What Is the Source of D Allulose？

The International Society for Rare Sugars (ISRS) defines rare sugars as monosaccharides and their derivatives that occur rarely in nature [1]. According to the ISRS definition, D-allulose is considered a rare sugar as a diastereoisomer at the C-3 position of D-fructose. D-allulose is a low-calorie sweetener. Taking a 100g/L sucrose solution as an example, D-allulose is 70% as sweet as sucrose [2], but only 0.  3% of the energy of sucrose[3]. At the same time, D-allulose has unique physiological functions. As an inhibitor of hepatic lipogenic enzymes and intestinal α-glucosidase[4], D-allulose is hardly metabolized and absorbed in the small intestine[5], which can further reduce postprandial hyperglycemia, improve insulin resistance and reduce the accumulation of body fat, which is of great benefit to both obesity and diabetes [6~7]. In addition, D-allulose has been declared “generally recognized as safe” (GRAS) by the US Food and Drug Administration (FDA) [8] and can be used in food and medicine.

D Allulose is a new rare monosaccharide that is safe for health. It has physiological properties such as reducing blood glucose response, reducing liver fat production, maintaining body weight, anti-oxidation and protecting blood vessels. D-Allulose is increasingly valued by researchers for its special nutritional and biological functions. Since the content of D-allulose in nature is very low, it is difficult to prepare it by chemical methods. At present, the major production method is the enantioselective isomerization between D-fructose and D-allulose[9~10]. The ketose 3-isomerase involved is a research hotspot, which can be obtained from different microorganisms. The most commonly used is the D-allulose 3-isomerase of Agrobacterium tumefaciens[11~12]  .

1  Research progress in production technology

1.  1  Chemical synthesis method

At first, D-allulose was synthesized by chemical methods. Bilik et al. [13] found that in an acidic aqueous solution, D-fructose can be converted to D-allulose under the catalytic action of molybdate ions.

In 1997, Donald [14] prepared D-allulose by chemically synthesizing 1,2:4,5-di-O-isopropylidene-β-D-fructopyranose. In addition, D-allo-ketonic acid can also be synthesized by boiling ethanol and triethylamine [15]. With the deepening of research, the chemical synthesis method has the disadvantages of high cost, dangerous operation, difficult process, complex purification, low yield, easy to cause environmental pollution, and its product safety needs to be studied. It is gradually being replaced by the bioconversion method.

1. 2    Bioconversion method

The bioconversion of D-allulose was first proposed by Professor Ken Izumori of Kagawa University in Japan. It uses hexitol as an intermediate to complete the bioconversion between rare hexoses, that is, the Izumoring bioconversion method [16]. It involves a diastereoisomerase (specific for diastereoisomerization of hydroxyl groups), a polyol dehydrogenase (catalyzing the reaction between ketose and sugar alcohol) and an aldopentose isomerase (aldopentose isomerization reaction) [17]. Among them, D-allulose 3-epimerase (DPE) can convert D-fructose and D-allulose.

1. 2. 1 D-Allulose 3-epimerase

D-Allulose 3-epimerase is the key enzyme for the conversion of D-fructose to D-allulose and is a member of the ketose 3-epimerase family of enzymes. In 1993, Izumori et al. [16] first isolated and purified a ketose isomerase from Pseudomonas cichorii. Its optimal product is D-tagatose, so it was named D-tagatose 3-epimerase (DTE).  DTE). In addition, Kim et al. [18] discovered an enzyme in Agrobacterium tumefaciens str. C58 that specifically catalyzes the conversion of D-fructose to D-allulose with a conversion rate of 32.9%. It was named D- allulose 3-epimerase. In recent years, D-allulose 3-epimerases from different microbial sources have been gradually discovered (see Table 1), and their properties have been studied.

1. 2. 2 Properties of D-allulose 3-epimerase

The properties of D-allulose 3-epimerase vary depending on the microbial source. As can be seen from Table 1, the optimum temperature for most D-allulose 3-epimerases is 50-70°C, and the optimum pH is 7.0-8.0. The optimum pH for the D-tagatose 3-epimerase of the Rhodobacteraceae is 9.0, while the optimum pH for the D-allulose 3-epimerase of the genus Doria is isomerase has an optimum pH of 6.0. In addition, the activity of the enzyme can be effectively increased by the addition of metal ions [27], and the optimum metal ion for D-allulose 3-epimerase is Mn2+ or Co2+.

(1) Effect of temperature on D-allulose 3-epimerase

Thermal stability is important for the industrial production of D-allulose. Generally speaking, in the production of the sugar industry, suitable high temperatures can improve the utilization rate of raw materials and the rate of bioconversion, reduce the viscosity of the solution, increase the solubility of reactants and products, and further increase the yield [28]. However, the heat stability of D-allulose 3-epimerase is poor, and its half-life is short [29], which restricts industrial production. Therefore, improving the heat stability of D-allulose 3-epimerase is necessary for the industrial production of allulose. Among these, random mutagenesis and rational protein design are typical methods for improving the thermal stability of enzymes in the field of protein engineering [30–32].

Choi et al. [19] used error-prone polymerase chain reaction (Error-prone PCR) and site-directed mutagenesis to obtain mutant strains S213C, I33L and I33LS213C of D-allulose 3-epimerase from Agrobacterium tumefaciens. Compared with the wild-type D-allulose 3-epimerase, the optimum temperature for the enzymatic activity of mutant strains S213C, I33L and I33LS213C increased by 2.5 °C, 5 °C and 7.5 °C, respectively, and the half-life at 50 °C increased by 3.3, 7.2 and 29. 9 times, and the apparent melting temperature increased by 3. 1℃, 4. 3℃ and 7. 6℃. The results showed that the thermal stability of the mutant strains of D-allulose 3-epimerase was significantly improved, among which the I33LS213C mutant strain may have the potential to produce D-allulose.

Meanwhile, Zhang et al. [33] obtained the Y68I mutant and G109P mutant by site-directed mutagenesis at tyrosine 68 and glycine 109 of the D-allulose 3-epimerase of Bacillus subtilis, respectively. Compared with the wild-type D-allulose 3-epimerase, the Y68I mutant showed the highest substrate binding affinity and catalytic efficiency, while the G109P mutant showed the highest thermal stability. In addition, a double-site Y68I/G109P mutant was also generated and showed good enzyme properties, as evidenced by: a 17.9% increase in the Michaelis constant (Km), a 1.2-fold increase in catalytic efficiency (Kcat/Km), and an increase in the half-life at 55°C from 156 min to 260 min, and the apparent melting temperature increased by 2.4 °C. This indicates that the Y68I/G109P mutant is suitable for the industrial production of D-allulose.

(2) Effect of pH on D-allulose 3-epimerase

The optimum pH value of D-allulose 3-epimerase is 7.0–9.0, which is in the alkaline range. However, production in the sugar industry is carried out under acidic conditions, as acidic conditions can reduce the formation of by-products and the browning reaction [34–35]. Therefore, the reaction pH of D-allulose 3-epimerase is not ideal for the bioconversion needs of the monosaccharide industry. The optimal reaction pH of the enzyme needs to be improved through genetic engineering in order to obtain a better product.

(3) The effect of metal ions on D-allulose 3-epimerase

Metal ions have a certain effect on D-allulose 3-epimerase. As can be seen from Table 1, the optimum metal ion for D-allulose 3-epimerase from Clostridium botulinum, D-allulose 3-epimerase from Clostridium cellulovorans, D-allulose 3-epimerase from Clostridium butyricum, D-allulose 3-epimerase from Doria formosa, and D-allulose 3-epimerase from Clostridium tricornutum is Co2+. isomerase's optimum metal ion is Co2+. The optimum metal ion for the D-allulose 3-epimerase from Agrobacterium tumefaciens, the D-tagatose 3-epimerase from Pseudomonas cepacia ST-24, the D-tagatose 3-epimerase from Sphingobium sp. and the D-allulose 3-epimerase from Streptococcus ruminantium is Mn2+.

For the D-tagatose 3-dehydrogenase from Burkholderia cepacia, Itoh et al. [17] showed that the activity does not require the assistance of metal ions, but the addition of metal ions, especially Mn2+, significantly increases the activity. In particular, Clostridium D-allo-hexose 3-epimerase has a strict metal ion dependence, requiring metal ions as cofactors to display activity. In the absence of ions, it is almost inactive, and it shows maximum activity in the presence of Co2+ [36]. In addition, it was found that the cellulase D-allulose 3-epimerase has extremely high thermal stability in the presence of Co2+ [36].

Patel et al. [37] used the yeast homologous protein Smt3 for N-terminal fusion to obtain Smt3 D-allulose-3 isomerase, and under the optimal reaction conditions, investigated the catalytic activity of divalent metal ions on Smt3 D-allulose-3 isomerase. The results showed that the activity of the enzyme was almost lost in the presence of Zn2+, Cu2+, and Ni2+. Ca2+ had a significant inhibitory effect on its activity, while Mg2+, Fe2+, and Ba2+ did not change the activity. On the contrary, Mn2+ and Co2+ could significantly increase the activity of the enzyme , even if the amount of Mn2+ in the assay reaction (0.025–0.1 mmol/L) is very low. Jia et al. [24] studied the effects of metal ions on Clostridium botulinum D-allo-keto-glucose 3-epimerase. The results showed that EDTA completely inhibited the activity of D-allo-keto-glucose 3-epimerase, and Zn2+, Mg2+, and Cu2+ inhibited part of the enzyme activity. In contrast, Co2+ and Mn2+ significantly increased the activity of the enzyme, especially Co 2+ can greatly enhance the activity of D-allulose 3-epimerase.

(4) The influence of other factors on D-allulose 3-epimerase

Except for the D-tagatose 3-epimerase of Pseudomonas citrea ST-24 and the D-tagatose 3-epimerase of Sphingobium sp. which have D-tagatose and D-fructose as their optimal products, respectively, most of the other allulose 3-epimerases have D-allulose as their optimal product. The equilibrium conversion rate between D-allulose and D-fructose is between 28% and 33% [38].

In addition, Kim et al. [39] showed that D-allulose has a high complexation ability with borate, which helps D-fructose further produce D-allulose. Lim et al. [40] used immobilized D-allulose 3-epimerase as a raw material to stably and highly produce D-allulose in the presence of borate. allulose. The main mechanism may be that the borate reacts with the carbohydrate to form a complex, and the complex interacts with the enzyme system to change the equilibrium of any reaction involving cis-diol carbohydrates through the difference in the binding affinity of the sugar, thereby achieving a high conversion rate [41~42].

2 Conclusion

At present, the main industrial enzyme used to produce D-allulose is D-allulose 3-epimerase, which has a high affinity and conversion rate for the substrate D-fructose. In order to further increase the yield of D-allulose, some researchers have used genetic engineering techniques to obtain D-allulose 3-epimerase with a higher conversion rate. Therefore, the safety of enzyme expression and secretion in microbial hosts needs to be further studied to avoid potential food safety problems. With the improvement of people's living standards and health awareness, as well as the deepening of experimental research, D-allulose will have a broader development prospect.

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