Efficient conversion of allitol to D-psicose by Bacillus pallidus Y25

An efficient method for conversion of allitol to D-psicose was achieved by a resting cell reaction of Bacillus pallidus Y25 for the first time. Notably, it was possible to produce D-allose and D-altrose from allitol directly via D-psicose by prolonging the reaction time. This method was applied for the preparation of D-psicose using the extract of Itea virginica as a starting material in this study. D-Psicose which is the absolutely key precursor for the production of other six carbon sugars could be obtained as the sole product at high yield.     

A novel inhibitory effect of D-allose on production of reactive oxygen species from neutrophils

The rare sugar D-allose produced from D-psicose using an immobilized L-rhamnose isomerase bioreactor was shown to have weak scavenging activity toward reactive oxygen species (ROS) and potent inhibitory effect on production of ROS from stimulated neutrophils. These findings may have important implications in understanding the ameliorative effect of D-allose in transplantation and ischemia/reperfusion injury.     

Mass production of D-psicose from d-fructose by a continuous bioreactor system using immobilized D-tagatose 3-epimerase

An improved process for the mass production of D-psicose from D-fructose was developed. A D-fructose solution (60%, pH 7.0) was passed at 45 degrees C through a column filled with immobilized D-tagatose 3-epimerase (D-TE) which was produced using recombinant Escherichia coli, and 25% of the substrate was converted to D-psicose. After epimerization, the substrate, D-fructose, was removed by treatment with baker's yeast. The supernatant was concentrated to a syrup by evaporation under vacuum and D-psicose was crystallized with ethanol. Approximately 20 kg of pure crystal D-psicose was obtained in 60 d.     

利用微生物酶法生产D-阿洛糖

近年来,D-阿洛糖由于其具有的许多药物活性（包括抗癌、抗肿瘤、抗炎、抗氧化、抗高血压、冷冻保护和免疫抑制等活性）,吸引了社会的极大关注.D-阿洛糖已经可以通过D-阿洛糖生产酶转化D-阿洛酮糖得到,常用的D-阿洛糖生产酶包括L-鼠李糖异构酶、核糖-5-磷酸异构酶和半乳糖-6-磷酸异构酶.描述了D-阿洛糖的性质和应用,回顾了D-阿洛糖生产酶的生物化学特性.此外还对几种D-阿洛糖生产酶的D-阿洛糖产物进行了比较.     

Direct production of allitol from D-fructose by a coupling reaction using D-tagatose 3-epimerase, ribitol dehydrogenase and formate dehydrogenase

Allitol was produced from D-fructose via a new NADH-regenerating enzymatic reaction system using D-tagatose 3-epimerase (D-TE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH). D-fructose was epimerized to D-psicose by the D-TE of Pseudomonas cichorii ST-24 and the D-psicose was subsequently reduced to allitol by the RDH of an RDH-constitutive mutant, X-22, derived from Klebsiella pneumoniae IFO 3321. NADH regeneration for the reduction of D-psicose by the RDH was achieved by the irreversible formate dehydrogenase reaction, which allowed the D-psicose produced from d-fructose to be successively transformed to allitol with a production yield from D-fructose of almost 100%. The reactions progressed without any by-product formation. After separation of the product from the reaction mixture by a simple procedure, a crystal of allitol was obtained in a yield exceeding 90%. This crystal was characterized and determined to be allitol by HPLC analysis, its IR and NMR spectra, its melting point, and optical rotation measurement.     

Inhibitory effect of d-allose on neutrophil activation after rat renal ischemia/reperfusion

D-Allose is one of the rare sugars produced from D-psicose. We examined whether d-allose reduces the extent of rat renal ischemia/reperfusion (I/R) injury by suppressing the activation of neutrophils. The renal concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1 and myeloperoxidase were significantly increased after renal I/R. These increases were significantly inhibited by D-allose administration. Furthermore, D-allose significantly inhibited the increase in the concentrations of blood urea nitrogen (BUN), creatinine, N-acetyl beta-D-glucosaminidase (NAG) and histopathologic changes after renal I/R. These findings strongly suggest that D-allose protects against I/R-induced renal injury by inhibiting the activation of neutrophils that play an important role in I/R-induced renal injury. These findings may have important implications in understanding the biologic functions of D-allose. D-Allose may prove useful in renal surgery and transplantation.     

Improved microcirculatory effect of D-allose on hepatic ischemia reperfusion following partial hepatectomy in cirrhotic rat liver

D-allose, one of the rare sugars produced from D-psicose, has been shown to be effective against reperfusion injury after ischemia and partial hepatectomy in cirrhotic rat liver by improving remnant liver blood flow and survival rates, and decreasing liver enzyme levels and liver tissue injury levels. These findings demand further study of the clinical implications of this sugar in view to the advancing fields of liver surgery and transplantation.     

乙醇体系中D-阿洛酮糖的结晶工艺优化

为开发一套D-阿洛酮糖的结晶工艺,本文以乙醇为结晶体系,分别对影响结晶过程的4个主要因素(包括D-阿洛酮糖溶液的密度、乙醇与D-阿洛酮糖的比例、结晶时间和结晶温度)进行了单因素实验,并在单因素实验的基础上,采用响应曲面法对结晶的工艺参数进行优化。结果表明,最优操作条件为:D-阿洛酮糖溶液密度为1.35 g/mL,乙醇与D-阿洛酮糖溶液比例为3.8:1,结晶时间为325 min,结晶温度为25℃。在此条件下,D-阿洛酮糖的初次结晶收率可达71.58%。以上结果可以得出结论:乙醇体系中可获得较高的D-阿洛酮糖结晶收率,本研究获得的模型可以用来优化D-阿洛酮糖在乙醇体系中的结晶过程。     

Chitosan productivity enhancement in Rhizopus oryzae YPF-61A by D-psicose

The effect of the rare sugar D-psicose on chitosan production by Rhizopus oryzae was studied. The fungus was not able to utilize D-psicose as a sole source of carbon, either for germination of the spores or for growth of the vegetative cells. In a medium containing a low amount of D-glucose, however, D-psicose supplementation between 5 and 12 g/l caused enhancement of the productivity of chitinous substances, especially chitosan, in the cell walls. Substantial changes in the chitosan molecule were not observed from FT-IR or 1H NMR data. It was inferred that a percentage age of the D-psicose added was transformed into another rare sugar tentatively identified as D-tagatose. It was concluded that D-psicose activates the synthesis of chitosan in the cell walls probably through its transformation into another sugar such as D-tagatose.     

Clostridium bolteae ATCC BAA-613 D-塔格糖3-差向异构酶的诱导表达、纯化及活性研究

D-塔格糖3-差向异构酶是生物法生产新型功能性因子D-阿洛酮糖最为有效的酶。一种新型的能够编码D-塔格糖3-差向异构酶的基因CLOBOL₀0069被克隆,它来源于Clostridium bolteae ATCC BAA-613。以pUC57为克隆载体,以pET-22b（+）为载体质粒,E.coli BL21（DE3）为宿主细胞,构建了基因重组工程菌。IPTG诱导剂诱导目的蛋白的表达;通过镍柱亲和层析,杂蛋白与目的蛋白得到了很好的分离。对纯化的重组蛋白样品进行SDS-PAGE分析,在约32ku处出现明显的特征条带。通过活性研究表明,Clostridium bolteae ATCC BAA-613 DTEase属于DTEase家族,并具有较高的生物转化率,反应10h后转化率达到20%。     

生物转化生成D-阿洛酮糖的类球红细菌的筛选

对鱼塘淤泥、水样中富集分离的27株光合细菌的静止细胞反应产物,采用高效液相色谱进行分析,从中筛选到1株D-阿洛酮糖产率较高的菌株,在形态和常规生理生化方面鉴定的基础上,结合16S rDNA序列分析鉴定为类球红细菌(Rhodobacter sphaeroides),定名为类球红细菌SK011。SK011利用D-果糖(36 g/L,pH7.5)为底物进行静止细胞转化,45℃,5 h,D-阿洛酮糖产率达到6.54%。     

Metabolic engineering pathways for rare sugars biosynthesis,physiological functionalities,and applications—a review

ABSTRACT

Biomolecules like rare sugars and their derivatives are referred to as monosaccharides particularly uncommon in nature. Remarkably, many of them have various known physiological functions and biotechnological applications in cosmetics, nutrition, and pharmaceutical industries. Also, they can be exploited as starting materials for synthesizing fascinating natural bioproducts with significant biological activities. Regrettably, most of the rare sugars are quite expensive, and their synthetic chemical routes are both limited and economically unfeasible due to expensive raw materials. On the other hand, their production by enzymatic means often suffers from low space-time yields and high catalyst costs due to hasty enzyme denaturation/degradation. In this context, biosynthesis of rare sugars with industrial importance is receiving renowned scientific attention, across the globe. Moreover, the utilization of renewable resources as energy sources via microbial fermentation or microbial metabolic engineering has appeared a new tool. This article presents a comprehensive review of physiological functions and biotechnological applications of rare ketohexoses and aldohexoses, including D-psicose, D-tagatose, L-tagatose, D-sorbose, L-fructose, D-allose, L-glucose, D-gulose, L-talose, L-galactose, and L-fucose. Novel in-vivo recombination pathways based on aldolase and phosphatase for the biosynthesis of rare sugars, particularly D-psicose and D-sorbose using robust microbial strains are also deliberated.     

D-Psicose,a Sweet Monosaccharide,Ameliorate Hyperglycemia,and Dyslipidemia in C57BL/6J db/db Mice

ABSTRACT: D-psicose has been implicated in glycemic control in recent animal and human studies. In this study, the effects of D-psicose on glycemic responses, insulin release, and lipid profiles were compared with those of D-glucose and D-fructose in a genetic diabetes model. C57BL/6J db/db mice were orally supplemented with 200 mg/kg BW of D-psicose, D-glucose, or D-fructose, respectively, while diabetes control or wild type mice were supplemented with water instead. D-psicose sustained weight gain by about 10% compared to other groups. The initial blood glucose level maintained from 276 to 305 mg/dL during 28 d in the D-psicose group, whereas a 2-fold increase was found in other groups (P < 0.05) among diabetic mice. D-psicose significantly improved glucose tolerance and the areas under the curve (AUC) for glucose among diabetes (P < 0.05), but had no effect on serum insulin concentration. The plasma lipid profile was not changed by supplemental monosacchrides, although the ratio of LDL-cholesterol/HDL-cholesterol was ameliorated by D-psicose. The administration of D-psicose reversed hepatic concentrations of triglyceride (TG) and total cholesterol (TC) by 37.88% and 62.89%, respectively, compared to the diabetes control (P < 0.05). The current findings suggest that D-psicose shows promise as an antidiabetic and may have antidyslipidemic effects in type 2 diabetes.     

Clostridium scindens ATCC35704中的D-塔格糖-3-差向异构酶的克隆表达、纯化及活性研究

D-阿洛酮糖作为一种新型低热量功能性甜味剂,可以通过D-塔格糖-3-差向异构酶家族,以D-果糖为底物C-3位异构化得到。一个新的来源于微生物Clostridium scindens ATCC 35704中ZP 0243228的D-塔格糖-3-差向异构酶基因(CS-DTE),通过克隆并成功导入E.coli BL21(DE3)中,构建了基因重组菌,诱导目的重组基因过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE分析,在约31 ku处出现显著的特征蛋白条带;通过对其活性检测表明,该重组酶分别以D-塔格糖和D-果糖为底物,可以生成D-山梨糖和D-阿洛酮糖,转化率分别为8.6%和27.9%。     

弗吉尼亚鼠刺叶片中稀少糖醇生物转化及分离制备

为探讨使用鼠刺植物制备稀少糖醇的可能性,以弗吉尼亚鼠刺叶片为原料,热水提取其所含的稀少糖及糖醇,使用产酸克雷伯氏菌静息细胞对提取物的单糖类物质进行生物转化,再利用絮凝、离子树脂色谱分离以及乙醇结晶连用手段对转化的提取物进行纯化,最后使用核磁共振谱对获得的稀少糖醇进行分析鉴定。液相色谱分析结果显示弗吉尼亚鼠刺叶片提取物含有稀少D-阿洛酮糖和蒜糖醇,其质量分数分别为27.86%和7.86%;利用制备的产酸克雷伯氏菌的静息细胞进行60 h催化反应后,提取物中86%的D-阿洛酮糖转化为蒜糖醇,其余被细胞代谢消耗。多种分离纯化手段的联合使用,能有效去除杂质和色素,获得蒜糖醇纯品。     

Potential inhibitory effects of D-allose, a rare sugar, on liver preneoplastic lesion development in F344 rat medium-term bioassay

D-allose, the C-3 epimer of d-glucose, is a monosaccharide present in minute quantities in nature and a rare sugar. The effects of D-allose on diethyl nitrosamine (DEN)-induced hepatocarcinogenesis were examined in male F344 rats by a rat medium-term bioassay based on the two-step model of hepatocarcinogenesis (experiment 1). In addition, a DNA microarray analysis was employed to clarify possible mechanisms of action of D-allose (experiment 2). The antioxidation potential of D-allose solution itself or of serum in rats treated with D-allose was also examined directly by measuring Cu(+)-reducing antioxidation power (experiment 3). Furthermore, to investigate the effects of D-allose in vivo under conditions of oxidative stress, it was administered with a choline-deficient, L-amino acid-defined diet (CDAA) in the medium-term liver carcinogenesis bioassay (experiment 4). Experiment 1 demonstrated no effects of D-allose on the development of glutathione S-transferase placental form (GST-P) positive foci in the liver. From DNA microarray analysis, several mRNA markers were found to be altered with functions related to apoptosis and cell proliferation (experiment 2), although D-allose itself and serum in vivo exhibited no antioxidation power directly (experiment 3). When D-allose was administered with the CDAA diet, decreases in the area and number of GST-P positive foci were noted with P values of 0.158 for area (%) and 0.061 for number (/cm(2)) (experiment 4). These results suggest the potential inhibitory effect of D-allose on liver carcinogenesis, particularly under oxidative stress conditions.     

Clostridium cellulolyticum D-塔格糖3-差向异构酶基因的克隆、表达及酶活性

D-塔格糖3-差向异构酶是生物法生产新型功能性因子D-阿洛酮糖最为有效的酶。作者克隆到一种新型的D-塔格糖3-差向异构酶基因,来源于微生物Clostridium cellulolyticumH10。以pET-22b(+)为载体质粒,E.coliBL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白质的过量表达;经亲和层析纯化的重组蛋白质样品进行SDS-PAGE分析,在约31 000处出现显著的特征蛋白质条带;活性检测结果表明:该重组酶具有较高的转化活性。     

Construction of a novel hydroxyproline-producing recombinant Escherichia coli by introducing a proline 4-hydroxylase gene

An Escherichia coli recombinant strain producing trans-4-hydroxy-L-proline (Hyp) was constructed by introducing a proline 4-hydroxylase gene into an L-proline-producing E. coli. Plasmid pPF1, which contains a gene encoding feedback resistant gamma-glutamyl kinase (proB74), was constructed and introduced into E. coli W1485 putA. The recombinant E. coli W1485 putA/pPF1 strain produced L-proline (1.2 g/l). The proline production by W1485 putA/pPF1 was converted to Hyp production by introducing pWFH1 which contains a proline 4-hydroxylase gene. E. coli W1485 putA which harbors pWFP1 carrying the proline 4-hydroxylase gene, proB74, and proA produced 25 g/l of Hyp in 96 h. A novel biosynthetic pathway of Hyp, which has not previously been produced in E. coli, was constructed in E. coli.     

Antioxidant properties of rare sugar D-allose: Effects on mitochondrial reactive oxygen species production in Neuro2A cells

The anti-oxidative activity of the rare sugar D-allose has recently been reported, but the mechanism is largely unclear. In this study, we evaluated the reactive oxygen species (ROS) scavenging activities of D-allose and then examined the effects of D-allose on ROS production in mitochondria to clarify the antioxidant properties of D-allose. While D-allose did not scavenge hydrogen peroxide and superoxide anions, it eliminated hydroxyl radicals to the same extent as D-glucose. Rotenone, an uncoupler of mitochondrial respiratory complex I, induces ROS production in mouse neuroblastoma Neuro2A cells in the presence of D-glucose. However, in the presence of D-allose, there was no change in the ROS levels in Neuro2A cells following rotenone treatment. Furthermore, treatment with D-allose attenuated the D-glucose-dependent ROS generation induced by rotenone. Whereas treatment with D-glucose enhanced ATP synthesis in Neuro2A cells, D-allose was less effective in producing intracellular ATP than D-glucose. Treatment with D-allose inhibited the ATP synthesis stimulated by D-glucose. These results suggest that D-allose suppresses ROS production in the mitochondria due to competition with D-glucose at the cellular level.     

Bioconversion of D-psicose to D-tagatose and D-talitol by Mucoraceae fungi

Rhizopus oryzae MYA-2483, which cannot utilize D-psicose as a sole source of carbon, converted D-psicose to two other compounds. These compounds were identified by NMR and IR as D-tagatose and D-talitol. In this study, we describe for the first time the bioconversion of D-psicose to D-tagatose. Various strains of Mucoraceae fungi, to which R. oryzae MYA-2483 belongs, exhibited conversion activity similar to that of R. oryzae MYA-2483. There is the possibility that a considerable number of fungi belonging to Mucoraceae possess such D-psicose conversion activity.