Rare Ketohexose Research Articles

Synthesis of natural/13C-enriched d-tagatose from natural/13C-enriched d-fructose

A concise, easily scalable synthesis of a rare ketohexose, d-tagatose, was developed, that is compatible with the preparation of d-[UL-13C6]tagatose. Epimerization of the widely available and inexpensive ketohexose d-fructose at the C-4 position via an oxidation/reduction (Dess-Martin periodinane/NaBH4) was a key step in the synthesis. Overall, fully protected natural d-tagatose (3.21 g) was prepared from d-fructose (9 g) on a 50 mmol scale in 23% overall yield, after five steps and two chromatographic purifications. d-[UL-13C6]Tagatose (92 mg) was prepared from d-[UL-13C6]fructose (465 mg, 2.5 mmol) in 16% overall yield after six steps and four chromatographic purifications.

High resolution and high throughput analytical methods for d-tagatose and process related impurities using capillary electrophoresis

d-tagatose is a low calorie multifunctional rare ketohexose sugar with sweetness similar to that of sucrose and it has high potential benefits for food and pharmaceutical industries. It is found in traces in some fruits as a natural component. In view of its high demand as a substitute for sugar, mass production of d-tagatose through enzymatic conversion of Lactose to d-tagatose is adopted. The existing HPLC method has limitations with respect sensitivity and resolution in quantification and monitoring of d-tagatose in the presence of its process related impurities. In the present investigation a new robust, fast and green analytical technique has been developed based on capillary electrophoresis (CE) for the separation and quantification of d-tagatose in presence of other sugars: Lactose, d-glucose, d-galactose and d-talose. Optimum conditions are found to be: Back Ground Electrolyte (BGE): 36 mM of Na2HPO4 and 130 mM of NaOH; pH: 12.6; voltage: +18 kV for high resolution and −18 kV for high throughput methods with direct UV-Detector at 265 nm. At these optimum conditions, good separation between the sugars is achieved in less than 20 min for high resolution and less than 4 min for high throughput methods. The developed methodology is validated as per ICHQ2R1 guide lines and successfully applied for monitoring d-tagatose during the enzymatic conversion of Lactose/d-galactose to d-tagatose and also to determine the unknown amounts of d-tagatose in crystallized samples and further, it is used in identifying the d-tagatose in fruits.

One-Pot Multienzyme Synthesis of Rare Ketoses from Glycerol.

A facile approach is introduced here for the synthesis of rare ketoses from glycerol and d-/l-glyceraldehyde (d-/l-GA). The reactions were carried out in a one-pot multienzyme fashion in which the only carbon source is glycerol. In the enzymatic cascade, glycerol is phosphorylated and then oxidized at C2 to afford dihydroxyacetone phosphate (DHAP), the key donor for enzymatic aldol reaction. Meanwhile, the primary alcohol of glycerol is also oxidized to give the acceptor molecule GA in situ (d- or l-isomer could be formed stereospecifically with either alditol oxidase or horse liver alcohol dehydrogenase). Different DHAP-dependent aldolases were used to generate the aldol adducts (rare ketohexose phosphates) with various stereoconfigurations and diastereomeric ratios. It is worth noting that the enzyme that catalyzes the phosphorylation reaction in the first step could also help recycle the phosphate in the last step to provide free rare sugar molecules. This study provides a useful method for rare ketose synthesis on a 100 mg to g scale, starting from relatively inexpensive materials which solved the problem of supplying both glycerol 3-phosphate and GA in our previous work. It also demonstrates an example of green synthesis due to highly efficient carbon usage and recycling of cofactors.

Biosynthesis of dendroketose from different carbon sources using in vitro and in vivo metabolic engineering strategies

BackgroundAsymmetric aldol-type C–C bond formation with ketones used as electrophilic receptor remains a challenging reaction for aldolases as biocatalysts. To date, only one kind of dihydroxyacetone phosphate (DHAP)-dependent aldolases has been discovered and applied to synthesize branched-chain sugars directly using DHAP and dihydroxyacetone (DHA) as substrate. However, the unstable and high-cost properties of DHAP limit large-scale application. Therefore, biosynthesis of branched-chain sugar from low-cost and abundant carbon sources is essential.ResultsThe detailed catalytic property of l-rhamnulose-1-phosphate aldolase (RhaD) and l-fuculose-1-phosphate aldolase (FucA) from Escherichia coli in catalyzing the aldol reactions with DHA as electrophilic receptors was characterized. Furthermore, we calculated the Bürgi–Dunitz trajectory using molecular dynamics simulations, thereby revealing the original sources of the catalytic efficiency of RhaD and FucA. A multi-enzyme reaction system composed of formolase, DHA kinase, RhaD, fructose-1-phosphatase, and polyphosphate kinase was constructed to in vitro produce dendroketose, a branched-chain sugar, from one-carbon formaldehyde. The conversion rate reached 86% through employing a one-pot, two-stage reaction process. Moreover, we constructed two artificial pathways in Corynebacterium glutamicum to obtain this product in vivo starting from glucose or glycerol. Fermentation with glycerol as feedstock produced 6.4 g/L dendroketose with a yield of 0.45 mol/mol glycerol, representing 90% of the maximum theoretical value. Additionally, the dendroketose production reached 36.3 g/L with a yield of 0.46 mol/mol glucose when glucose served as the sole carbon resource.ConclusionsThe detailed enzyme kinetics data of the two DHAP-dependent aldolases with DHA as electrophilic receptors were presented in this study. In addition, insights into this catalytic property were given via in silico simulations. Moreover, the cost-effective synthesis of dendroketose starting from one-, three-, and six-carbon resources was achieved through in vivo and in vitro metabolic engineering strategies. This rare branched-chain ketohexose may serve as precursor to prepare 4-hydroxymethylfurfural and branched-chain alkanes using chemical method.

Improved rheological properties of chicken egg frozen gels fortified by ᴅ-ketohexoses

The aim of this study was to examine the rheological properties of chicken egg gels containing 10% ᴅ-ketohexose (ᴅ-fructose, ᴅ-allulose, ᴅ-tagatose, and ᴅ-sorbose) and to compare the effects of ketohexoses on freeze-thawing of egg gels with ᴅ-glucose (Glc), sucrose (Suc), and trehalose (Tre). In both whole egg (WE) and egg white (EW) gels, ketohexose-gels had higher breaking stress than Glc-, Suc-, and Tre-gel (WE gels, 15.6–35.3%; EW gels, 7.6–26.1%). The ketohexose-gels of WE showed similar viscoelastic properties and water-holding capacity to Glc-, Suc-, and Tre-gel. Ketohexose-gels of EW possessed higher viscosity and higher water-holding capacity than the other sugar-gels. Ketohexoses more effectively suppressed the syneresis and physical properties of egg gels by freeze-thawing than Glc, Suc, and Tre did. The application of ketohexoses to egg gel products can improve the quality of frozen foods that contain egg gels. Practical applications Sugar is extensively used for producing egg-based gel products because sugar contributes to the stabilization of food structure and adds sweetness. Sugar also prevents deterioration of the physical properties of frozen foods from freezing and thawing. Interest in the rare ketohexoses ᴅ-allulose (Alu) and ᴅ-tagatose (Tag) is growing in food industry, because Alu and Tag have some health benefits, such as prevention of postprandial blood glucose level and suppression of adiposity. The utilization of rare ketohexoses as food ingredients should improve the rheological and functional properties of food products, including those of frozen foods.

Food properties of egg white protein modified by rare ketohexoses through Maillard reaction

Summaryd‐psicose (Psi), a rare ketohexose, improves food properties of proteins through the Maillard reaction (MR) much more than d‐fructose (Fru). This encouraged us to investigate the improvement of food properties of proteins through MR with various rare ketohexoses (d‐psicose, Psi; d‐tagatose, Tag; and d‐sorbose, Sor). The food properties of egg white protein (EWP) after reaction with Psi, Tag, Sor and Fru were studied. Psi‐EWP (43.3 μmol TE g−1) and Tag‐EWP (43.4 μmol TE g−1) had the highest antioxidant activity, followed by Sor‐EWP (38.4 μmol TE g−1) and Fru‐EWP (38.9 μmol TE g−1). MR enhanced the breaking stress of heat‐induced EWP gels by 224–267%. The breaking stress was higher in Psi‐EWP (81.1 kN m−2), Tag‐EWP (80.0 kN m−2) and Sor‐EWP (77.8 kN m−2) than in Fru‐EWP (71.6 kN m−2). Furthermore, the foaming capacity was highest in Psi‐EWP and Sor‐EWP. Rare ketohexoses improved food properties of EWP more than Fru. Overall, Psi offered the greatest functional improvement.

Biosynthesis of rare hexoses using microorganisms and related enzymes.

Rare sugars, referred to as monosaccharides and their derivatives that rarely exist in nature, can be applied in many areas ranging from foodstuffs to pharmaceutical and nutrition industry, or as starting materials for various natural products and drug candidates. Unfortunately, an important factor restricting the utilization of rare sugars is their limited availability, resulting from limited synthetic methods. Nowadays, microbial and enzymatic transformations have become a very powerful tool in this field. This article reviews the biosynthesis and enzymatic production of rare ketohexoses, aldohexoses and sugar alcohols (hexitols), including D-tagatose, D-psicose, D-sorbose, L-tagatose, L-fructose, 1-deoxy-L-fructose, D-allose, L-glucose, L-talose, D-gulose, L-galactose, L-fucose, allitol, D-talitol, and L-sorbitol. New systems and robust catalysts resulting from advancements in genomics and bioengineering are also discussed.

Identification and characterization of a novel l-arabinose isomerase from Anoxybacillus flavithermus useful in d-tagatose production

D-Tagatose is a highly functional rare ketohexose and many attempts have been made to convert D-galactose into the valuable D-tagatose using L-arabinose isomerase (L-AI). In this study, a thermophilic strain possessing L-AI gene was isolated from hot spring sludge and identified as Anoxybacillus flavithermus based on its physio-biochemical characterization and phylogenetic analysis of its 16s rRNA gene. Furthermore, the gene encoding L-AI from A. flavithermus (AFAI) was cloned and expressed at a high level in E. coli BL21(DE3). L-AI had a molecular weight of 55,876 Da, an optimum pH of 10.5 and temperature of 95°C. The results showed that the conversion equilibrium shifted to more D-tagatose from D-galactose by raising the reaction temperatures and adding borate. A 60% conversion of D-galactose to D-tagatose was observed at an isomerization temperature of 95°C with borate. The catalytic efficiency (k (cat) /K (m)) for D-galactose with borate was 9.47 mM(-1) min(-1), twice as much as that without borate. Our results indicate that AFAI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for D-galactose, suggesting its great potential for producing D-tagatose.

Characteristics and Antioxidant Activity of Maillard Reaction Products from Psicose‐Lysine and Fructose‐Lysine Model Systems

D-Psicose, an epimer of D-fructose isomerized at C-3 position, is a rare ketohexose that is thought to be beneficial for obese people and diabetic patients as a noncaloric sweetener. In the present study, model Maillard reaction products were obtained from D-psicose (or D-fructose) and L-lysine heating at 120 °C up to 8 h with the initial pH 9.0. The changes in pH, UV-vis absorbance, and free amino groups during the reaction were detected. Moreover, the antioxidant potential of the Maillard reaction products at different intervals was investigated. Although there was almost no difference in the oxygen radical absorbance capacity, the Maillard reaction products from psicose performed better than that from fructose in the radical-scavenging activity of 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 1, 1,-diphenyl-2-picryl-hydrazyl. The reducing power of the Maillard reaction products from psicose was also stronger than that from fructose. These results indicated that psicose played an effective role in the Maillard reaction and its Maillard reaction products could act as potential antioxidants in food industry.

A comprehensive HPLC analytical system for the identification and quantification of hexoses that employs 2-aminobenzamide coupling

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural distributions and biological functions remain to be clarified. To establish a robust analytical system that can separate, identify and quantify rare sugars, 12 d-hexoses-including five rare aldohexoses and three rare ketohexoses-were labelled with 2-aminobenzamide (2-AB), and the fluorescently tagged monosaccharides were separated by high-performance liquid chromatography (HPLC). Because the ketohexoses were much less reactive than were the aldohexoses, the reaction conditions were optimized to achieve the maximum yields (>75%) for both aldohexoses and ketohexoses. The calibration curve determined for the rare ketohexose, d-psicose (Psi), was linear between 1 pmol and 1 micromol and had a correlation coefficient of 0.9999. Using the developed method, the psicose content in a leaf of Itea virginica, in which the presence of psicose has previously been reported, was found to be 2.7 mg psicose/g leaf. The result proved feasibility of the method even for natural products. Because the system is a comprehensive tool, it should help answer questions concerning the biosyntheses and functions of rare hexose sugars.

Preventive effect of d-psicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury in rat

To investigate the preventive effects of d-psicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury, prepubertal male Sprague–Dawley rats were exposed to DEHP via their diet or orally, while under treatment with d-psicose. The rats given a diet-containing 1% DEHP alone for 7–14 days showed severe testicular atrophy accompanied by aspermatogenesis. On the other hand, those given the diet plus 2% but not 1% d-psicose-supplemented water for 14 days did not develop testicular atrophy, and exhibited an almost complete spermatogenesis. There was no significant difference in plasma mono-(2-ethylhexyl) phthalate (MEHP) levels between the d-psicose-free and d-psicose-treated groups. The testicular malondialdehyde (MDA) level after a single oral administration of 2 g/kg of DEHP showed a similar pattern of increase to the plasma MEHP level and peaked in 24 h suggesting a close and dose-dependent relation between plasma MEHP and testicular reactive oxygen species (ROS) levels. Pretreatment with d-psicose at a concentration of 2% and 4% resulted in an almost complete but not absolute suppression of testicular MDA production among rats administered 2 g/kg of DEHP. The microarray analysis showed the induction of oxidative stress related genes including the thioredoxin, glutathione peroxidase 1 and 2, glutaredoixn 1 after 24 h of the DEHP treatment in the testis. These results show that d-psicose prevents DEHP-induced testicular injury by suppressing the generation of ROS in the rat testis. This effect may be due to the direct scavenging by d-psicose of ROS generated in the testis.

Enzymatic procedures in the preparation of regioprotected d-fructose derivatives

A combination of different lipases from Pseudomonas cepacia, Candida antarctica B, Candida rugosa and Mucor miehei, aided the regioesterification of the free fructose allowing the synthesis of 1,6-di- O-acetyl- d-fructofuranose, 1,4,6-tri- O-acetyl- d-fructofuranose, 1,6-di- O-acetyl-4- O-benzoyl- d-fructofuranose and 1,6-di- O-benzoyl- d-fructofuranose. Using C. antarctica B and C. rugosa lipases the alcoholysis of fructose peracetate (α, β-form) has furnished 1,2,3,4-tetra- O-acetyl-α- d-fructofuranose and 2,3,4,6-tetra- O-acetyl-β- d-fructofuranose. 1,4,6-Tri- O-acetyl- d-fructofuranose was successfully employed to produce a rare ketohexose, namely d-psicose.

Antioxidative Activity and Gelling Rheological Properties of Dried Egg White Glycated with a Rare Keto‐hexose through the Maillard Reaction

ABSTRACT: The functional properties of egg white (EW) glycated with a rare D‐psicose and 2 alimentary sugars (D‐fructose and D‐glucose) were investigated. Glycation led not only to the formation of cross‐links but also to the formation of antioxidative substances to EW, the extent of which depended on the sugar used, modification, and incubation time. The temporal development of browning and fluorescence of psicose‐EW was relatively faster than those of fructose‐EW and glucose‐EW. The psicose‐EW showed the greater 2,3‐bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5carboxanilide (XTT) reducibility, 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid radical scavenging activity, and gelling rheological properties. These results indicated that psicose‐EW as a food ingredient might have the ability to improve gelling behavior and decrease oxidation reactions during food processing and storage.

Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties.

The effect of nonenzymatic glycation on the structural changes and gelling properties of hen ovalbumin (OVA) through the Maillard reaction was studied. OVA was incubated at the dry state with a rare ketohexose (D-psicose, Psi) and two alimentary sugars (D-fructose, Fru; D-glucose, Glc) at 55 degrees C and 65% relative humidity. To evaluate the modification of OVA by different reducing sugars during the glycation process, the extent of the Maillard reaction, aggregation processes, structural changes, and gelling behaviors were investigated. Reactivity of Psi with the protein amino groups was much lower than that of both Fru and Glc, whereas Psi induced production of browning and fluorescent substances more strongly than the two alimentary sugars did. Furthermore, OVA showed an increased tendency toward multimeric aggregation upon modifying with Psi through covalent bond. The modified OVAs with reducing sugar were similar to nonglycated control sample in Fourier transform infrared (FT-IR) characteristics, but significantly decreased in intensity of tryptophan-related fluorescence. The results indicate that although glycation brought about similar changes in the secondary structure without great disruption of native structure, its influence on the side chains of protein in tertiary structure could be different. Breaking strength of heat-induced glycated OVA gels with Psi was markedly enhanced by the Maillard reaction. These results suggest that Psi had a strong cross-linking activity with OVA; consequently, the glycated OVA with Psi could improve gelling properties under certain controlled conditions.

Bioproduction strategies for rare hexose sugars.

A new strategy for the bioproduction of all ketohexoses was developed using hexitols as intermediates. Biocatalysts used to employ the strategy were D-tagatose 3-epimerase, which epimerizes ketohexoses at the C-3 position, and oxidoreductases, which catalyze oxidation-reduction reactions between ketohexoses and the corresponding hexitols. Arranging all the ketohexoses and hexitols in a symmetric ring and connecting them with 20 biochemical reactions, I was able to construct a design for the bioproduction of all the rare ketohexoses. Various aldose isomerases transform ketohexoses into the corresponding aldohexoses, so the strategy is useful for the bioproduction of all the rare hexose sugars. Furthermore, the design revealed that there are four routes to the L-hexose world from the D-hexose one.

Production of l-psicose from allitol by Gluconobacter frateurii IFO 3254

A rare ketohexose, l-psicose, was produced from allitol by Gluconobacter frateurii IFO 3254. The transformation reaction was carried out at 30°C with shaking using the washed cells. The conversion rate was about 98% when 10% allitol was used. The cells grown on tryptic soy broth containing 1% glycerol were found to have the best conversion potential. Cells stored at −20°C for 10 d showed almost the same transformation activity as intact cells.