D-阿洛酮糖酶法生产的研究进展

稀有单糖D-阿洛酮糖是D-果糖的C-3差向异构体,主要通过D-塔格糖3-差向异构酶或D-阿洛酮糖-3-差向异构酶对D-果糖进行异构化获得。D-阿洛酮糖不仅可以作为食品添加剂和膳食补充剂,而且具有改善胰岛素抵抗、增强抗氧化和降血糖控制等多种生理功能。因此,D-阿洛酮糖作为传统高能量糖如蔗糖、果糖等的健康替代品,具有重要的研究价值。作者综述了D-阿洛酮糖的理化性质、来源、体内代谢、生理功能、应用和最新生产技术,讨论了D-阿洛酮糖生产中存在的问题及解决方案。针对低废物生成、低能耗、高糖产量等特点,提出了一种绿色、可循环利用的D-阿洛酮糖生产工艺技术。     

L-鼠李树胶糖-1-磷酸醛缩酶立体选择性的半理性改造合成D-阿洛酮糖

稀有糖D-阿洛酮糖在食品、医药、保健等领域显示了巨大的应用潜能,然而以较高的收率大量生产D-阿洛酮糖依然面临着挑战。前期研究表明,属于磷酸二羟基丙酮(dihydroxyacetone phosphate,DHAP)依赖型醛缩酶家族的L-鼠李树胶糖-1-磷酸醛缩酶(Rha D)在以D-甘油醛为醛受体时,失去了对该醛受体的立体选择性,生成D-阿洛酮糖和D-山梨糖两种稀有糖,两者比例接近1∶1。为了对Rha D醛缩酶的立体选择性进行优化使其专一性地生产D-阿洛酮糖,对Rha D醛缩酶的152位酪氨酸进行了19种其他氨基酸的定点饱和突变,发现Rha DY152A突变体倾向于生成D-阿洛酮糖,D-阿洛酮糖和D-山梨糖的比例从最初的1∶1显著提高到8∶1,为下一步对Rha D醛缩酶分子改造进而定向合成单一产物D-阿洛酮糖提供理论依据。     

D-阿洛酮糖生物合成研究进展

D-阿洛酮糖具有低热量、低吸收特点，是目前最具市场和研究价值的具有保健功能的稀有糖，其安全性已经得到包括美国食品药品监督管理局在内的多国监管机构的认可，可以用于食品。文章简述了D-阿洛酮糖的生理功能及应用，介绍了D-阿洛酮糖生物合成过程中涉及的生物酶、生物转化过程、产品分离纯化和结晶的方法，讨论分析了当前D-阿洛酮糖生产过程的瓶颈问题，并提出可能的解决措施。     

D-阿洛酮糖的专利技术综述

D-阿洛酮糖作为一种新型低热量功能性甜味剂,是自然界中存在量极少的稀有糖,其具有调节血糖、减少能量摄入等生理功能,广泛应用于食品、医药、化妆品、高分子等领域,近年来成为甜味剂的研究热点之一。文章主要综述了D-阿洛酮糖的全球、国内专利申请情况、专利技术发展情况以及重点申请人分析,以期为国内研发机构和相关企业在D-阿洛酮糖领域的创新研发方向、专利保护策略以及知识产权布局提供有益的参考和建议。     

生物转化法产D-阿洛糖的分离纯化

利用重组菌E.coli BL21/p ET-22b-tl-rpib,将原料D-阿洛酮糖生物转化为具有抑癌效果的稀有糖D-阿洛糖。经液相色谱和串联质谱联用技术(LC-MS/MS)鉴定,反应混合液中25%的D-阿洛酮糖转化成了D-阿洛糖。等电聚焦法测定了重组蛋白的等电点为6.3,并成功运用到产物混合液的去蛋白沉淀中。经过预处理的混合糖液,通过DTFCa2+型离子交换树脂实现了分离纯化。最佳分离条件为:柱温60℃,进样量4m L,流速1m L/min。     

功能性甜味剂D-阿洛酮糖研究进展

高糖摄入引起的糖尿病、高血压等疾病严重危害着人类的生命健康,这一形势也促使人们对天然、低热量糖替代品的开发日益关注。作为一种近年发现的具有特殊生理功效的稀少糖,D-阿洛酮糖具有与蔗糖相近的口感及容积特性,被称为食品中蔗糖"理想替代品"。D-阿洛酮糖可以在D-阿洛酮糖3-差向异构酶的催化作用下进行生物合成,已被FDA批准为一般公认安全。D-阿洛酮糖因其优良的性能逐渐成为食品、保健和医疗领域的研究热点。本文综述了D-阿洛酮糖的理化性质、生理功能,及其最新的化学合成和生物制备方法,为D-阿洛酮糖在各大领域的应用做理论铺垫。     

稀有糖D-阿洛糖性质及生物法生产研究进展

D-阿洛糖是一种稀有的己醛糖,具有抗癌等众多生理功能,成为近年来的研究热点。其生物法生产多是利用L-鼠李糖异构酶、核糖-5-磷酸异构酶等酮醛糖异构酶以D-阿洛酮糖为底物,催化可逆的异构化反应获得。该文对D-阿洛糖的性质、应用和代谢,生物法生产相关酶的性质及应用等内容进行综述,在此基础上提出了要实现D-阿洛糖大规模生产必需解决的问题。     

D-阿洛酮糖及其合成研究进展

D-阿洛酮糖是一种具有保健功能的己酮糖,可以预防糖尿病、维持正常血脂水平,避免传统甜味剂对人体造成的代谢负担,具有重要的应用价值,但在自然界中含量稀少,近年来涌现多种D-阿洛酮糖的合成方法。文章围绕D-阿洛酮糖的功能、化学合成法和生物酶异构化法等内容开展综述,重点阐述了D-阿洛酮糖3-差向异构酶的来源及性质、结构及催化机制、分子改造、多酶偶联、食品级表达等方面的研究进展,旨在推进生物转化制备D-阿洛酮糖的工业发展,为保障公众营养健康提供新思路。     

D-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中的表达

D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase, DPE)可以催化D-果糖转化为稀有糖D-阿洛酮糖,是生产D-阿洛酮糖过程中的关键酶。为了提高DPE的表达水平,该研究以枯草芽孢杆菌(Bacillus subtilis) WB800为宿主菌,异源表达Clostridium scindens ATCC 35704来源的DPE。首先,构建不同单启动子和串联启动子介导DPE表达的重组菌,通过摇瓶培养,发现由单启动子P_hag介导的重组菌株经发酵后酶活力最高,最高酶活力为19.62 U/mL,是P₄₃介导的原始菌株酶活力的1.3倍。最后对P_hag的核糖体结合位点(ribosome binding site, RBS)序列进行突变,突变后的菌株酶活力再次提高了29.4%,是原始菌株酶活力的1.69倍。该研究结果为工业化生产DPE提供了方法学参考。     

D-阿洛酮糖合成及偶联酿酒酵母发酵的研究

该研究利用含有组成型启动子的质粒pET-20b载体,在大肠杆菌（Escherichia coli）BL21（DE3）中对3种D-阿洛酮糖3-差向异构酶进行异源表达,其后以D-果糖为底物进行静息细胞转化。结果表明,重组菌在摇床30 ℃、200 r/min转速下发酵48 h,能够利用D-果糖为底物生产D-阿洛酮糖,转化率分别达到27.56%、23.99%和25.98%。为降低D-果糖对D-阿洛酮糖纯化过程中的影响,在产糖的过程后偶联酿酒酵母（Saccharomyces cerevisiae）好氧发酵过程,消耗掉混合糖液中的D-果糖,结果显示转化24 h后D-果糖去除率达到94.22%,该研究为下游D-阿洛酮糖的分离和纯化提供了新的思路。     

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.     

Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization

Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.     

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.     

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.     

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.     

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.     

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%。     

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.     

Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma (PC12) cells

We evaluated the neuroprotective effects of D-psicose, one of the rare sugars, on 6-hydroxydopamine (6-OHDA)-induced apoptosis in catecholaminergic PC12 cells, the in vitro model of Parkinson's disease (PD). Apoptotic characteristics of PC12 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. The results showed that D-psicose at a concentration of 50 mM, exerted significant protective effects against the 6-OHDA (200 muM)-induced PC12 cell apoptosis, while other sugars had little or no protective effects. We have observed a significant increase in the level of intracellular glutathione after 24 h in 6-OHDA (200 muM) treated cells, while a decrease in the level was observed at 3 h and 6 h. Also, a synergistic exposure to D-psicose and 6-OHDA for 24 h showed a significant increase in intracellular glutathione level. Therefore, these results suggest that D-psicose may play a potential role as a neuroprotective agent in the treatment of neurodegenerative diseases by inducing an up-regulation of intracellular glutathione.