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。     

鱼类对糖的代谢

糖类在鱼体内的代谢包括分解、合成、转化和输送等环节。摄入的糖类在鱼体消化道内被淀粉酶、麦芽糖酶分解成单糖,然后被吸收。吸收后的单糖在肝脏及其他组织进一步氧化分解,并释放出能量,或被用于合成糖原、体脂、氨基酸,或参与合成其他生理活性物质。糖原是糖类在体内的储存形式,葡萄糖氧化分解是供给鱼类能量的重要途径,血糖（葡萄糖）则是糖类在体内的主要运输形式。从上述糖代谢情况来看,鱼类对饲料糖的代谢与陆上动物并无二致。但鱼类被视为对糖的利用能力低下”一,这促使人们去探讨鱼类糖代谢的特殊机制。现就这方面的工作回顾总结如下：     

蓝藻光驱固碳合成糖类物质的技术研究进展*

糖类物质在食品、医药、日化、发酵领域有着广泛应用,对人类健康和社会发展有着重要意义。发展新型糖类物质合成技术有利于解决传统植物生物质“采集-炼制”产糖模式所面临的高成本、长周期、时空限制等风险和问题。蓝藻是一类重要的光自养微生物,也是极具潜力的新型微生物光合平台,发展蓝藻光驱固碳产糖技术有望实现二氧化碳向特定糖类产物的一站式定向转化,实现糖类物质合成的模式变革。糖类物质本身在蓝藻天然光合代谢网络中发挥重要作用,特别是卡尔文循环、糖原代谢、相容性物质代谢等几个重要生理模块的运转都是以不同糖类物质的转化来驱动的;而合成生物技术的发展又为光合产糖网络重塑和扩展注入了新的驱动力,在产品类型、合成模式及生产效率上显著提升了蓝藻光驱固碳产糖技术的发展和应用潜力。针对蓝藻光驱固碳产糖技术的发展应用,从模式、策略、产物等不同维度总结了相关进展和风险挑战,并对其未来前景和方向进行了展望。     

酶法生产稀少糖的研究进展

稀少糖是指自然界中存在稀少的单糖及其衍生物,可应用于食品、制药和营养等许多领域,同时也可以作为各种天然产品和候选药物的原料。目前大多数稀少糖因其在自然界中含量稀少而非常昂贵,而且这些稀少糖的化学合成原料价格高昂,不利于其大量生产。由于酶催化反应具有反应条件温和、特异性强、效率高、可持续性强等优点,稀少糖酶法生物合成已成为这一领域的有力工具。文中就稀少糖,包括D-阿洛酮糖、D-塔格糖、D-山梨糖、L-果糖、D-阿洛糖等其他稀少糖的生物学功能及应用,以及稀少糖相关酶的研究和酶法生产进行综述。     

植物原生质膜的糖转运蛋白

高等植物光合细胞同化的糖类由质外体途径穿过原生质膜转运时,利用Ｈ＾＋－ＡＴＰ形成的质了左糖逆电化学势进行共转运是由糖转运蛋白介导的。文中对近年来植物原生质膜单糖和蔗糖转运蛋白的克隆、鉴定及特性,糖转运蛋白组织定位和表达及其与光合同化物运和分配的关系等同研究进展作了介绍。     

生物合成稀有人参皂苷的研究进展*

人参皂苷是我国传统中药人参的主要活性物质,稀有人参皂苷相较人参皂苷具有更好的生物活性,也更利于身体吸收,具有镇静催眠、促进细胞分化增殖、抗肿瘤、降血糖、提升免疫力等作用。然而,稀有人参皂苷结构复杂且在人参中含量极低,限制了其开发利用。随着生物技术的发展,利用生物法合成稀有人参皂苷成为本领域的研究热点。因此,对近年来生物合成稀有人参皂苷研究进行汇总梳理,总结稀有人参皂苷的主要种类结构及近年来生物转化法和异源合成法合成稀有人参皂苷的研究进展,生物转化法汇总了以人参皂苷为底物的转化生物,异源合成法总结人参皂苷的生物合成途径及形成结构多样化人参皂苷的酶。对生物合成稀有人参皂苷存在的问题进行了讨论,同时展望了其前景以及未来研究方向,以期为从事人参研究者提供更多生物线索和制备策略。     

果树中糖类代谢和调控研究

糖是生物体内主要的碳源,是光合作用的主要产物,可为生物体提供能量,在植物的生长发育过程中起重要作用。本文综述了近年来关于糖类与果树生长发育及品质形成方面的相关研究进展,重点介绍了糖类运输、积累与基因表达、糖信号传导和糖类调控网络等方面的研究进展,并对今后利用分子生物学手段进行果实品质改良等方面的研究方向进行了展望。     

嗜糖黄杆菌β-葡萄糖苷酶的功能及在稀有人参皂苷制备中的应用

【背景】有些稀有皂苷具有较好的药理活性,寻找活性高和专一性好的糖苷酶可能实现稀有皂苷的定向制备。嗜糖黄杆菌中含有丰富且未被表征的糖苷酶基因是寻找新酶的潜在来源。【目的】从嗜糖黄杆菌中发现活性高和专一性好的糖苷酶,用于制备稀有人参皂苷。【方法】重组表达嗜糖黄杆菌中15个假定的葡萄糖苷酶基因,系统研究重组酶的性质和功能,筛选可用于制备稀有皂苷的酶,利用薄层层析法和高效液相色谱法鉴定转化产物。【结果】从嗜糖黄杆菌中获得3种活性较好的β-葡萄糖苷酶,即SA2629、SA0236和SA2851。其中,SA2629具有最高的比酶活(78.7U/mg)和催化效率[kcat=(27.38±1.40)s^-1],且SA2629能同时水解人参皂苷C-20位上的β-1,6-葡萄糖苷键和C-3位直接与苷元相连的葡萄糖苷键。SA2851和SA0236只对C-20位上的β-1,6-葡萄糖苷键具有水解活性,其中SA0236活力高。将SA2629和SA0236与课题组前期获得的一种β-1,2-葡萄糖苷酶分别组合,可以将高含量人参皂苷Rb1完全转化成稀有皂苷CK和F2。【结论】获得了可用于制备稀有人...     

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%。     

糖生物学和糖组学

糖生物学和糖组学,虽然都是研究糖类在机体中的作用,但是,它们分别有不同的视角。糖生物学是沿袭了糖化学、糖生物化学发展而来,着重研究糖类和蛋白质的相互作用;而糖组学则源于基因组学,以基因编码糖基转移酶为起始,由这些酶得到糖组,进而开展糖组的研究。     

Izumoring: a strategy for bioproduction of all hexoses

Izumoring is a new concept for the bioproduction of all hexose sugars - 16 aldohexoses, eight ketohexoses and 10 hexitols -- using enzymatic and microbiological reactions. The biocatalysts necessary for the strategy were (i) d-tagatose 3-epimerase [which epimerizes all ketohexoses at C-3 of the sugar], (ii) polyol dehydrogenases [which catalyze oxidation-reduction reactions between ketohexoses and the corresponding hexitols], and (iii) aldose isomerases [which catalyze isomerization reactions between aldohexoses and corresponding aldoketoses]. All ketohexoses, aldohexoses and hexitols may be arranged in a symmetric ring connected by the biochemical reactions, allowing the design for the bioproduction of all 34 hexose sugars. The ring shows there are four entrances to the l-hexose world from the natural d-hexoses. The Izumoring ring shows clearly the relationship and the position of all 34 six carbon sugars.     

Enzymatic approaches to rare sugar production

Rare sugars have recently attracted much attention because of their potential applications in the food, nutraceutical, and pharmaceutical industries. A systematic strategy for enzymatic production of rare sugars, named Izumoring, was developed > 10 years ago. The strategy consists of aldose-ketose isomerization, ketose C-3 epimerization, and monosaccharide oxidation-reduction. Recent development of the Izumoring strategy is reviewed herein, especially the genetic approaches to the improvement of rare sugar-producing enzymes and the applications of target-oriented bioconversion. In addition, novel non-Izumoring enzymatic approaches are also summarized, including enzymatic condensation, phosphorylation-dephosphorylation cascade reaction, aldose epimerization, ulosonic acid decarboxylation, and biosynthesis of rare disaccharides.     

Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn–Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose–aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.     

Functional expression of L-fucokinase/guanosine 5'-diphosphate-L-fucose pyrophosphorylase from Bacteroides fragilis in Saccharomyces cerevisiae for the production of nucleotide sugars from exogenous monosaccharides

The biosynthesis of glycoconjugates requires the relevant glycosyltransferases and nucleotide sugars that can act as donors. Given the biological importance of posttranslational glycosylation, a facile, robust and cost-effective strategy for the synthesis of nucleotide sugars is highly desirable. In this study, we demonstrate the synthesis of nucleotide sugars from corresponding monosaccharides in a highly efficient manner via metabolic engineering, using an enzymatic approach. This method exploits l-fucokinase/guanosine 5'-diphosphate (GDP)-l-fucose (L-Fuc) pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts l-Fuc into GDP-L-Fuc via an L-Fuc-1-phosphate intermediate. Because L-Fuc and d-arabinose (D-Ara) are structurally similar, it is assumed that the biosynthesis of GDP-D-Ara in a recombinant Saccharomyces cerevisiae strain harboring the FKP gene can occur through a mechanism akin to that of GDP-L-Fuc via the salvage pathway. Thus, we reasoned that by exogenously supplying different monosaccharides structurally related to L-Fuc, it should be possible to produce the corresponding nucleotide sugars with this recombinant yeast strain, regardless of internal acquisition of nucleotide sugars through expression of additive enzymes in the de novo pathway.     

Quantitative determination of wood-derived soluble oligosaccharides by HPLC

Soluble oligosaccharide carbohydrates from the partial enzymatic hydrolysis of pulp were degraded to monosaccharides by enzymatic hydrolysis using a complete mixture of cellulolytic and hemicellulolytic enzymes and subsequently analysed by HPLC. With this method it was possible to obtain a quantitative estimate of both the 5- and 6-carbon sugars comprising the soluble oligosaccharides.     

The “epimerring” highlights the potential of carbohydrate epimerases for rare sugar production

Rare sugars can find applications in various industrial sectors and, therefore, hold significant economic value. Due to their low natural abundance, efficient production processes are needed to enable their commercial exploitation. About a decade ago, the available biosynthetic routes were summarized in the so-called “Izumoring”, which mainly comprised reactions catalysed by keto-aldol isomerases and oxidoreductases. Although just a single epimerase specificity (acting on the 3-position of ketoses) was included, these enzymes hold the potential to truly revolutionize the field as they offer shortcuts in conversion processes. For example, C2-epimerases could replace double isomerization reactions, whereas C4/5-epimerases could form a new bridge between d- and l-sugars as alternative to the current two-step oxidoreduction reaction. Here, we present the “Epimerring” to highlight the potential of new epimerases that can still be discovered and/or engineered, which may open doors to new and improved synthesis routes for rare sugars. Several efforts and options in the search of such biocatalysts are shortly summarized.     

Enzymatic synthesis of hydrophilic and hydrophobic derivatives of natural phenolic acids in organic media

The enzymatic esterification of natural phenolic antioxidants such as cinnamic acid and benzoic acid derivatives, with aliphatic alcohols, monosaccharides as well as alkylglucosides, using various lipases and esterases in non-aqueous media, was investigated. Reaction rate and esterification yield seems to be linked to the structural characteristics of the substrates (aromatic acids and alcohols or sugars) used.     

Production of 2,3-butanediol by Klebsiella pneumoniae grown on acid hydrolyzed wood hemicellulose

Summary Previously steam explosion had been used to enhance the enzymatic hydrolysis of lignocellulosic substrates to glucose. The conditions for pretreating aspen wood chips were optimized so that highest amounts of undegraded hemicellulose could be obtained after washing the steam exploded chips. The hemicellulose rich water soluble fractions showing highest pentosan yields were then acid hydrolysed to their composite sugars. Approximately 65–75% of the total reducing sugars detected in the wood hydrolysates were in the form of monosaccharides with D-xylose being the major component. Klebsiella pneumoniae was grown in media containing these wood hydrolysates as the substrate and 2,3-butanediol yields of 0.4–0.5 g per g of monosaccharide utilised were obtained.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.