海藻糖的生产制备及其应用前景

海藻糖是一种广泛分布于细菌、真菌和动植物体内的双糖。在生物体内 ,它不仅作为结构成分和能量物质存在 ,而且在热击和脱水等协迫条件下 ,对生物体和生物大分子起着良好的非特异性保护作用。由于其独特的生物学功能 ,它在食品、分子生物学、医药、化妆品、农业等方面具有广阔的应用前景。简述海藻糖的生产制备、应用研究及其前景展望。     

Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function

Yeasts and filamentous fungi are endowed with two different trehalose-hydrolysing activities, termed acid and neutral trehalases according to their optimal pH for enzymatic activity. A wealth of information already exists on fungal neutral trehalases, while data on localization, regulation and function of fungal acid trehalases have remained elusive. The gene encoding the latter enzyme has now been isolated from two yeast species and two filamentous fungi, and sequences encoding putative acid trehalase can be retrieved from available public sequences. Despite weak similarities between amino acids sequences, this type of trehalase potentially harbours either a transmembrane segment or a signal peptide at the N-terminal sequence, as deduced from domain prediction algorithms. This feature, together with the demonstration that acid trehalase from yeasts and filamentous fungi is localized at the cell surface, is consistent with its main role in the utilisation of exogenous trehalose as a carbon source. The growth on this disaccharide is in fact pretty effective in most fungi except in Saccharomyces cerevisiae. This yeast species actually exhibits a "Kluyver effect" on trehalose. Moreover, an oscillatory behaviour reminiscent of what is observed in aerobic glucose-limited continuous cultures at low dilution rate is also observed in batch growth on trehalose. Finally, the S. cerevisiae acid trehalase may also participate in the catabolism of endogenous trehalose by a mechanism that likely requires the export of the disaccharide, its extracellular hydrolysis, and the subsequent uptake of the glucose released. Based on these recent findings, we suggest to rename "acid" and "neutral" trehalases as "extracellular" and "cytosolic" trehalases, which is more adequate to describe their localization and function in the fungal cell.     

Trehalose targets Nrf2 signal to alleviate d-galactose induced aging and improve behavioral ability

As an important factor leading to aging and chronic diseases, oxidative stress has become a hot research topic. Trehalose is a natural sugar widely found in many edible plants, animals and natural microorganisms, and recent studies have suggested that trehalose is an antioxidant, although its underlying molecular mechanism is unclear. Therefore, we evaluated the protective mechanism of trehalose against oxidative stress-induced senescence. In the mouse model of d-galactose (D-gal) induced aging, we found that trehalose significantly reversed the learning and memory impairment caused by D-gal and improved the ability to explore unknown things, which was associated with a significant reduction in brain tissue damage. Further studies have shown that trehalose activates the expressions of downstream target genes HO-1, NQO1, SOD, GSH and CAT by promoting the nuclear translocation of Nrf2 in the liver. The detoxification ability of organs is increased, antioxidant enzyme activity is enhanced, lipid peroxidation is reduced, and the secretion of inflammatory factors TNF-α, IL-1β, il-6 is decreased. In conclusion, trehalose play an anti-aging role by activating genes related to Nrf2 pathway.     

Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase

This paper questions what types of molecular transformation are involved in the conversion of maltose to trehalose by trehalose synthase from Thermus caldophilus GK24. The reverse reaction pathway has been examined with the aid of alpha,alpha-(2,4,6,6',2',4',6",6"'-(2)H(8))trehalose (1). The mass data of the isolated reaction products clearly indicate that deuterated glucose is confined only to substrate molecules, and thus the reversible enzymatic conversion of trehalose into maltose proceeds through an intramolecular pathway.     

Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf

Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi‐arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG‐mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose‐6‐phosphate synthase (TPS), trehalose‐6‐phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress‐treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW^?1, and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW^?1 on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata.     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

Electroporation of maize embryogenic calli with the trehalose-6-phosphate synthase gene from Arabidopsis thaliana

Trehalose is a non-reducing disaccharide of glucose that occurs in a large number of organisms, playing an important role in desiccation and heat stress protection. Trehalose accumulation has proven to be an effective way of increasing drought tolerance in both model plants such as tobacco and important crops such as potato or rice. In this work we aim to genetically engineer maize with the Arabidopsis thaliana trehalose phosphate synthase gene (AtTPS1), involved in trehalose biosynthesis via electroporation. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene Bar as a selective agent was inserted in the plasmid vector pGreen0229 and used to transform maize inbred line Pa91 via electroporation. Fifteen putative transgenic plants (T0 generation) were obtained. Transgene integration in T0 plants was analyzed by Southern-blot analysis. T0 plants had normal phenotypes, although smaller than wild type plants. Contrary to wild type plants, when sexual organs emerged, tassels appeared at least 15 days earlier than ears in the same plant, rendering impossible the self-pollination of the T0 plant. These plants were then crossed with wild type plants and in some cases T1 seeds were obtained. T1 seeds presented deformities, especially the lack of endosperm, but it was still possible to germinate some of these seeds. The so obtained plants were tested by Northern blot but no AtTPS1 gene expression was detected, a fact possibly due to the incomplete insertion of the AtTPS1 gene or an extremely low gene expression level.     

过表达兴安落叶松TPP基因增强拟南芥对盐胁迫的耐受能力*

兴安落叶松(Larix gmelinii)是极为重要的造林针叶树种,具有早期速生、抗逆性强、生态效益好等特点。海藻糖参与调控干旱、寒冷、盐害等多种逆境胁迫,海藻糖-6-磷酸磷酸酶(TPP)是海藻糖合成通路的重要酶。从兴安落叶松逆境胁迫转录组中筛选到LgTPPI.1基因全长序列,克隆了其编码区(CDS),构建了重组载体并获得过表达LgTPPI.1拟南芥纯合株系。结果表明,LgTPPI.1 CDS全长1 236 bp,共编码411个氨基酸;LgTPPI.1基因的mRNA在根和茎中表达水平较低,在针叶中表达水平较高;过表达LgTPPI.1基因拟南芥在盐胁迫下海藻糖含量显著提高、脯氨酸和抗氧化酶类活性增加、胁迫响应标记基因表达上调、对盐胁迫的耐受性增强。这些结果表明,裸子植物利用与被子植物类似的海藻糖通路来耐受非生物胁迫。为后续进一步解析落叶松中海藻糖合成相关基因的功能,揭示裸子植物中针叶树对逆境的响应机制奠定基础。     

Apatite nanoparticles mediate intracellular delivery of trehalose and increase survival of cryopreserved cells

Cryopreservation of tissue cells is an important method to maintain cell viability and cellular function. However, cell viability and function are less than ideal by conventional cell cryopreservation methods, which may result in apoptosis and necrosis of cells in cryopreservation. Trehalose plays a role in maintaining cell structure and protecting cells from stress. However, owing to the difficulty in transport of trehalose across the cell membrane, its antifreeze effect is limited. A large amount of trehalose (up to 237 ± 8.5 mM) can be delivered to smooth muscle cells incubated in a medium containing trehalose and apatite nanomaterials at 37 °C for 6 h. Our data showed that trehalose was efficiently delivered intracellularly with the aid of nanoparticles (NP), with a loading efficiency up to 137.3 ± 34.5%, thus allowing for cryopreservation of LMC with nontoxic sugar as the sole cryoprotectant. Colloidal bioelastic apatite NP were used as bioactive promoters for the cryopreservation of tissue cells with trehalose. The addition of apatite NP in the medium substantially increased aortic smooth muscle cell cryosurvival, up to 83.6% (30% improvement over control without NP), a level comparable to that associated with the traditional Me₂SO cryoprotective regimen. Furthermore, the cytotoxicity of nanocapsules in the intracellular delivery of trehalose was negligible. This method provides a new option to enhance the activity of valvular cells for cryopreservation.     

Over-expression of BvMTSH,a fusion gene for maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase,enhances drought tolerance in transgenic rice

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]     

New insights on trehalose: a multifunctional molecule

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.     

Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Counteraction of trehalose on urea-induced protein unfolding: Thermodynamic and kinetic studies

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00 mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20 mol/L) in 8.00 mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30 mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.     

Role of trehalose in the spores of Streptomyces

Abstract Dormant spores of Streptomyces antibioticus contain large amounts of trehalose (11–12% of dry weight) and can be subjected to a dehydration treatment without a significant loss of viability. Loss of dehydration resistance coincided with a decrease in the trehalose level of the spores, under different conditions of incubation. The viability of dehydration-sensitive cells was enhanced by the presence of exogenous trehalose during dehydration. The morphology and functional activity of isolated membranes of S. antibioticus can be retained when dehydrated in the presence of trehalose. It is suggested that, in dormant spores of S. antibioticus , trehalose may serve to protect cellular components during dehydration by acting as a substitute for water.     

ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA

Rhizoctonia solani AG-1 IA is the causal agent of rice sheath blight (RSB) and causes severe economic losses in rice-growing regions around the world. The sclerotia play an important role in the disease cycle of RSB. In this study, we report the effects of reactive oxygen species (ROS) and trehalose on the sclerotial development of R. solani AG-1 IA. Correlation was found between the level of ROS in R. solani AG-1 IA and sclerotial development. Moreover, we have shown the change of ROS-related enzymatic activities and oxidative burst occurs at the sclerotial initial stage. Six genes related to the ROS scavenging system were quantified in different sclerotial development stages by using quantitative RT-PCR technique, thereby confirming differential gene expression. Fluorescence microscopy analysis of ROS content in mycelia revealed that ROS were predominantly produced at the hyphal branches during the sclerotial initial stage. Furthermore, exogenous trehalose had a significant inhibitory effect on the activities of ROS-related enzymes and oxidative burst and led to a reduction in sclerotial dry weight. Taken together, the findings suggest that ROS has a promoting effect on the development of sclerotia, whereas trehalose serves as an inhibiting factor to sclerotial development in R. solani AG-1 IA.     

Osmometric behavior of mouse oocytes in the presence of different intracellular sugars

In order to successfully cryopreserve oocytes using low concentrations of intracellular sugars, it is important to characterize their osmotic response in the presence of these intracellular sugars. In the present study, murine (B6D2F1) oocytes were microinjected with 0.8M glucose, trehalose or stachyose solutions to achieve an intracellular concentration equivalent to 0.1M, and then exposed to hypertonic solutions of increasing strength by supplementing an isotonic solution with 0.2, 0.3, 0.5, and 0.7 M of trehalose. Analysis of volumetric response of microinjected oocytes showed that the oocytes behaved as ideal osmometers in the presence of intracellular sugars and satisfied the Boyle van't Hoff relationship. Extrapolation of the osmotically inactive fraction (V macro b) from the Boyle van't Hoff relationship yielded values of 0.188+/-0.028, 0.212+/-0.042, 0.197+/-0.044, and 0.211+/-0.042 for control, glucose, trehalose and stachyose-injected oocytes, respectively. The present data revealing osmometric behavior of mouse oocytes in the presence of different intracellular sugars are important for the optimization of cryopreservation protocols using sugars.     

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.