Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by β-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).     

Thermal Destabilization of Stem Bromelain by Trehalose

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5° and 7°C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60°C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.     

Overexpression and characterization of a thermostable trehalose synthase from Meiothermus ruber

A thermostable trehalose synthase (TreS) gene from Meiothermus ruber CBS-01 was cloned and overexpressed in Escherichia coli. The purified recombinant TreS could utilize maltose to produce trehalose, and showed an optimum pH and temperature of 6.5 and 50°C, respectively. Kinetic analysis showed that the enzyme had a twofold higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, indicating maltose as the preferred substrate. The TreS also had a weak hydrolytic property with glucose as the byproduct, and glucose was a strong competitive inhibitor of the enzyme. The maximum production of trehalose by the enzyme reached 65% at 20°C. The most importantly the enzyme could maintain very high activity (above 90%) at pH 4.0–8.0 and 60°C 5 h. These results provided that the stable TreS was suitable for the industrial production of trehalose from maltose in a one-step reaction.     

Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [²H₂]trehalose combined with unlabeled trehalose was converted to [²H₂]maltose and maltose, but without any production of [²H]maltose or [²H]trehalose and with no incorporation of exogenous [²H₇]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.     

Multiple Effects of Viscosity,Water Activity and Glass Transition Temperature on Peroxidase Activity in Binary and Ternary Carbohydrate Solutions

The individual and combined effects of water activity (a_w), bulk viscosity and glass transition temperature (Tg’) on the activity of horseradish peroxidase (HRP) in buffered sugars (glucose, trehalose and maltose) and maltodextrin solutions were investigated. Viscosity was the most important factor in the inhibition of HRP activity; however, when Tg’ was changed by the using solutes with different molecular weight, it became a key factor in the modulation of enzyme activity. Viscosity being equal, the sugar addition to maltodextrin solution lowered a_w and lowered Tg’ causing an increase of the enzymatic activity. Nevertheless, an inhibition of the HRP activity occurred when a_w values of 0.87 were reached due to the addition of glucose, which, among the tested sugars, showed the lowest molecular weight. Among disaccharides, maltose was more effective than trehalose in impairing the enzyme activity both in binary and ternary systems, and this is due to a non competitive biochemical inhibition exerted by this sugar on HRP. When compared to glucose, maltose and trehalose were more effective in reducing HRP activity only in the low viscosity range whilst in the high viscosity range (1–4 10^?6 m² s^?1) glucose, despite its lower Tg’ value, was slightly more efficient than disaccharides due to its a_w lowering effect.     

Vacuum foam drying for preservation of LaSota virus: Effect of additives

The purpose of this research was to apply vacuum foam drying (VFD) for processing of LaSota virus and to screen formulation additives for its stability. The aqueous dispersion of harvest containing sucrose or trehalose in combination with additive (monosaccharides, polymers, N-Z-amine) was prepared. The diluted dispersions in vials were vacuum concentrated, foamed to form a continuous structure, and vacuum dried. The products were evaluated for foam characteristics, residual moisture, virus titer, x-ray diffraction pattern, and stability profile. The foamability increased with solid content in solutions. The foamability of sucrose was enhanced with incorporation of N-Z-amine (10% and 15% wt/vol) and polyvinyl pyrrolidone (PVP K30, 3% wt/vol). The fructose- or galactose-containing mixtures were deposited irregularly on the vial surface. The virus titer increased with disaccharides in the formulation. Sucrose provided better protection than trehalose. Unlike lyophilization, N-Z-amine with sucrose protected the virus from Millard’s Browning. Amino acids do not have a catalytic effect on hydrolysis of sucrose during VFD. Monosaccharides were ineffective. A synergistic effect of PVP K30 or polyethylene glycol 6000 (3% wt/vol) with N-Z-amine provided the maximum virus titer (6.97 and 7.15, respectively). This formulation retained the desired virus potency at 5°, 25°, and 40°C. The diffraction pattern revealed that a threshold concentration of N-Z-amine was required for inhibiting crystallization of sucrose during VFD. VFD was successfully applied to produce a solid LaSota formulation. The products were amorphous and did not devitrify on storage. Published: July 21, 2006     

Induction of an Increase in Nerve Growth Factor Content in the Cell-conditioned Medium of Mouse L-M Cells by Basic Peptides

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu²⁺ , Hg²⁺, Zn²⁺, and Tris.     

The uptake and hydrolysis of disaccharides by fast-and slow-growing species of Rhizobium

Slow growing strains of rhizobia appear to lack both uptake systems and catabolic enzymes for disaccharides. In the fast-growing strains of rhizobia there are uptake mechanisms and catabolic enzymes for disaccharide metabolism. In Rhizobium leguminosarum WU 163 and WU235 and R. trifolii WU290, sucrose and maltose uptake appears to be constitutive whereas in R. meliloti WU60 and in cowpea Rhizobium NGR234 uptake of these disaccharides is inducible. There is evidence that there are at least two distinct disaccharide uptake systems in fast-growing rhizobia, one transporting sucrose, maltose and trehalose and the other, lactose. Disaccharide uptake is via an active process since uptake is inhibited by azide, dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone but not by arsenate. Bacteroids of R. leguminosarum WU235 and R. lupini WU8 are unable to accumulate disaccharides.     

Purification and properties of invertase from a glutamate-producing bacterium

Invertase was purified from the cell extracts of the glutamic acid bacterium (Brevibacterium divaricatum) by ammonium sulfate fractionation, batch theatment with DEAE-cellulose, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was proved to be almost homogeneous by polyacrylamide gel electrophoresis.The molecular weight of the enzyme was estimated to be 92,000 by both gel filtration and SDS-polyacrylamide gel electrophoresis methods. The optimum pH and temperature for the activity were 6.8 and 40°C. The enzyme was highly specific to sucrose as substrate, having only 10% as much activity toward raffinose as that toward sucrose, and being inert toward other disaccharides: maltose, trehalose, lactose, melibiose and cellobiose. The K_m value for sucrose was 0.19 M. The enzyme required phosphate or arsenate ions for activity. Monovalent or divalent Cu ions and sulfhydryl reagents inhibited the enzyme.     

Quinoxalines Derived from Disaccharides with O-Phenylenediamine under Weakly Acidic Reflux Conditions

Quinoxaline derivatives from disaccharides (isomaltose, maltose, laminaribiose, trehalose and sucrose) and o-phenylenediamine (OPD) under weakly acidic reflux conditions were subjected to GLC and GC-MS analyses.

Three high-molecular-weight quinoxaline derivatives (IA-I, -II and -III) were obtained from isomaltose in a yield of 90%, indicating no splitting of the glucosidic linkages. Both low- and high-molecular-weight quinoxaline derivatives were obtained from 5 mm maltose. From 50 mm maltose, a low-molecular-weight quinoxaline, GA-I, was obtained predominantly. Quinoxalines from laminaribiose were both high (LA-I and -II) and low (ATBQ, GA-I, G-l and GA-II) molecular-weight derivatives, in yields of 18% and 48%, respectively. LA-I and -II were stereoisomers, at the C-3 position, of laminaribiose. Trehalose gave no quinoxalines, and sucrose gave only small amounts of low-molecular-weight quinoxalines.

From these results, we found that 1,6-linkages of the disaccharides used are most resistant to the OPD degradation, followed by the 1,4- and 1,3-linkages, in that order. Possible pathways for the formation of quinoxalines are proposed.     

Temperature dependent vibrational modes of glycosidic bond in disaccharide sugars

We studied the temperature dependent vibrational modes of the glycosidic bond in trehalose, sucrose, and maltose at wavenumbers ranging from 1000 to 1200 cm(-1). We found that the slope of temperature dependent Raman shifts of the glycosidic bond in trehalose and sucrose changed at temperatures around 120 degrees C, indicating a bond length or a bond angle (dihedral and torsional angles) change. However, we did not observe any slope change in maltose because the melting temperature of maltose is very close to 120 degrees C. We also found, at temperatures below 120 degrees C, that Raman shifts of the vibrational modes of the glycosidic bond in trehalose showed the strongest temperature dependence among the three disaccharides.     

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.     

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at −80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.     

Evaluating potential sugar food sources from the olive grove agroecosystems for Prays oleae parasitoid Chelonus elaeaphilus

Chelonus elaeaphilus Silvestri (Hymenoptera: Braconidae) is a host-specific parasitoid of the olive moth, Prays oleae (Bernard), that can cause parasitism rates of up to 80% in Mediterranean olive groves. A laboratory study was carried out to assess the potential of sugars provided by wild plant species in olive grove agroecosystem to enhance the fitness of C. elaeaphilus. Insects were reared in a climate-controlled chamber at 25?±?2°C, 60?±?5% relative humidity (RH) with a photoperiod of 16:8 (L:D) h. Five naturally occurring wild plant nectar sugars (sucrose, fructose, glucose, maltose and mannose) were tested for their effect on insect longevity. The nectar sugar content of sucrose, fructose and glucose in 12 selected olive grove agroecosystem plant species was analysed and categorised on the basis of sugar ratios. Female insect longevity was increased when they were fed on both sucrose and glucose compared to either maltose or fructose, suggesting that sucrose-dominant nectars would bene?t this parasitoid. Sucrose was predominant in the nectar of five of the studied plant species (Silene gallica, Borago officinalis, Echium plantagineum, Lavandula stoechas and Lonicera hispânica). The results are discussed in terms of potential enhancement of the biological control of P. oleae.     

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

Thermal stability of <Emphasis Type="Italic">α</Emphasis>-amylase in aqueous cosolvent systems

The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T _m )_app and activation energy (E _a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T _m )_app were 20°C, 14°C, 13°C and 9°C, respectively. The E _a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.     

Viability and thermal stability of a strain of Saccharomyces cerevisiae freeze-dried in different sugar and polymer matrices

The viability and thermal stability of a freeze-dried yeast strain were studied in relation to some physical properties of the matrices in which the cells were freeze-dried. Samples of inoculum with solutions of the matrix components [polyvinylpyrrolidone (PVP), maltose, trehalose, maltodextrins, or mixtures of maltodextrin and trehalose] and controls without matrices were freeze-dried and then equilibrated at several relative humidities. Viability was determined before and after freeze-drying and after heat treatment (100 min at 70 °C). Freeze-drying with trehalose, PVP, maltose or 1.8-kDa maltodextrin, and mixtures of maltodextrin/trehalose increased viability in comparison with controls. The 3.6-kDa maltodextrin was ineffective at protecting the cells during freeze-drying. The glass transition temperature (T _g), which depends on moisture content, was indicated as a possible factor to determine the stability of labile materials. Protective effects of the excipients during thermal treatment were analysed in relation to the physical changes (collapse or structural shrinkage) which were dependent on the T _g of the systems. The presence of a certain amount of amorphous disaccharides during freeze-drying and heating was found to be a critical factor for ensuring cell viability, which was protected even in rubbery (above T _g) matrices. Received: 4 December 1998 / Received last revision: 2 March 1999 / Accepted: 14 March 1999     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Viability of dry Trichoderma harzianum spores under storage

Spores of the potential biocontrol agent Trichoderma harzianum P1 were prepared without (M1) and with heat shock (40?°C for 90?min) after fermentation (M2), filtered into a paste and dried over silica gel. M1 and M2 exhibited high viability (55%) and similar initial trehalose contents (4.0 and 5.4%, respectively) after slow drying. No significant differences in viability were found between treatments during storage for 110 days under different temperatures, T (8, 33 and 42?°C) and water activities, a _w (0.03, 0.33 and 0.75). Viability of spores, after storage at a _w =0.03 were 100 and 70% for 8 and 33?°C, respectively. During storage, decrease in trehalose content and viability was faster at a _w =0.75 and 42?°C. Loss of viability was modeled by a first order kinetic model depending on 1/T and a _w . M2 (with heat shock) showed slightly higher trehalose contents than M1 which resulted in 100% viability after 52 days at 8?°C.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.