稀有糖的生物转化生产策略：Izumoring方法

稀有糖是在自然界中存在但含量极少的一类单糖及衍生物,其在膳食、保健、医药等领域中发挥着重要的功能。本文综述了一种稀有糖的生物转化生产策略----Izumoring方法,即利用D-塔格糖3-差向异构酶、醛糖异构酶和多元醇脱氢酶等进行所有单糖及糖醇之间的相互转化;利用该原则,分别构建了己糖类、戊糖类和丁糖类的Izumoring转化策略,并可获得所有稀有糖的酶反应和生物转化生产途径。同时,展望了稀有糖生物转化生产的研究趋势。     

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.     

Prebiotic sugar synthesis: Hexose and hydroxy acid synthesis from glyceraldehyde catalyzed by iron(III) hydroxide oxide

Summary Iron(III) hydroxide oxide [Fe(OH)O] efficiently catalyzed the condensation of 25 MM dl-glyceraldehyde to ketohexoses at 25°C (pH 5–6). At 16 days the yields were sorbose (15.2%), fructose (12.9%), psicose (6.1%), tagatose (5.6%), and dendroketose (2.5%) with 19.6% of triose unreacted. Analysis at 96 days showed no decomposition of hexoses. Under these conditions Fe(OH)O also catalyzed the isomerization and rearrangement of glyceraldehyde to dihydroxyacetone and lactic acid, respectively. In these reactions, about 10% of the glyceraldehyde was oxidized to glyceric acid with concurrent reduction of the iron(III) to iron(II). The partial reduction of Fe(OH)O did not noticeably reduce its ability to catalyze hexose synthesis. The relationship of these results to prebiotic sugar synthesis is discussed.     

Tritiated borohydride reduction of carbohydrates: reduction of individual monosaccharides, alone or in groups, results in highly divergent specific activities.

Reduction of carbohydrates by tritiated borohydride resulted in the production of alditols or glycosides with characteristically divergent specific radioactivities. Simultaneous reduction of individual sugars in the presence of a reference standard, talose, permitted the assignment of a unique specific radioactivity with respect to talitol as 100%. A variety of structures was examined, including neutral hexoses, free and acetylated aminosugars, ketohexoses and glycosides containing a fixed pyranose ring adjacent to a carbonyl group. In the latter case, the resulting steric hindrance severely restricted the incorporation of tritium. In both of the ketohexoses tested, the minor product of the two epimeric alditols exhibited the higher specific radioactivity. In all cases, reduction produced a characteristic and reproducible specific activity in which the values varied from 51 to 182% of that found for talose. These results are interpreted on the basis of generalizations concerning mechanism and predictive value.     

Solvent accessibility to flavin in oxynitrilase

Oxynitrilase containing 2-thioFAD [C(2) = S] in place of FAD exhibits catalytic activity similar to that of native enzyme. Reaction of methyl methanethiolsulfonate with 2-thioFAD bound to oxynitrilase results in the formation of the corresponding flavin disulfide [C(2)-SSCH3]. Normal flavin [C(2) = O] is formed by reacting 2-thioFAD oxynitrilase with m-chloroperoxybenzoate or H2O2. Both reactions proceed via a spectrally detectable flavin 2-S-oxide intermediate [C(2) = S+-O-], but sizable amounts of this intermediate accumulate only in the m-chloroperoxybenzoate reaction (about 40%). While similar reactions have been reported with free 2-thioflavin, kinetic and other data indicate that the oxynitrilase reactions occur with intact enzyme. This shows that the 2-position of the pyrimidine ring in the bound coenzyme is accessible to solvent. The data are consistent with previous studies on the reaction of peroxides with oxynitrilase-bound 5-deazaFAD which show that the pyrimidine ring is accessible at position 4. Analogous studies indicate that the pyrimidine ring is buried in the case of flavin bound to lactate oxidase, since the data indicate that both positions 2 and 4 are inaccessible to solvent.     

Polymer-mediated cyclodehydration of alditols and ketohexoses

The polymer PEDOT⁺ (1 or 2) mediates a cyclodehydration reaction with alditols 3, 5, 7, 9, in hydrocarbon solvents, to give cyclic ethers 4, 6, 8, or 10, respectively, in high yield with a trivial isolation protocol. Polymers 1 or 2 also mediate the cyclodehydration of ketohexoses such as d-fructose, but not aldohexoses, to the important industrial intermediate 5-hydroxymethylfurfural (17), under milder conditions when compared to reactions mediated by mineral acids. A cascade reaction with ketohexoses is observed in toluene via cyclodehydration followed by Friedel–Crafts alkylation of the initially formed benzylic alcohol to give 16.     

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.     

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.     

Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in maize

Starch biosynthesis during pollen maturation is not well understood in terms of genes/proteins and intracellular controls that regulate it in developing pollen. We have studied two specific developmental stages: "early," characterized by the lack of starch, before or during pollen mitosis I; and "late," an actively starch-filling post-pollen mitosis I phase in S-type cytoplasmic male-sterile (S-CMS) and two related male-fertile genotypes. The male-fertile starch-positive, but not the CMS starch-deficient, genotypes showed changes in the expression patterns of a large number of genes during this metabolic transition. In addition to a battery of housekeeping genes of carbohydrate metabolism, we observed changes in hexose transporter, plasma membrane H(+)-ATPase, ZmMADS1, and 14-3-3 proteins. Reduction or deficiency in 14-3-3 protein levels in all three major cellular sites (amyloplasts [starch], mitochondria, and cytosol) in male-sterile relative to male-fertile genotypes are of potential interest because of interorganellar communication in this CMS system. Further, the levels of hexose sugars were significantly reduced in male-sterile as compared with male-fertile tissues, not only at "early" and "late" stages but also at an earlier point during meiosis. Collectively, these data suggest that combined effects of both reduced sugars and their reduced flux in starch biosynthesis along with a strong possibility for altered redox passage may lead to the observed temporal changes in gene expressions, and ultimately pollen sterility.     

Functional and structural responses of liver alcohol dehydrogenase to lysine modifications

Acetylation, glycosylation, and methylation, which modify lysine residues of horse liver alcohol dehydrogenase, have been investigated. Acetylation reacted with approximately two-third of the total lysines to induce the greatest structural changes of the enzyme. Glycosylation modified only one lysine residue selectively with indiscernible structural changes. The glycosylation effect was very specific with respect to diastereoisomers for aldopentoses, aldohexoses, and ketohexoses. Methylation produced the largest enhancement in the oxidative activity, which is related to the stability of the modified enzyme to prolonged modification and thermal denaturation. Kinetic studies revealed that a change in the maximal velocity was primarily responsible for the observed activity differences in the modifications.     

Reactivity of some sugars and sugar phosphates towards gold(III) in sodium acetate-acetic acid buffer medium

The kinetics of the oxidation of some aldoses and aldose phosphates have been studied spectrophotometrically in sodium acetate-acetic acid buffer medium at different temperatures. The reactions are first order with respect to [Au(III)] and [substrate]. Both H+ and Cl- ions retard the reaction. The reactions appear to involve different gold(III) species, viz. AuCl4-, AuCl3(OH2) and AuCl3(OH)- . The results are interpreted in terms of the probable intermediate formation of free radicals and Au(II). Aldoses react with gold(III) in the order: triose > tetrose > pentose > hexose. The sugar phosphates react with gold(III) at a faster rate than the parent sugars except glucose-1-phosphate, which reacts at slower rates than glucose. A tentative reaction mechanism leading to the formation of products has been suggested.     

Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.     

Metal-ion catalysis of aldose-ketose isomerizations in acidic solutions

A comparative study has been made of the ability of metal ions to catalyze the Lobry de Bruyn-Alberda van Ekenstein transformation. An analytical enzymic assay system, which is highly sensitive and selective, was developed to monitor the rates of isomerization of hexose and triose phosphates. The metal ions catalyze the isomerization of hexose phosphates by virtue of their Lewis acid character with the first transition series being most effective. The metal ions also appear to have no ability to direct the course of the reactions. Amino acids also catalyze the isomerization apparently by virtue of the free carboxylate anion.     

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [¹⁴C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [¹⁴C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.     

Hexitols as major intermediates of glucose assimilation by mycelium of Puccinia graminis

Mycelium of Puccinia graminis was grown for 4 d on 200 mM D-[U-¹⁴C]glucose followed by a cold chase for 30 h. Analysis of cellular metabolites during the chase indicated significant turnover only in carbohydrates soluble in 80% (w/v) ethanol. A kinetic analysis of the depletion of [¹⁴C] in pools of free sugars and sugar alcohols indicated that the trehalose pools and a small proportion (12–16%) of the mannitol and glucitol pools did not turn over, whilst pools of glucose, fructose, and the remainder of the hexitols became totally,depleted of label during the chase. Because the [¹⁴C] was totally lost from the pools of glucose and fructose prior to the hexitols, it was deduced that both of these hexoses were precursors of the hexitols. Estimation of the carbon fluxes through pools indicated that 52, 36 and 16% of the carbon from glucose was assimilated via glucitol, fructose and mannitol respectively, demonstrating that glucitol could not have originated from fructose as sole precursor. After offering D-[U-¹⁴C]glucitol, [¹⁴C] was assimilated into trehalose phosphate, glucans and amino acids, but not into free glucose or fructose. These data indicate that hexitols are quantitatively important intermediates during the assimilation of glucose by Puccinia graminis.     

The pentose phosphate pathway in rabbit liver. Studies on the metabolic sequence and quantitative role of the pentose phosphate cycle by using a system in situ

1. The reactions of the pentose phosphate cycle were investigated by the intraportal infusion of specifically labelled [(14)C]glucose or [(14)C]ribose into the liver of the anaesthetized rabbit. The sugars were confined in the liver by haemostasis and metabolism was allowed to proceed for periods up to 5min. Metabolism was assessed by measuring the rate of change of the specific radioactivity of CO(2), the carbon atoms of glucose 6-phosphate, fructose 6-phosphate and tissue glucose. 2. The quotient oxidation of [1-(14)C]glucose/oxidation of [6-(14)C]glucose as measured by the incorporation into respiratory CO(2) was greater than 1.0 during most of the time-course and increased to a maximum of 3.1 but was found to decrease markedly upon application of a glucose load. 3. The estimate of the pentose phosphate cycle from C-1/C-2 ratios generally increased during the time-course, whereas the estimate of the pentose phosphate cycle from C-3/C-2 ratios varied depending on whether the ratios were measured in glucose or hexose 6-phosphates. 4. The distribution of (14)C in hexose 6-phosphate after the metabolism of [1-(14)C]ribose showed that 65-95% of the label was in C-1 and was concluded to have been the result of a rapidly acting transketolase exchange reaction. 5. Transaldolase exchange reactions catalysed extensive transfer of (14)C from [2-(14)C]glucose into C-5 of the hexose 6-phosphates during the entire time-course. The high concentration of label in C-4, C-5 and C-6 of the hexose 6-phosphates was not seen in tissue glucose in spite of an unchanging rate of glucose production during the time-course. 6. It is concluded that the reaction sequences catalysed by the pentose phosphate pathway enzymes do not constitute a formal metabolic cycle in intact liver, neither do they allow the definition of a fixed stoicheiometry for the dissimilation of glucose.     

Production of 6-phenylacetylene picolinic acid from diphenylacetylene by a toluene-degrading Acinetobacter strain

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.     

Novel selective biocatalytic deacetylation studies on dihydropyrimidinones

Five racemic ethyl 4-(3/4-acetoxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylates have been synthesized by acetylation of corresponding hydroxy analogues, which in turn have been synthesized under microwave condition by multi-component Biginelli cyclocondensation of ethyl acetoacetate, urea and the corresponding hydroxybenzaldehydes in the presence of ferric chloride. These dihydropyrimidinones have been subjected to biocatalytic resolution using acetoxyl group on the phenyl ring as remote handle; Novozyme^®-435, an immobilized lipase from Candida antarctica in THF:DIPE has been found to catalyze the deacetylation reactions in an enantioselective fashion.     

Hexose transport in L6 rat myoblasts. I. Rate-limiting step, kinetic properties, and evidence for two systems

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2-Deoxy-D-glucose (2-DOG) and 2-deoxy-2-fluoro-D-glucose (2FG) were used as analogues to investigate the rate-limiting step of hexose uptake into the cell. Virtually all of the 2-DOG or 2FG taken up into the cell was found to be in the phosphorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate-limiting step. The inhibitory effect of various glucose analogues on 2-DOG and 3-O-methyl-D-glucose (3-OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2-DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3-OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.     

Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading Acinetobacter Strain

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.