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%。     

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.     

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%.     

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。     

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.     

Effects of dietary D-psicose on diurnal variation in plasma glucose and insulin concentrations of rats

The effects of supplemental D-psicose in the diet on diurnal variation in plasma glucose and insulin concentrations were investigated in rats. Forty-eight male Wistar rats were divided into four groups. Each group except for the control group was fed a diet of 5% D-fructose, D-psicose, or psico-rare sugar (3:1 mixture of D-fructose and D-psicose) for 8 weeks. Plasma glucose levels were lower and plasma insulin levels were higher at all times of day in the psicose and psico-rare sugar groups than in the control and fructose groups. Weight gain was significantly lower in the psicose group than in the control and fructose groups. Liver glycogen content, both before and after meals was higher in the psicose group than in the control and fructose groups. These results suggest that supplemental D-psicose can lower plasma glucose levels and reduce body fat accumulation. Hence, D-psicose might be useful in preventing postprandial hyperglycemia in diabetic patients.     

D-阿洛酮糖的功能及其生物合成研究进展

随着肥胖、糖尿病等代谢性疾病的发病率在全球范围急剧上升,人们对食品营养和健康等问题日益关注。D-阿洛酮糖作为重要的天然稀有己酮糖,不仅保持良好的甜度,而且具有降血糖、降血脂、抗氧化等功效,逐渐成为食品、保健和医疗领域的研究热点。文中阐述了D-阿洛酮糖的主要生理功能,综述了D-阿洛酮糖的生物合成研究进展及其酮糖3-差向异构酶的晶体结构,为筛选D-阿洛酮糖的产生菌株及提高合成酶的热稳定性和转化率提供理论指导,以满足工业化生产的需求。     

Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase

Isomaltulose is a natural isomer of sucrose. It is widely used as a functional sweetener with promising properties, including slower digestion, lower glycemic index, prolonged energy release, lower insulin reaction, and less cariogenicity. It has been approved as a safe sucrose substitute by the Food and Drug Administration of the US; Ministry of Health, Labor and Welfare of Japan; and the Commission of the European Communities. This article presents a review of recent studies on the properties, physiological effects, and food application of isomaltulose. In addition, the biochemical properties of sucrose isomerases producing isomaltulose are compared; the heterologous expression, fermentation optimization, structural determination, and catalysis mechanism of sucrose isomerase are reviewed; and the biotechnological production of isomaltulose from sucrose is summarized.     

A study of the changes in the physiological properties of Dhar Yeast by growing it in cultures containing sucrose,ethyl alcohol or methyl alcohol as source of carbon food

Summary On growing yeast in cultures containing different sources of carbon food as sucrose, ethyl alcohol and methyl alcohol, great changes in its physiological properties are observed. When these mutated yeasts are grown in similar cultures containing sucrose as carbon source, changes effected previously decrease to a minimum although a distinct residual effect is seen in all the above cases even after a month.     

The mechanism of the human intestinal sucrase action

Highly purified sucrase accepts as a substrate α-anomers within D-glucopyranosides. Hydrolysis of sucrose and palatinose processds with the net retention of configuration at the C-1 of D-glucopyranose. The fission of the glycosidic bond takes place between the C-1 of the D-glucose ring and the glycosidic oxygen; therefore D-fructose is released as β-D-fructofuranose from sucrose. During hydrolysis, transglycosylation takes place and a few oligosaccharides are formed and in the presence of methanol α-methyl-D-glucopyranoside is also produced. Non-stoichiometric amounts of D-glucose and D-fructose are produced from sucrose, turanose and palatinose and less D-glucose than expected is present. Formation of sucrose from D-glucose and D-fructose was not observed.     

Rheological characteristics of heat-induced custard pudding gels with high antioxidative activity

Custard pudding gels were prepared from fresh whole egg, milk and sugar. The effects of D-psicose (Psi), a non-calorie rare hexose, on the antioxidative activity and rheological properties of the custard pudding gels were investigated at different temperatures for comparison with those of control sugars (sucrose, Suc; D-fructose, Fru). The rheological behavior of the heat-induced pudding gels was evaluated by using breaking and creep tests. During the heat-induced gel formation, custard pudding containing Psi (15%, wt/wt) demonstrated a stronger breaking strength and higher viscoelasticity than those containing Fru and Suc. The thermodynamic parameters obtained from DSC indicated that the egg white (EW) proteins were made less thermally stable when heated in the presence of Psi than in the presence of Fru and Suc. These findings are consistent with enhanced aggregation of the EW solution in the presence of Psi. Furthermore, the Psi pudding gels possessed higher antioxidative activity than the control sugar pudding gels by using an analysis of the scavenging activity on DPPH radicals and the ferric-reducing antioxidative power. These results suggest that Psi favored cross-linking of Psi-protein molecules through the Maillard reaction which increased the formation of intermediate products to improve functionality. Custard pudding containing Psi could therefore be an effective functional sweet desert with high antioxidative activity and the outstanding gelling characteristics.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

Inulin and its enzymatic production by inulosucrase: Characteristics,structural features,molecular modifications and applications

Inulin, a natural fructan, cannot be hydrolyzed by digestive enzymes in the human body and plays a role as a dietary fiber and prebiotic. Due to its versatile physicochemical properties and physiological functions, inulin has been widely applied in food, pharmaceuticals, and many other fields. The microorganism-derived inulin-forming enzyme inulosucrase (ISase) (EC: 2.1.4.9) can biosynthesize higher-molecular-weight inulin than plants using sucrose as the sole substrate, and the enzyme also shows transfructosylation activity toward other saccharide acceptors. In this article, the properties, functions, and applications of inulin are overviewed. The biosynthesis of inulin by ISase is addressed, including ISase characteristics, structural features, molecular modifications and applications.     

链霉菌来源D-甘露糖异构酶的性质及其在制备D-甘露糖中的应用

[背景]D-甘露糖具有多种功能活性,在食品、医药、饲料等行业应用广泛.D-甘露糖异构酶可以催化D-果糖与D-甘露糖之间的相互转化,在D-甘露糖的酶法制备中具有应用潜力.[目的]克隆一个链霉菌(Streptomyces sp.)来源的D-甘露糖异构酶基因(sssMIaseA)并在大肠杆菌中表达,研究其酶学性质,并用于制备...     

Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases

Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses of the ant Lasius niger to various compounds perceived as sweet in humans: a structure-activity relationship study

A behavioural study on the ant Lasius niger was performed by observing its feeding responses to 85 compounds presented in a two-choice situation (tested compound versus water control or sucrose solution). Among these compounds, only 21 were phagostimulating: six monosaccharides (D-glucose, 6-deoxy-D-glucose, L-galactose, L-fucose, D-fructose, L-sorbose), four derivatives of D-glucose (methyl alpha-D-glucoside, D-gluconolactone and 6-chloro- and 6-fluoro-deoxy-D-glucose), five disaccharides (sucrose, maltose, palatinose, turanose and isomaltose), one polyol glycoside (maltitol), three trisaccharides (melezitose, raffinose and maltotriose) and two polyols (sorbitol and L-iditol). None of the 16 non-carbohydrate non-polyol compounds tested, although perceived as sweet in humans, was found to be active in ants. The molar order of effectiveness of the major naturally occuring compounds (melezitose > sucrose = raffinose > D-glucose > D-fructose = maltose = sorbitol) is basically different from the molar order of their sweetness potency in humans (sucrose > D-fructose > melezitose > maltose > D-glucose = raffinose = sorbitol). On a molar basis melezitose is in L. niger about twice as effective as sucrose or raffinose, while D-glucose and D-fructose are three and four times less effective, respectively, than sucrose or raffinose. From a structure-activity relationship study it was inferred that the active monosaccharides and polyols should interact with the ant receptor through only one type of receptor, through the same binding pocket and the same binding residues, via a six-point interaction. The high effectiveness of melezitose in L. niger mirrors the feeding habits of these ants, which attend homopterans and are heavy feeders on their honeydew, which is very rich in this carbohydrate.     

Current studies on physiological functions and biological production of lactosucrose

Lactosucrose (O-β-d-galactopyranosyl-(1,4)-O-α-d-glucopyranosyl-(1,2)-β-d-fructofuranoside) is a trisaccharide formed from lactose and sucrose by enzymatic transglycosylation. This rare trisaccharide is a kind of indigestible carbohydrate, has good prebiotic effect, and promotes intestinal mineral absorption. It has been used as a functional ingredient in a range of food products which are approved as foods for specified health uses in Japan. Using lactose and sucrose as substrates, lactosucrose can be produced through transfructosylation by β-fructofuranosidase from Arthrobacter sp. K-1 or a range of levansucrases, or through transgalactosylation by β-galactosidase from Bacillus circulans. This article presented a review of recent studies on the physiological functions of lactosucrose and the biological production from lactose and sucrose by different enzymes.     

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%.     

A specific receptor site for glycerol, a new sweet tastant for Drosophila: structure-taste relationship of glycerol in the labellar sugar receptor cell

Glycerol, a linear triol, is a sweet tastant for mammals but it has not previously been recognized to stimulate the sense of taste in insects. Here we show by electrophysiological experimentation that it effectively stimulates the labellar sugar receptor cell of Drosophila. We also show that in accord with the electrophysiological observations, the behavioral feeding response to glycerol is dose dependent. 3-Amino-1,2-propanediol inhibited the response of the sugar receptor cell to glycerol, specifically and competitively, while it had almost no effect on responses to sucrose, D-glucose, D-fructose and trehalose. In the null Drosophila mutant for the trehalose receptor (DeltaEP19), the response to glycerol showed no change, in sharp contrast with a characteristic drastic decrease in the response to trehalose. The glycerol concentration-response curves for I-type and L-type labellar hairs were statistically indistinguishable, while those for sucrose, D-glucose, D-fructose and trehalose were clearly different. These all indicate the presence of a specific receptor site for glycerol. The glycerol site was characterized by comparing the effectiveness of various derivatives of glycerol. Based on this structure-taste relationship of glycerol, a model is proposed for the glycerol site including three subsites and two steric barriers, which cannot accommodate carbon-ring containing sugars such as D-glucose.     

Isolation and Identification of a Pathogen of Silkworm Bombyx mori

A pathogenic bacterial strain, ST-1, was isolated from a naturally infected silkworm. The strain was identified on the basis of its physiological and biochemical properties and the results of sequence analysis of its 16S rRNA gene. The results of the 16S rRNA gene sequence analysis revealed that ST-1 shared the highest sequence identity (more than 99%) with Pseudomonas chlororaphis subsp. aurantiaca. ST-1 bacteria were gram-negative and 0.7-0.9 × 1.3-1.5 μm long, short rods with rounded ends. The strain could utilize sodium citrate, malonate, D-glucose, sucrose, D-fructose, D-mannose, and L-arabinose. Pathogenicity of ST-1 for silkworm could be depicted as a linear regression of the logarithm (y) of ST-1 concentration against probability (x) (y = 0.4040 + 0.0600x). The median lethal concentration (LC(50)) was 2.12 × 10(4) cfu/ml. In conclusion, ST-1 was identified as Ps. chlororaphis subsp. aurantiaca. This is the first report that Ps. aurantiaca is a pathogen for silkworm Bombyx mori.