reuse. This study provides a promising method for DAE immobilization, underscoring the potential of using biosilica-based organic-inorganic composite carriers for the development of robust enzyme systems, thereby advancing the production of value-added food ingredients like D-allulose.

Biochemical characterization, structure-guided mutagenesis, and application of a recombinant D-allulose 3-epimerase from Christensenellaceae bacterium for the biocatalytic production of D-allulose.

D-Allulose has become a promising alternative sweetener due to its unique properties of low caloric content, moderate sweetness, and physiological effects. D-Allulose 3-epimerase (DAEase) is a promising enzyme for D-Allulose production. However, the low catalytic efficiency limited its large-scale industrial applications. To obtain a more effective biocatalyst, a putative DAEase from Christensenellaceae bacterium (CbDAE) was identified and characterized. The recombinant CbDAE exhibited optimum activity at pH 7.5°C and 55°C, retaining more than 60% relative activity from 40°C to 70°C, and the catalytic activity could be significantly increased by Co2+ supplementation. These enzymatic properties of purified CbDAE were compared with other DAEases. CbDAE was also found to possess desirable thermal stability at 55°C with a half-life of 12.4h. CbDAE performed the highest relative activity towards D-allulose and strong affinity for D-fructose but relatively low catalytic efficiency towards D-fructose. Based on the structure-guided design, the best double-mutation variant G36N/W112E was obtained which reached up to 4.21-fold enhancement of catalytic activity compared with wild-type (WT) CbDAE. The catalytic production of G36N/W112E with 500g/L D-fructose was at a medium to a higher level among the DAEases in 3.5h, reducing 40% catalytic reaction time compared to the WT CbDAE. In addition, the G36N/W112E variant was also applied in honey and apple juice for D-allulose conversion. Our research offers an extra biocatalyst for D-allulose production, and the comprehensive report of this enzyme makes it potentially interesting for industrial applications and will aid the development of industrial biocatalysts for D-allulose.

Bioproduction of Rare d-Allulose from d-Glucose via Borate-Assisted Isomerization.

d-Allulose is a low-calorie functional rare sugar with excellent processing suitability and unique physiological efficacy. d-Allulose is primarily produced from d-fructose through enzymatic epimerization, facing the constraints of a low conversion yield and high production cost. In this study, a double-enzyme cascade system with tetraborate-assisted isomerization was constructed for the efficient production of d-allulose from inexpensive d-glucose. With the introduction of sodium tetraborate (STB), capable of forming complexes with diol-bearing sugars, the conversion yield of d-allulose from d-glucose substantially escalated from the initial 17.37% to 44.97%. Furthermore, d-allulose was found to exhibit the most pronounced binding affinity for STB with an association constant of 1980.51 M-1, notably surpassing that of d-fructose (183.31 M-1) and d-glucose (35.37 M-1). Additionally, the structural analysis of the sugar-STB complexes demonstrated that d-allulose reacted with STB via the cis 2,3-hydroxyl groups in the α-furanose form. Finally, the mechanism underlying STB-assisted isomerization was proposed, emphasizing the preferential formation of an allulose-STB complex that effectively shifts the isomerization equilibrium to the allulose side, thereby resulting in high yield of d-allulose. Such an STB-facilitated isomerization system would also provide a guidance for the cost-effective synthesis of other rare sugars.

Strategy for production of high-purity rare sugar D-allulose from corn stover

Here, we report a strategy for producing high-value and high-purity rare sugar D-allulose from inexpensive corn stover. To eliminate the negative impact of xylan hydrolysis products on the production of D-allulose, NH4Cl treatment was employed to thoroughly remove xylan from corn stover. By using 0.6 M NH4Cl to treat the corn stover at 160 °C for 1 h, 97.2% of xylan was removed. After the pretreatment, 92% of cellulose in the corn stover was degraded into glucose by CTec2 and 17.9% (close to the theoretical conversion rate) of glucose was further transformed into D-allulose by D-Tagatose 3-epimerase and D-glucose isomerase. After separation and purification through MCK-35 calcium ion resin, followed by freeze-drying, the obtained solid D-allulose had a purity of 99.4%. This study establishes a low-cost and affordable source for D-allulose production, while simultaneously presenting a new direction for enhancing the high-value utilization of lignocellulosic biomass.

Transporter mining and metabolic engineering of Escherichia coli for high-level D-allulose production from D-fructose by thermo-swing fermentation.

D-Allulose is an ultra-low-calorie sweetener with broad market prospects in the fields of food, beverage, health care, and medicine. The fermentative synthesis of D-allulose is still under development and considered as an ideal route to replace enzymatic approaches for large-scale production of D-allulose in the future. Generally, D-allulose is synthesized from D-fructose through Izumoring epimerization. This biological reaction is reversible, and a high temperature is beneficial to the conversion of D-fructose. Mild cell growth conditions seriously limit the efficiency of producing D-allulose through fermentation. FryABC permease was identified to be responsible for the transport of D-allulose in Escherichia coli by comparative transcriptomic analysis. A cell factory was then developed by expression of ptsG-F, dpe, and deletion of fryA, fruA, manXYZ, mak, and galE. The results show that the newly engineered E. coli was able to produce 32.33±1.33gL-1 of D-allulose through a unique thermo-swing fermentation process, with a yield of 0.94±0.01gg-1 on D-fructose.

Surface activation of hierarchically porous diatomite modified with polyethyleneimine for immobilizing d-allulose 3-epimerase

d-Allulose, a low-calorie functional sweetener popular among the obese population, can be biosynthesized using d-allulose 3-epimerase (DAE). However, there are currently no commercially viable immobilized DAE enzymes on the market for the large-scale production of d-Allulose. In this study, immobilizing the DAE enzyme was developed based on polymer-activated diatomite with surface enhancement using crosslinkers. Specifically, the amino groups of polyethyleneimine were successfully carried on the porous diatomite and then activated by glutaraldehyde to form a biopolymer coating. The obtained carrier was firmly covalently linked to the DAE enzyme via the Schiff-base reaction. The constructed immobilized DAE with a “core-shell” structure showed a high specific activity of 2088.93 ± 9.85 U/g and an excellent conversion rate of 34.4%. This biocatalyst was stable within a wide temperature and pH range. It also showed outstanding thermal stability, the half-lives of the immobilized DAE were 109 and 124 times that of the free DAE at 55 °C and 60 °C, respectively. Moreover, the immobilized DAE remained at about 81.33% of the initial enzyme activity after 8 recycles. The results of this study may contribute to the cost reduction of d-Allulose industrial production and provide a potential immobilized enzyme strategy for future large-scale manufacturing of d-Allulose.

Establishment of the Biotransformation of D-Allulose and D-Allose Systems in Full-Red Jujube Monosaccharides.

In order to reduce sucrose content in jujube juice and prepare a jujube juice beverage rich in rare sugars, jujube juice was used as raw material for multienzyme catalysis in this study. The effects of single factors such as substrate, pH, DPE and L-RI addition ratio, enzyme treatment temperature, and metal ions on sucrose conversion and D-allulose formation in jujube juice were investigated. Changes in glucose, D-allulose, and D-allose contents in jujube juice before and after enzyme conversion were analyzed by high-performance liquid chromatography (HPLC). The results showed that 'Xiangfenmuzao' was more suitable for subsequent double enzyme coupling reactions in different varieties of jujube juice at different periods. Factors such as pH, DPE and L-RI enzyme ratio, temperature, and treatment time had significant effects on sucrose conversion and D-allulose production in 'Xiangfenmuzao' juice (p < 0.05). When the ratio of DPE and L-RI was 1:10, pH was 7.5, and the temperature was 60 °C for 7 h, the fructose content in the full-red stage jujube juice of 'Xiangfenmuzao' and 'Jinsixiaozao' decreased gradually, and the final yield was about 53%. The yield of D-allulose was about 29%, and the yield of D-allulose was about 17%. In this study, DPE and L-RI were used to treat whole red jujube juice, which could effectively reduce sucrose content in jujube juice and obtain a functional jujube juice beverage that is low in calories and rich in rare sugar.

The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production.

The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose.

Structural and Functional Features of Ketose-3-Epimerases and Their Use for D-Allulose Production

Structural and Functional Features of Ketose-3-Epimerases and Their Use for D-Allulose Production