定向进化提高来源于Arthrobacter ramosus的MTHase的热稳定性

麦芽寡糖基海藻糖水解酶(mahosyhrehalose hydrolase,MTHase)是以淀粉或麦芽糊精为底物制备海藻糖的关键酶之一.来源于Arthrobacter ramosus的MTHase,表达量好,比活高,但热稳定性差,限制了其工业化应用.采用定向进化技术,筛选得到L137M和A216T两个突变体,在60℃...     

Biochemical aspects of basidiospore maturation in Agaricus bisporus at various temperatures

Phosphatidylethanolamine is the main phospholipid of Agaricus bisporus basidiospores obtained under sterile conditions from young basidiomes with closed partial veils. Storing the basidiospores for five months at room temperature resulted in a complete loss of their germinating capacity. Conversely, storing them at a low temperature increased their germination rate by 15-20%. At both temperature levels, the phosphatidylcholine ratio significantly increased during storage to the level found in mature basidiospores. In addition, a drastic (8-10-fold) decrease in trehalose content occurred after two months of storage at room temperature. The trehalose content decreased only 1.5-fold at low temperatures. The involvement of trehalose and lipids in the retention of spore viability is discussed.     

Metabolic regulation of the trehalose content of vegetative yeast.

We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast. When cells of S. cerevisiae M1 growing exponentially at 30 degrees C were shifted to 45 degrees C for 20 min, or to 39 degrees C for 40 min, the concentration of trehalose increased by about 25-fold; an effect reversed upon lowering the temperature to 30 degrees C. This was compared to the more than 50-fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase. Whereas the latter was paralleled by a 12-fold increase in the activity of trehalose-6-phosphate synthase, no significant change in the activities of trehalose-synthesizing and -degrading enzymes was measured under heat shock conditions. Accordingly, cycloheximide did not prevent the heat-induced accumulation of trehalose. However, the concentrations of the substrates for trehalose-6-phosphate synthase, i.e. glucose-6-phosphate and UDP-glucose, were found to rise during heat shock by about 5-10-fold. Since the elevated levels of both sugars are still well below the Km-values determined for trehalose-6-phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions. A similar increase in the steady-state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP. This suggests that temperature-dependent changes in the kinetic parameters of glycolytic enzymes vary in steady-state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis. Trehalose, glucose-6-phosphate, UDP-glucose, and ATP, were all found to increase during the 40 min heat treatment at 39 degrees C. Since this also occurs in a mutant lacking the heat shock-induced protein HSP104 (delta hsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions. However, mutant delta hsp104, in contrast to the parental wild-type, was sensitive towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S. cerevisiae from the damage caused by a 50 degrees C treatment.     

Biochemical aspects of basidiospore maturation in <Emphasis Type="Italic">Agaricus bisporus</Emphasis> at various temperatures

Phosphatidylethanolamine is the main phospholipid of Agaricus bisporus basidiospores obtained under sterile conditions from young basidiomes with closed partial veils. Storing the basidiospores for five months at room temperature resulted in a complete loss of their germinating capacity. Conversely, storing them at a low temperature increased their germination rate by 15–20%. At both temperature levels, the phosphatidyl-choline ratio significantly increased during storage to the level found in mature basidiospores. In addition, a drastic (8–10-fold) decrease in trehalose content occurred after two months of storage at room temperature. The trehalose content decreased only 1.5-fold at low temperatures. The involvement of trehalose and lipids in the retention of spore viability is discussed.     

Cloning and characterization of a gene encoding trehalose phosphorylase (TP) from Pleurotus sajor-caju

Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.     

Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast. Synergetic effect between trehalose and phospholipid environment.

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.     

Induction of the lambda receptor is essential for effective uptake of trehalose in Escherichia coli.

Trehalose transport in Escherichia coli after growth at low osmolarity is mediated by enzyme IITre of the phosphotransferase system (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). The apparent Km (16 microM) of trehalose uptake is low. Since trehalose is a good source of carbon and the apparent affinity of the uptake system is high, it was surprising that the disaccharide trehalose [O-alpha-D-glucosyl(1-1)-alpha-D-glucoside] has no problems diffusing through the outer membrane at high enough rates to allow full growth, particularly at low substrate concentrations. Here we show that induction of the maltose regulon is required for efficient utilization of trehalose. malT mutants that lack expression of all maltose genes, as well as lamB mutants that lack only the lambda receptor (maltoporin), still grow on trehalose at the usual high (10 mM) trehalose concentrations in agar plates, but they exhibit the half-maximal rate of trehalose uptake at concentrations that are 50-fold higher than in the wild-type (malT+) strain. The maltose system is induced by trehalose to about 30% of the fully induced level reached when grown in the presence of maltose in a malT+ strain or when grown on glycerol in a maltose-constitutive strain [malT(Con)]. The 30% level of maximal expression is sufficient for maximal trehalose utilization, since there is no difference in the concentration of trehalose required for the half-maximal rate of uptake in trehalose-grown strains with the wild-type gene (malT+) or with strains constitutive for the maltose system [malT(Con)]. In contrast, when the expression of the lambda receptor is reduced to less than 20% of the maximal level, trehalose uptake becomes less efficient. Induction of the maltose system by trehalose requires metabolism of trehalose. Mutants lacking amylotrehalase, the key enzyme in trehalose utilization, accumulate trehalose but do not induce the maltose system.     

Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers

The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration.     

Both heat-shock and cold-shock influence trehalose metabolism in an entomopathogenic nematode

Heat-shock response is highly conserved in animals and microorganisms, and it results in the synthesis of heat-shock proteins. In yeast, heat-shock response has also been reported to induce trehalose accumulation. We explored the relationship between heat- (35 C) or cold-shock (1 and 10 C) and trehalose metabolism in the entomopathogenic nematode, Heterorhabditis bacteriophora. Because both heat- and cold-shocks may precede desiccation stress in natural soil environments, we hypothesized that nematodes may accumulate a general desiccation protectant, trehalose, under both situations. Indeed, both heat- and cold-shocks influenced trehalose accumulation and activities of enzymes of trehalose metabolism in H. bacteriophora. Trehalose increased by 5- and 6-fold in heat- and cold-shocked infective juveniles, respectively, within 3 hr of exposure, compared with the nematodes maintained at 25 C (culture temperature). The activity of trehalose-6-phosphate synthase (T6PS), an enzyme involved in the synthesis of trehalose, also significantly increased in both heat- and cold-shocked nematodes during the first 3 hr of exposure. Generally, the trehalose levels and activities of T6PS declined to their original levels within 3 hr when nematodes were transferred back to 25 C. In both heat- and cold-shocked nematodes, trehalase activity decreased significantly within the first 3 hr and generally returned to the original levels within 3 hr when these nematodes were transferred back to 25 C. The results demonstrate that the trehalose concentrations in H. bacteriophora are influenced by both heat- and cold-shocks and are regulated by the action of 2 trehalose-metabolizing enzymes, T6PS and trehalase. The accumulated trehalose may enhance survival of nematodes under both cold and warm conditions, but it may also provide simultaneous protection against desiccation that may result from subsequent evaporation or freezing. This is the first report of the relationship between trehalose metabolism and heat-shock for the Nematoda.     

The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at -8, -6, -4, -2, 0, 2, or 4 degrees C with different concentrations of methanol (0.5-7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at -8 and -6 degrees C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 degrees C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 degrees C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24h of storage at the effective temperatures (-4, -2, 0, 2, and 4 degrees C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 degrees C. At 0 degrees C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at -2 and 4 degrees C, the best results were with 0.1 M trehalose plus 3.0 M methanol.     

Improving the freeze tolerance of bakers' yeast by loading with trehalose

We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.     

Impacts of trehalose and l-proline on the thermodynamic nonequilibrium phase change and thermal properties of normal saline

Understanding the phase change behavior and thermal properties of cryoprotective agents (CPAs) in biological solutions is essential for enhancing the success of cryopreservation and biobanking. In this study, the phase change behavior and thermal properties of normal saline added with trehalose or l-proline were investigated using differential scanning calorimeter (DSC) and cryomicroscope during freezing and warming. The addition of trehalose or l-proline can eliminate the eutectic formation in normal saline. Trehalose had significantly lower latent heat release than l-proline does at a high concentration of 1 M (P < 0.05), while unfrozen water content of trehalose is significantly lower than that of l-proline at all the concentrations (P < 0.05). It was also found that addition of 0.2 M, 0.3 M and 1 M trehalose can achieve partial vitrification in normal saline and that the glass transition temperature rises along with the increase in concentrations of trehalose. However, no vitrification was observed in normal saline with l-proline at any concentrations. Besides, rates of ice crystal growth in normal saline added with trehalose are slower than those in normal saline with l-proline at the same concentrations. These results suggest that both trehalose and l-proline can act as CPAs by avoiding eutectic formation and inhibiting ice formation in normal saline for cell cryopreservation. It could be useful for CPA selection and designing in the future.     

Isolation and partial characterization of trehalase from Ascaris muscle.

The tissues of female Ascaris suum were assayed for alpha,apha'-glucoside 1-D-glucohydrolase (trehalase) activity. A soluble from of the enzyme was isolated from muscle tissue and purified approximately 37-fold. The enzyme was specific for trehalose as substrate. The pH optimum for enzymatic activity was found to be 6.0, and the apparent Km for trehalose was estimated to be 2.1 x 10-4 M. The product of the reaction was identified as D-glucose by chemical, chromatographic and enzymatic methods.     

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.     

Factors affecting the survival of Bradyrhizobium applied in liquid cultures to soya bean [Glycine max (L.) Merr.] seeds

AIMS: To determine the impact of medium composition, bacterial strain, trehalose accumulation, and relative humidity during seed storage on the survival of Bradyrhizobium japonicum on soya bean [Glycine max (L.) Merr.] seeds. METHODS AND RESULTS: Bacteria in liquid cultures were applied to seeds, and the number of survivors was quantified after 2, 24, 48, or 96 h. Addition of yeast extract to a defined medium increased on-seed survival 50- to 80-fold. Addition of 40 mmol l(-1) of NaCl to the medium doubled or tripled the accumulation of trehalose in cells and increased survival several fold, and the addition of both salt and trehalose had an additive effect. There was a threefold difference among strains in survival, and survival of the various strains was significantly correlated with differences in the accumulation of trehalose. The correlation between trehalose accumulation by bacteria and survival was also highly significant in other experiments. Studies in controlled humidity environments showed 100-fold or more differences in survival. CONCLUSIONS: The consistently significant correlation of trehalose content of cells with survival on seed suggests that trehalose is an important component of the survival mechanisms. When some of the factors (salt and trehalose in the medium plus humidity control) were studied in combination, several 100-fold increases in survival of bacteria on seeds were recorded. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible by manipulation of several parameters--strain selection, salt and trehalose content of the medium, control of relative humidity--to achieve substantial improvements in survival of Bradyrhizobium on soya bean seeds.     

Trehalose synthase converts glycogen to trehalose

Trehalose (alpha,alpha-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose <--> trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [(3)H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca(2+), but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (> or = 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.     

Mechanism for Recognition of an Unusual Mycobacterial Glycolipid by the Macrophage Receptor Mincle

Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1–1Glcα headgroup is liganded to a Ca²⁺ in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.     

The effect of sucrose and trehalose on viability of one- and two-cell rabbit embryos

The effect of sucrose and trehalose on the viability of one- and two-cell rabbit embryos was investigated. A significant decrease in the viability of one- and two-cell embryos exposed for 30 min. at 20 degrees C was observed. At 38 degrees C none of the two-cell embryos in a sucrose solution survived after 30 min exposure, while approximately 50% of the embryos survived in a trehalose solution. The cleveage rate in culture of two-cell embryos exposed both to 2.0 M or 1.45 M trehalose was significantly lower in comparison with the control group. However the survival rate after transfer of two-cell embryos exposed to 1.45 M trehalose solution at 20 degrees C remained the same as that of the control group.     

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.