新型低热量甜味剂——D-阿洛酮糖的研究进展

寻找适合糖尿病人服用的甜味剂具有重要的经济价值。D-阿洛酮糖是D-果糖的C3差向异构体,是一种稀有糖。作为一种新型低热量甜味剂,D-阿洛酮糖近年来日益引起人们的重视。本文对国际上近年来发表的关于D-阿洛酮糖的药理活性资料进行了系统的总结。D-阿洛酮糖几乎不提供热量,具有显著的降血糖、降血脂效果,对糖尿病动物的胰岛β细胞有明显的保护作用。此外,D-阿洛酮糖无毒副作用,是一种食用安全的糖。这些研究结果说明:D-阿洛酮糖在预防和治疗糖尿病方面具有极大的潜力,可以作为糖尿病人食用的新型甜味剂,具有良好的市场前景。     

双酶偶联转化果糖制备含有稀少糖的混合糖液

酶转化法是功能性稀少糖生产的重要途径,但单一稀少糖转化酶的转化率普遍较低。文中提出构建双酶偶联转化系统提高转化效率的思路,即利用D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE)和L-鼠李糖异构酶(L-rhamnose isomerase,L-RhI)双酶偶联反应,催化D-果糖生成D-阿洛酮糖和D-阿洛糖等功能性稀少糖。DPE和L-RhI加酶量的比例为1∶10,其中DPE的浓度为0.05 mg/mL;转化反应的最佳温度为60℃,最适pH为9.0。当D-果糖浓度为2%时,反应10 h达到平衡,此时D-阿洛酮糖和D-阿洛糖的产量分别为5.12和2.04 g/L。利用文中提出的双酶偶联系统可以将果葡糖浆等富含果糖的低附加值原料转化为含有功能性稀少糖的高附加值混合糖液。     

以D-果糖为原料利用新型异构酶转化生产D-阿洛糖

稀少糖是自然界中含量稀少、化学合成困难的一类低热量单糖。D-阿洛糖是一种重要的稀少己醛糖,其具有减少活性自由基、抑制癌细胞增殖等独特的生理学功能。因此,以微生物发酵生产D-阿洛酮糖-3-差向异构酶(DPE)和L-鼠李糖异构酶(L-RhI)转化生产D-阿洛糖,成为近几年来国际研究的热点之一。文中分别克隆了来源于解纤维梭菌Clostridium cellulolyticum H10的DPE基因以及来源于枯草芽胞杆菌Bacillussubtilis 168的L-RhI基因,并分别使其在宿主菌B.subtilis及大肠杆菌Escherichia coli BL21(DE3)中得到了表达。进一步利用镍亲和层析和阴离子交换色谱等手段对这两种酶进行了纯化,并对这两种纯化后酶的转化能力进行了分析测定。结果表明,以D-果糖为原料利用两种异构酶依次转化获得D-阿洛酮糖及D-阿洛糖,其两步转化效率分别为27.34%和34.64%。     

生物法合成稀有己酮糖的研究进展

稀有糖是一类在自然界中存在但含量很低、同时具有重要生理功能的一类单糖及其衍生物,在膳食、保健、医药等领域中发挥着重要的作用。此外稀有糖还可以作为多种天然产物和药物的合成前体。然而稀有糖的合成成本较高,大大制约了其广泛应用。当前利用微生物和酶转化法合成稀有糖成为一种强有利的工具。综述了生物法合成稀有己酮糖(包括D-塔格糖、D-山梨糖、D-阿洛酮糖、L-塔格糖、L-果糖、L-山梨糖和1-脱氧-L-果糖等)的研究进展,探讨了稀有己酮糖合成策略的研究趋势。     

来源于瘤胃菌Ruminococcus sp.的D-阿洛酮糖3-差向异构酶的底物结合位点分析

【目的】研究来源于瘤胃菌Ruminococcus sp.的D-阿洛酮糖3-差向异构酶的底物结合机制。【方法】通过同源模拟和同源序列比对,筛选与其底物结合相关的关键位点,进而通过定点突变构建突变体并对其动力学性质进行研究。【结果】筛选得到关键位点Y6和A109,构建了突变体Y6F、Y6I、A109P及A109L。【结论】Y6既与底物结合又与催化能力相关,其-OH只与底物结合相关,芳香环则与催化能力和结合能力均相关;而A109则只是底物结合的位点。该研究结果为D-阿洛酮糖3-差向异构酶的催化机理研究及分子改造提供了借鉴。     

一种自溶芽孢杆菌环状糊精葡聚糖转移酶的提纯和性质,以及选择性形成β-环状糊精

环状糊精(CD)是一种带α-1,4键的均一的环状寡糖,通常由6—12个D-糖苦基残基组成。由6,7,8个D-糖苦基残基组成的CDs分别称做α-CD,β-CD和γ-CD。这些CDs可用于食品添加剂和制药剂等。它可以包装挥发性或不稳定物质,或形成稳定的包合配合物。     

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D-阿拉伯糖结晶影响因素分析

D-阿拉伯糖是多种功能性糖合成的中间体,其纯度高低决定了功能性糖转化率的高低,所以得到高纯度的D-阿拉伯糖尤为重要。通过对D-阿拉伯糖结晶温度、搅拌速度、结晶液中离子含量等因素进行试验,确定了采用梯度降温形式、搅拌速度控制在5 r/min,离子含量控制在100 μs/cm以下能够得到纯度达99.8%的D-阿拉伯糖晶体,实验结果为后续功能性糖的高效转化奠定了基础。     

利用代谢工程构建D-甘露醇生产菌株

D-甘露醇广泛应用于食品、制药、化学品工业等领域。从野生型大肠杆菌出发,将来自假肠膜明串珠菌Leuconostoc pseudomesenteroides ATCC 12291菌株的甘露醇脱氢酶与果糖转运蛋白编码基因整合到大肠杆菌ATCC 8739的染色体中,并失活其他的发酵途径 (丙酮酸甲酸裂解酶、乳酸脱氢酶、富马酸还原酶、乙醇脱氢酶、甲基乙二醛合成酶和丙酮酸氧化酶) ,构建了一株遗传稳定的D-甘露醇生产菌株。使用无机盐培养基和葡萄糖果糖作为混合碳源,厌氧发酵6 d,D-甘露醇产量达1.2 mmol/L。基于细胞生长和D-甘露醇合成的偶联,进一步通过代谢进化技术提高细胞合成D-甘露醇的生产能力。经过80代的驯化,D-甘露醇产量提高了2.6倍,甘露醇脱氢酶的活性提高了2.8倍。构建获得的遗传稳定的工程菌能直接发酵糖生产D-甘露醇,不需添加抗生素、诱导剂和甲酸,在工业化生产时有一定优势。     

产酮产碱菌E54生产D-异抗坏血酸钠工艺优化研究的新进展

D-异抗坏液酸钠是一种优良的食品抗氧化剂,工业上大多采用间接发酵法生产,即通过细菌发酵由D-葡萄糖生成前体——2-酮基-D-葡萄糖酸钙,再经甲酯化和化学转化得到D-异抗坏血酸钠。此项工艺虽已很成熟,但目前生产中发酵过程多采用假单孢菌,投糖浓度仅能达到16～18％,且存在着易为噬菌体和杂菌感染的不足。     

Recent advances on applications and biotechnological production of d-psicose

D-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of D-psicose. In addition, the biochemical properties of D-tagatose 3-epimerase family enzymes and the D-psicose-producing enzyme are compared, and the biotechnological production of D-psicose from D-fructose is reviewed.     

Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose

The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50 degrees C and pH 8.0 with Mn2+. The turnover number (k(cat)) and catalytic efficiency (k(cat)/Km) of the enzyme for D-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30 degrees C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50 degrees C after 100 min, corresponding to a conversion yield of 32.9%.     

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

D-Psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)₈ TIM barrel fold with a Mn²⁺ metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/ products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexosebound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.     

Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.     

Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T _m) and the urea midtransition concentration (C _m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T _m and C _m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.     

X-ray structures of the <Emphasis Type="Italic">Pseudomonas cichorii</Emphasis> D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.