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Enzymatic preparation of rare sugars as an alternative to traditional sweeteners is an effective strategy to achieve a low-calorie healthy diet. Ribose-5-phosphate isomerase B (RpiB) is a key enzyme in the non-oxidative branch of the catalytic pentose phosphate pathway. Here, we investigated the potential of Curtobacterium flaccumfaciens ZXL1 (C. flaccumfaciens ZXL1) derived RpiB (CfRpiB) in D-allose preparation. The optimal reaction conditions for recombinant CfRpiB were found experimentally to be pH 7.0, 55 °C, and no metal ions. The kinetic parameters Km, kcat, and catalytic efficiency kcat/Km were 320 mM, 4769 s−1, and 14.9 mM−1 s−1 respectively. The conversion of D-allulose by purified enzyme (1 g L−1 ) to D-allose was 13% within 1 h. In addition, homology modeling and molecular docking were used to predict the active site residues: Asp13, Asp14, Cys72, Gly73, Thr74, Gly77, Asn106, and Lys144. [ABSTRACT FROM AUTHOR]

l -Rhamnose isomerase (l -RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l -RhI (Ts RhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R Ts RhIs can increase the preference against d -allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R Ts RhIs against d -allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l -rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R Ts RhIs were noted to have the altered substrate specificity, and I102Q Ts RhI has the highest catalytic efficiency against d -allose presumably through the formation of an additional hydrogen bond with d -allose. The purified wild-type and mutant Ts RhIs were further used to produce d -allose from 100 g/L d -fructose in the presence of d -allulose 3-epimerase, and the yields can reach as high as 22 % d -allulose and 12 % d -allose upon equilibrium. I102Q Ts RhI takes only around half of the time to reach the same 12 % d -allose yield, suggesting that this mutant enzyme has a potential to be applied in d -allose production. • Residue Ile102 of Ts RhI was changed to other polar or charged residues. • Mutant I102N, I102Q, and I102R Ts RhIs have the altered substrate specificity. • I102Q Ts RhI might form an additional hydrogen bond with d -allose. • I102Q Ts RhI takes around half time to reach equilibrium for d -allose production. • I102Q Ts RhI has a potential to be used in d -allose production. [ABSTRACT FROM AUTHOR]

A putative l-rhamnose isomerase (l-RI) gene from Paenibacillus baekrokdamisoli was expressed as a recombinant enzyme in Escherichia coli BL21(DE3) and characterized as a producer of d-allose from d-allulose. Recombinant l-RI from P. baekrokdamisoli was homogeneously purified on SDS-PAGE with a 46 kDa molecular mass and specific activity of 1.27 U/mg by His-Trap affinity chromatography. The enzyme was estimated to be a tetramer in enzyme active form because its molecular mass was determined to be approximately 190 kDa by Gelfiltration chromatography. In the isomerization reaction between d-allose and d-allulose, recombinant l-RI exhibited the highest activity at pH 8.0 and 60°C in the presence of 0.5 mM Mn2+. The half-lives of the enzyme at 50, 55, 60, 65, 70, and 75°C were 417, 57, 27, 20, 3.3, and 0.2 h, respectively. The Michaelis-Menten constants (Km), turnover numbers (kcat), and catalytic efficiencies (kcat/Km) of the enzyme for d-allose and d-allulose were 33 mM, 13.79 s−1, and 0.4 mM−1s−1 and 45.24 mM, 6.58 s−1, and 0.14 mM−1s−1, respectively. The enzyme showed isomerization activity for aldoses with right-handed configuration of hydroxyl group at the C-2 and C-3 positions, such as l-mannose, l-lyxose, d-gulose, d-allose, and d-ribose. During production of d-allose from d-allulose, the enzyme produced 125 g/L of d-allose from 500 g/L of d-allulose in 3 h with 41.6 g/L/h productivity with 104 U/mL enzyme. We first reported the l-RI from the Paenibacillus genus, and the results suggested that the P. baekrokdamisolil-RI could be applied as a d-allose producer. [ABSTRACT FROM AUTHOR]

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

D-Allose and D-allulose are two important rare natural monosaccharides found in meager amounts. They are considered to be the ideal substitutes for table sugar (sucrose) for, their significantly lower calorie content with around 80 % and 70 % of the sweetness of sucrose, respectively. Additionally, both monosaccharides have gained much attention due to their remarkable physiological properties and excellent health benefits. Nevertheless, D-allose and D-allulose are rare in nature and difficult to produce by chemical methods. Consequently, scientists are exploring bioconversion methods to convert D-allulose into D-allose, with a key enzyme, L-rhamnose isomerase (L-RhIse), playing a remarkable role in this process. This review provides an in-depth analysis of the extractions, physiological functions and applications of D-allose from D-allulose. Specifically, it provides a detailed description of all documented L-RhIse, encompassing their biochemical properties including, pH, temperature, stabilities, half-lives, metal ion dependence, molecular weight, kinetic parameters, specific activities and specificities of the substrates, conversion ratio, crystal structure, catalytic mechanism as well as their wide-ranging applications across diverse fields. So far, L-RhIses have been discovered and characterized experimentally by numerous mesophilic and thermophilic bacteria. Furthermore, the crystal forms of L-RhIses from E. coli and Stutzerimonas/Pseudomonas stutzeri have been previously cracked, together with their catalytic mechanism. However, there is room for further exploration, particularly the molecular modification of L-RhIse for enhancing its catalytic performance and thermostability through the directed evolution or site-directed mutagenesis. • D-Allose and D-allulose are rare sugars and isomers of each other. • D-Allose and D-allulose have great importance in the field of food and pharmaceutical. • L-Rhamnose isomerase can catalyze various aldoses into their respective ketoses. • L-Rhamnose isomerase can produce high amount of D-allose by using D-allulose as a substrate. • L-Rhamnose isomerase can also catalyze the reversible isomerization between many other rare sugars. [ABSTRACT FROM AUTHOR]