De- and rehydration behavior of alpha,alpha-trehalose dihydrate under humidity-controlled atmospheres

Effects of humidity were investigated on de- and rehydration behavior of alpha,alpha-trehalose dihydrate (T(h)) throughout simultaneous measurements of differential scanning calorimetry and X-ray diffractometry (DSC-XRD) and simultaneous thermogravimetry and differential thermal analysis (TG-DTA). When T(h) was heated from room temperature under dry nitrogen atmosphere, a metastable anhydrous crystal (T(alpha)) was formed at 105 degrees C after dehydration of T(h). The resulting T(alpha) melted at 125 degrees C and became amorphous, followed by cold crystallization from 150 degrees C giving rise to a stable anhydrous crystal T(beta). Under a highly humid atmosphere, on the other hand, T(beta) was formed at 90 degrees C directly as a result of T(h) dehydration. T(alpha) was readily rehydrated and turned back to T(h) when nitrogen gas with low water vapor pressure of 2.1kPa was admitted, whereas high water vapor pressure up to 7.4kPa was required for rehydration of T(beta) into T(h). This study provided a picture of pathways that link various solid forms of trehalose, taking into account the effects of a humid environment.     

Monitoring Phase Transformations in Intact Tablets of Trehalose by FT-Raman Spectroscopy

The purpose of this study is to monitor phase transformations in intact trehalose tablets using FT-Raman spectroscopy. Tablets of trehalose dihydrate, amorphous trehalose (obtained by freeze-drying aqueous trehalose solutions), and anhydrous trehalose (β-trehalose) were prepared. The tablets were exposed to different conditions [11% and 0% RH (60°C); 75% RH (25°C)] and monitored periodically over 96 h using Raman spectroscopy. Within 96 h of storage, the following phase transformations were observed: (1) trehalose dihydrate → β-trehalose (11% RH, 60°C), (2) trehalose dihydrate → α-trehalose (0% RH, 60°C), (3) β-trehalose → trehalose dihydrate (75% RH, 25°C), and (4) amorphous trehalose → trehalose dihydrate (75% RH, 25°C). FT-Raman spectroscopy was a useful technique to identify the solid form and monitor multiple-phase transformations in intact trehalose tablets stored at different conditions.     

Reversible dehydration of trehalose and anhydrobiosis: from solution state to an exotic crystal?

Physico-chemical properties of the trehalose-water system are reviewed with special reference to the transformations that may shed light on the mechanism of trehalose bio-protection. Critical analysis of solution thermodynamics is made in order to scrutinize trehalose properties often called 'anomalous' and to check the consistency of literature results. Discussion on the conversion between the solid state polymorphic forms is given, with a special emphasis of the transformations involving the newly identified anhydrous crystalline form of alpha,alpha-trehalose, TRE(alpha). This exotic crystal is almost 'isomorphous' with the dihydrate crystal structure, and possesses the unique feature of reversibly absorbing water to produce the dihydrate, without changing the main structural features. The reversible process could play a functional role in the well-known ability of this sugar to protect biological structures from damage during desiccation. The final aim of the paper is to add some new insights into and to reconcile previous hypotheses for the peculiar 'in vivo' action of trehalose.     

Trehalose amorphization and recrystallization

The stability of the amorphous trehalose prepared by using several procedures is presented and discussed. Amorphization is shown to occur by melting (T(m)=215 degrees C) or milling (room temperature) the crystalline anhydrous form TRE-beta. Fast dehydration of the di-hydrate crystalline polymorph, TRE-h, also produces an amorphous phase. Other dehydration procedures of TRE-h, such as microwave treatment, supercritical extraction or gentle heating at low scan rates, give variable fractions of the polymorph TRE-alpha, that undergo amorphization upon melting (at lower temperature, T(m)=130 degrees C). Additional procedures for amorphization, such as freeze-drying, spray-drying or evaporation of trehalose solutions, are discussed. All these procedures are classified depending on the capability of the undercooled liquid phase to undergo cold crystallization upon heating the glassy state at temperatures above the glass transition temperature (T(g)=120 degrees C). The recrystallizable amorphous phase is invariably obtained by the melt of the polymorph TRE-alpha, while other procedures always give an amorphous phase that is unable to crystallize above T(g). The existence of two different categories is analyzed in terms of the transformation paths and the hypothesis that the systems may exhibit different molecular mobilities.     

Dependence of trehalose protective action on the initial phase state of dipalmitoylphosphatidylcholine bilayers

Dipalmitoylphosphatidylcholine (DPPC) bilayers hydrated in the presence of trehalose were equilibrated at various temperatures (4, 20, and 60 degrees C) corresponding to the crystalline Lc, gel L beta', and liquid-crystalline L alpha phases, respectively, and then desiccated at these temperatures or freeze-dried at -80 degrees C to ca. DPPC dihydrate. The thermotropic behavior of the resulting DPPC/trehalose mixtures was investigated by differential scanning calorimetry and found to be dependent not only on the trehalose concentration but also on the phase state of the hydrated bilayers prior to their drying. Trehalose was most effective when the desiccation was carried out from the L alpha phase at 60 degrees C. In this case, one trehalose molecule per two DPPC molecules was sufficient to depress the melting temperature from values typical of DPPC dihydrate to 45 degrees C. Trehalose's influence decreased when dried from the L beta' phase and was significantly less pronounced when dried from the Lc phase. These data show that trehalose's protective influence depends on the initial phase state of the lipid bilayer and reaches its maximum in the liquid-crystalline state. The possible role of this effect in anhydrobiosis is pointed out.     

Trehalose uptake through P2X7 purinergic channels provides dehydration protection

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.     

Studies of the Crystallization of Amorphous Trehalose Using Simultaneous Gravimetric Vapor Sorption/Near IR (GVS/NIR) and “Modulated” GVS/NIR

The purpose of this research was to investigate the influence of changes in the amorphous state on the crystallization of trehalose. Amorphous trehalose is known to stabilize biomaterials; hence, an understanding of crystallization is vital. Amorphous trehalose, prepared by spray-drying, was exposed to either a single step (0–75%) in relative humidity (RH) or to modulated 0–75–0% RH to cause crystallization. For the single-step experiment, two samples crystallized in a predictable manner to form the dihydrate. One sample, while notionally identical, did not crystallize in the same way and showed a mass loss throughout the time at 75% RH, with a final mass less than that expected for the dihydrate. The idiosyncratic sample was seen to have a starting near infrared (NIR) spectra similar to that exhibited by anhydrous crystalline trehalose, implying that short-range order in the amorphous material (or a small amount of crystalline seed, not detectable using powder X-ray diffraction) caused the sample to fail to form the dihydrate fully when exposed to high RH. The modulated RH study showed that the amorphous material interacted strongly with water; the intensity of the NIR traces was not proportional to mass of water but rather the extent of hydrogen bonding. Subsequent crystallization of this sample clearly was a partial formation of the dihydrate, but with the bulk of the sample then shielded such that it was unable to show significant sorption when exposed to elevated RH. It has been shown that the nature of the amorphous form will alter the way in which samples crystallize. With oscillation in RH, it was possible to further understand the interactions between water and amorphous trehalose.     

Infrared spectroscopic study on the properties of the anhydrous form II of trehalose. Implications for the functional mechanism of trehalose as a biostabilizer

FTIR spectra were obtained for several different states of trehalose including dihydrate crystal, anhydrous form II (designated by Gil, A. M.; Belton, P. S.; Felix V. Spectrochim. Acta 1996, A52, 1649-1659), anhydrate crystal, dried melt, amorphous solid and aqueous solution. From the observation of the symmetric and antisymmetric stretch vibrations of the glycosidic linkage, it is found that this sugar assumes at least three types of backbone conformations. Among them, the conformation with C(2) symmetry is characterized as 'open state', which means that the sugar easily absorbs water molecules. The conformation of the sugars in anhydrous form II and in freeze-dried trehalose is shown to be in the open state. Next, the hygroscopic properties of the anhydrate, form II and the amorphous solid are compared based on their IR spectra. Interestingly, form II alone is converted to the original dihydrate in a week under mild environmental-like conditions: relative humidity of 40% and room temperature. These results suggest the possibility that form II plays a role in avoiding the devitrification of the sugar glass. Finally, we discuss the role of form II in preserving freeze-dried biomaterials.     

Innovative crystal transformation of dihydrate trehalose to anhydrous trehalose using ethanol

The crystal transformation of dihydrate trehalose to anhydrous trehalose was investigated using ethanol and a new type of crystal particle with porous structure could be obtained. The specific surface area of the anhydrous crystal transformed at 50 degrees C was 3.3 m(2)/g, with a median pore diameter of 0.21 microm, and void volume of 0.22 mL/g. The crystal transformation was monitored by measuring the crystal moisture content. The crystal transformation rates could be correlated with the Avrami equation, using the mechanism parameter n=11.5, suggesting that the change of surface area occurred during crystal transformation from dehydrate to anhydrous trehalose. The apparent activation energy of the crystal transformation was 132 kJ/mol.     

Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.     

Amorphous trehalose dihydrate by cryogenic milling

Trehalose dihydrate is a safe, naturally occurring disaccharide used as a food ingredient and pharmaceutical excipient. It has been reported that room temperature milling does not lead to the formation of amorphous trehalose dihydrate. This paper reports the behaviour of trehalose dihydrate upon milling at cryogenic temperatures as studied by DSC, TGA, XRPD and Raman spectroscopy. We have demonstrated that the crystal to glass transformation for trehalose dihydrate is possible using cryogenic milling. This is the first reported example of cryogenic milling (a mild and widely applicable technique) applied to generating amorphous hydrates.     

Combined effects of trehalose and cations on the thermal resistance of beta-galactosidase in freeze-dried systems

The purpose of this study was to investigate the combined effects of trehalose and cations on the preservation of beta-galactosidase in freeze-dried systems and their relationship to physical properties. Differential scanning calorimetry was employed to measure the glass transition temperature (T(g)) and the endothermal peak area, related to the amount of crystalline trehalose dihydrate present in the samples. In systems in which the trehalose matrix was humidified to conditions which allowed a high proportion of trehalose to crystallize, the enzyme was rapidly inactivated upon heating at 70 degrees C. In these conditions the addition of CsCl, NaCl and particularly KCl or MgCl(2), improved the enzyme stability with respect to that observed in matrices containing only trehalose. For a given moisture content, addition of salts produced very little change on the glass transition temperature; therefore the protective effect could not be attributed to a higher T(g) value. The crystallization of trehalose dihydrate in the humidified samples was delayed in the trehalose/salt systems (principally in the presence of Mg(2+)) and a parallel improvement of enzyme stability was observed.     

Catalytic versatility of trehalase: synthesis of alpha-D-glucopyranosyl alpha-D-xylopyranoside from beta-D-glucosyl fluoride and alpha-D-xylose

Trehalase was previously shown (see ref. 5) to hydrolyze alpha-D-glucosyl fluoride, forming beta-D-glucose, and to synthesize alpha, alpha-trehalose from beta-D-glucosyl fluoride plus alpha-D-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. alpha-D-Glucopyranose and alpha-D-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with beta-D-glucosyl fluoride. As little as 0.2mM added alpha-D-glucose (0.4mM alpha-D-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mM beta-D-glucosyl fluoride at 0 degrees. Digests of beta-D-glucosyl fluoride plus alpha-D-xylose yielded the alpha, alpha-trehalose analog, alpha-D-glucopyranosyl alpha-D-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when beta-D-glucosyl fluoride is the donor, and for water when alpha-D-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.     

Trehalose maintains phase separation in an air-dried binary lipid mixture

Mixing and thermal behavior of hydrated and air-dried mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-d70-sn-glycero-3-phosphocholine (DSPCd-70) in the absence and presence of trehalose were investigated by Fourier transform infrared spectroscopy. Mixtures of DLPC:DSPCd-70 (1:1) that were air-dried at 25 degrees C show multiple phase transitions and mixed phases in the dry state. After annealing at high temperatures, however, only one transition is seen during cooling scans. When dried in the presence of trehalose, the DLPC component shows two phase transitions at -22 degrees C and 75 degrees C and is not fully solidified at -22 degrees C. The DSPCd-70 component, however, shows a single phase transition at 78 degrees C. The temperatures of these transitions are dramatically reduced after annealing at high temperatures with trehalose. The data suggest that the sugar has a fluidizing effect on the DLPC component during drying and that this effect becomes stronger for both components with heating. Examination of infrared bands arising from the lipid phosphate and sugar hydroxyl groups suggests that the strong effect of trehalose results from direct interactions between lipid headgroups and the sugar and that these interactions become stronger after heating. The findings are discussed in terms of the protective effect of trehalose on dry membranes.     

Supercritical CO(2) fluid extraction of crystal water from trehalose dihydrate. Efficient production of form II (T(alpha)) phase

Form II is a kind of metastable crystalline form of trehalose anhydrate, and it is easily converted to the dihydrate crystal by absorbing water in moist atmosphere at room temperature (Akao et al., Carbohydr. Res. 2001, 334, 233-241). It can be utilized as an edible and nontoxic desiccant, and thus its efficient production from the dihydrate is significant from a viewpoint of industrial applications. In this study, we attempt to extract crystal water from the dihydrate using supercritical CO(2). We examine the dependence of extraction efficiency on the extraction time, the temperature and pressure of the fluid. Then, FTIR measurements are carried out to detect the extracted water and to identify the polymorphic phase of the sugar sample after the extraction treatment. In particular, the so-called first derivative euclidean distance analysis for IR spectra is shown to be quite useful for the structural identification. Consequently, we demonstrate that form II is produced from the dihydrate through supercritical CO(2) fluid extraction if appropriate temperature and pressure conditions (around 80 degrees C and 20 MPa) are maintained.     

Cholesterol crystalline polymorphism and the solubility of cholesterol in phosphatidylserine

There is a marked hysteresis between the heating and cooling polymorphic phase transition of anhydrous cholesterol. At a scan rate of 0.05 degrees C/min the difference in transition temperatures between heating and cooling scans is approximately 10 degrees C. This phenomenon also occurs with mixtures of cholesterol with phosphatidylserine and can result in an underestimation of the amount of crystalline cholesterol in a sample that has not been cooled sufficiently. With 1-palmitoyl-2-oleoyl phosphatidylserine and 1-stearoyl-2-oleoyl phosphatidylserine the cholesterol crystallites form while the lipid remains in the L(alpha) phase. Sonication of dimyristoyl phosphatidylserine with a 0.4 mol fraction cholesterol results in the loss of cholesterol crystallite diffraction, but only a partial loss of the polymorphic transition detected by calorimetry. We therefore conclude that the thermal history of the sample can have profound effects on the appearance of the polymorphic phase transition of cholesterol by differential scanning calorimetry. Depending on the morphology of the vesicles, diffraction methods may underevaluate the amount of cholesterol crystallites present.     

Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii

A previous paper indicated that corynomycolates synthesized by the fluffy layer fraction prepared from Corynebacterium matruchotii cells appeared exclusively as alpha-trehalose 6-monocorynomycolate (TMM) (T. Shimakata, K. Tsubokura, T. Kusaka, and K. Shizukuishi, 1985, Arch. Biochem. Biophys. 238, 497-508). In the present communication, the role of trehalose in the synthesis and subsequent metabolism of corynomycolic acids was reexamined. Consequently the following facts were clarified: (i) trehalose 6-phosphate (T-6-P), but not trehalose, stimulated corynomycolate synthesis from palmitate in the presence of ATP; the immediate product was TMM, which showed a rapid turnover. Since the turnover was blocked by addition of alpha-trehalose, only TMM accumulated among corynomycolate-containing substances. These results strongly suggested that T-6-P is an essential component as the acceptor in corynomycolate-synthetic system; (ii) TMM was the precursor not only to alpha-trehalose 6,6'-dicorynomycolate (TDM) and free corynomycolic acids but also to cell wall corynomycolate; (iii) addition of alpha-trehalose blocked the transfer of the corynomycolate moiety from TMM to cell wall corynomycolate, TDM, and free corynomycolic acids to a similar extent. These results clearly indicate that trehalose plays an essential role in the metabolism of corynomycolate after Claisen condensation and subsequent reduction in C. matruchotii.     

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Schizophyllum communealpha,alpha-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into alpha-d-glucose 1-phosphate and alpha-d-glucose. Recruitment of phosphate by the free enzyme induces alpha,alpha-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km=21,000 m(-1).s(-1)) was dramatic (>or=10(7)-fold) in purified Arg507-->Ala (R507A) and Lys512-->Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4-, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with alpha,alpha-trehalose (Ki=0.4 microm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki=300 microm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward alpha,alpha-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively.     

Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.