Transfructosylation of Thiol Group by β-Fructofuranosidases

β-Fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers’ yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-furctofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the β-fructofranosidase.     

Galactosylation of thiol group by beta-galactosidase

beta-Galactosidase catalyzed beta-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-beta-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzae beta-galactosidase hydrolyzed p-nitrophenyl S-beta-D-galactoside most rapidly among several beta-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolor enzyme was as effective as the A. oryzae enzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulans galactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8-2.8 M did not affect the synthetic yield of the thiogalactoside so greatly.     

Dependence of wheat callus growth,differentiation and mineral content on carbohydrate supply

The effects of different carbon sources on the growth, differentiation and mineral content of wheat callus were investigated. Callus originating from immature embryos showed optimal growth produced the highest ratio of shoots when it was cultured on the medium containing 0.058 M sucrose. Higher carbohydrate concentrations reduced both shoot formation and growth. On the other hand, when sucrose concentration was less than 0.029 M neither differentiation nor greening was observed. Mannitol had a stimulating effect on shoot formation when the medium containing 0.029 M sucrose was supplemented by mannitol to get the final concentration of 0.058 M. The respiration rate increased along with increasing concentration of sucrose and glucose, and reached a maximum in the case of sucrose at the concentration of 0.263 M. On the addition of different concentrations of mannitol to a 0.058 M sucrose medium the respiration remained essentially unchanged. The mineral content of the tissue cultures also depended on the carbohydrate concentration. The water content decreased with increasing carbohydrate concentrations and among carbohydrates examined, sucrose was the most effective. The nitrogen and potassium contents of the calli reached their maximum values at 0.117 M–0.175 M carbohydrate content. The highest phosphorus contents were detected at 0.350 M–0.468 M carbohydrates. Phosphorus proved to be the most sensitive to osmotic changes.     

Enzymatic synthesis of N-acetylglucosaminyl-cyclodextrin by the reverse reaction of N-acetylhexosaminidase from jack bean

Novel heterobranched cyclodextrins (CDs), N-acetylglucosaminyl-cyclodextrins (GlcNAc-CD), were synthesized from a mixture of GlcNAc and alpha, beta, or gamma CD by the reverse reaction of N-acetylhexosaminidase from jack bean. Optimum pH and temperature for the production of GlcNAc-alpha CD by N-acetylhexosaminidase were pH 4.9 and 50-70 degrees C, respectively. The maximum yield of GlcNAc-alpha CD was 17.5% (mol/mol) at the concentration of 1 M GlcNAc and 0.25 M alpha CD. The reverse reaction product, GlcNAc-alpha CD, was separated into two peaks by HPLC analysis on the ODS column. Their structures were identified as 6-O-beta-D-N-acetylglucosaminyl-alpha CD and 2-O-beta-D-N-acetylglucosaminyl-alpha CD by FAB-MS and NMR spectroscopies. N-Acetylhexosaminidase from jack bean also synthesized N-acetylgalactosaminyl-alpha CD from N-acetylgalactosamine and alpha CD.     

The acylation of L-glycerol-3-phosphate by microsomes from rat brain: effects arising from the properties of the reaction medium

Abstract— The velocity of the reaction catalysed by acyl-CoA: l -giycerol-3-phosphate acyltransferase (EC 2.3.1.15) of microsomes from rat brain was affected by the nature of the buffering agent, the ionic strength and the sucrose concentration of the reaction medium. The enzyme was inhibited by buffers based on trimethyl-pyridine, diethyl barbituric acid, and boric acid. Buffers based on N-ethyl morpholine, potassium phosphate, sodium arsenate, imidazole, tris and triethanolamine were not inhibitory. Dithiothreitol protected the enzyme and produced maximal activity at levels in the reaction medium between 0.2 and 2.8 mM. Optimum ionic strength was determined by varying the concentration of a potassium phosphate buffer and in this medium the optimum ionic strength was about 0.2 M. In other studies with sodium formate, potassium acetate and other salts there was a broad plateau of activity in a range about 0.2 M. A study of pH vs. activity with two different buffering agents at constant ionic strength showed a broad maximum of activity from pH 7.2 to pH 7.8. The velocity of the reaction could be further increased by the inclusion of 0.25 M-sucrose in the reaction medium in the presence of 0.2 M salts. The sucrose effect produced maximum velocities at sucrose concentrations ranging from 0.2 to 0.6 M. The studies reported here indicate that the activity of the enzyme is dependent upon the state of hydration of the microsomal membranes and in part on the ability of the enzyme or membrane to cope with large micelles of S-palmityl-CoA.     

Multiple steady states in a continuous stirred tank reactor: an experimental case study for hydrolysis of sucrose by invertase

The purpose of this work was to validate experimentally that multiple steady states may be achieved in a continuous stirred tank reactor (CSTR) during hydrolysis of sucrose by invertase. Experiments were done with four initial sucrose concentrations (0.1, 0.175, 0.584 and 1 M) to study their effect on residual sucrose and reaction rate at steady state. Two different steady states (S=0.7 M, r=9×10⁻⁴ mol/l min and S=0.135 M, r=1.54×10⁻³ mol/l min) were found depending on initial concentration of sucrose in the reactor. Two stable steady states were possible in a CSTR using invertase for the hydrolysis of sucrose. A third possible steady state can be derived theoretically, but it should be a metastable condition because any small disturbance in the system will result in transitory states stabilizing at sugar concentrations of either 0.135 or 0.7 M.     

Caution in the use of 2-iminothiolane (Traut's reagent) as a cross-linking agent for peptides. The formation of N-peptidyl-2-iminothiolanes with bombesin (BN) antagonist (D-Trp(6),Leu(13)-psi[CH(2)NH]-Phe(14))BN(6-14) and D-Trp-Gln-Trp-NH(2).

During a study aimed at generating a bispecific molecule between BN antagonist (D-Trp(6),Leu(13)-psi[CH(2)NH]-Phe(14))BN(6-14) (Antag1) and mAb22 (anti-FcgammaRI), we attempted to cross-link the two molecules by introducing a thiol group into Antag1 via 2-iminothiolane (2-IT, Traut's reagent). We found that reaction of Antag1 with 2-IT, when observed using HPLC, affords two products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesized a model peptide, D-Trp-Gln-Trp-NH(2) (TP1), the N-terminal tripeptide of Antag1. Reaction of TP1 with 2-IT for 5 min gave products 1a and 3a; the concentration of 1a decreased with reaction time, whereas that of 3a increased. Thiol 1a, the expected Traut product, was identified by collecting it in a vial containing N-methylmaleimide and then isolating the resultant Michael addition product 2a, which was confirmed by mass spectrometry. Thiol 1a is stable at acidic pH, but is unstable at pH 7.8, cyclizes and loses NH3 to give N-TP1-2-iminothiolane (3a), ES-MS (m/z) [602.1 (M+H)(+)], as well as regenerating TP1. Repeat reaction with Antag1 and 2-IT allowed us to isolate N-Antag1-2-iminothiolane (3b), FAB-MS (m/z) [1212.8 (M+H)(+)] and trap the normal Traut product 1b as its N-methylmaleimide Michael addition product 2b, ES-MS (m/z) [1340.8 (M+H)(+)]. Thiol 1b is also stable at acidic pH, but when neutralized is unstable and cyclizes, forming 3b and Antag1.     

NMR for direct determination of K(m) and V(max) of enzyme reactions based on the Lambert W function-analysis of progress curves

(1)H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K(m) and V(max), were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters K(m) and V(max) were fitted to obtain the experimental progress curve and resulted in K(m)=28mM and V(max)=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K(m)=379μM and k(cat)=0.04s(-1). The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

Effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

The effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana L. were investigated. Maximal betacyanin accumulation was observed at 88 m M sucrose on cell number basis and at 175 m M sucrose on fresh weight basis. This is because cell size decreased as the initial sucrose concentration was increased. Supplementary studies using mannitol indicated that sucrose itself caused increased cell number and that cell size was affected by both sucrose concentration and water potential. Betacyanin accumulation per cell and per fresh weight at a constant concentration of sucrose (88 m M ) decreased with decreasing water potential. When sucrose concentration increased at a constant water potential (–0.7 MPa), betacyanin accumulation per fresh weight increased up to 88 m M and remained at constant level at higher concentrations, while betacyanin accumulation per cell decreased remarkably, due to a dramatic increase in cell number.     

Glucosylation of acetic acid by sucrose phosphorylase

Transglucosylation from sucrose to acetic acid by sucrose phosphorylase (EC 2.4.1.7) was studied. 1-O-Acetyl-alpha-D-glucopyranose was isolated as the main product of the enzyme reaction. We also compared the pH-dependence of transglycosylation catalyzed by sucrose phosphorylase toward carboxyl and hydroxyl groups. With hydroquinone as an acceptor molecule, the transfer ratio of glucose residue was higher at neutral pH. This pH-activity profile was similar to that of the phosphorolysis of sucrose by sucrose phosphorylase, but with acetic acid as an acceptor molecule, the transfer ratio of glucose residue was higher at low pH. These findings suggest that the undissociated carboxyl group is essential to the acceptor molecule for the transglycosylation reaction of sucrose phosphorylase. In a sensory test, the sour taste of acetic acid was markedly reduced by glucosylation. The threshold value of the sour taste of acetic acid glucosides was approximately 100 times greater than that of acetic acid.     

Synthesis and characterization of fructooligosaccharides using levansucrase with a high concentration of sucrose

A method for synthesizing branched fructo-oligosaccharides (BFOS) with a high concentration of sucrose (1–3 M) was developed using levansucrase prepared fromLeuconostoc mesenteroides B-1355C. The degree of polymerization of oligosaccharides synthesized according to the present method ranged from 2 to over 15. The synthesized BFOS were stable at a pH ranges of 2 to 4 under 120°C. The percentage of BFOS in the reaction digest was 95.7% (excluding monosaccharides; 4.3% was levan). BFOS reduced the insoluble glucan formation byStreptococcus sobrinus on the surfaces of glass vials or stainless steel wires in the presence of sucrose. They also reduced the growth and acid productions ofS. sobrinus. Oligosaccharides can be used as sweeteners for foods such as beverages requiring thermo-and acid-stable properties and as potential inhibitors of dental caries. The first two authors contributed equally to this work.     

Kinetics of sucrose conversion to fructo-oligosaccharides using enzyme (invertase) under free condition

The study reports the synthesis of fructo-oligosaccharide (FOS) from sucrose using invertase derived from Saccharomyces cerevisiae. The reaction was conducted in a batch mode under free enzyme condition. Fructo-oligosaccharide formation was detected at a high sucrose concentration of over 200 g/L. The investigation was extended to study the effect of different parameters such as initial sucrose concentration (ISC), pH, and enzyme concentration. A maximum FOS yield of 10 % (dry basis) was observed using 525 g/L of ISC, with 6 U/mL of the enzyme, and pH 5.5 at 40 °C. 1-Kestose was the major product of among different forms of FOS. The FOS yield increased with an increase in sucrose concentration up to 525 g/L, beyond which it started to decrease. However, the maximum FOS yield was not affected by the increasing concentration of the enzyme beyond a certain level (2 U/mL). Furthermore, the activity of enzyme slightly increased with an increase in the pH up to 6, and thereafter it declined. Addition of glucose decreased the FOS yield because of enzyme inhibition. A five-step, ten-parameter model was developed, for which the simulation was performed in COPASI. The results predicted by the model were consistent with the experimental data.     

Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors

Summary Previously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injections of 50 l samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucroce analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10–50 m°C for concentrations of 0.05–0.30 M glucose or sucrose present in the original non-diluted samples.The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of scurose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant.On-line control of the effluent concentration of lucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomuter. Preset concentration of glucose in the effluent could be maintained over an extended period of time espite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance.     

Taste preferences in Parkinson's disease patients

Parkinson's disease (PD) can affect both sensory and motor function.Sensory dysfunction has been reported in the visual, olfactoryand somatosensory systems. Although several experimental animalstudies report a change in sucrose preference following treatmentwith dopamine antagonists, there have been no reports of gustatorydysfunction in PD patients. The present study was undertakento determine if PD patients (n = 25) varied in their preferenceratings for sweet stimuli compared to an age- and sex-matchedcontrol group (n = 16). Subjects rated their degree of liking(preference) for seven concentrations of sucrose and NaCl usinga 6-point fixed category scale. An ANOVA indicated that PD patientsdid not differ significantly from the control group in theirpreferences for NaCl. The preference concentration functionfor sucrose, however varied between the PD and control groups.The preference curve for the normal group was an inverted U-shapedcurve which peaked at 0.3 M sucrose. In contrast, the preferencecurve for the PD patients was a monotonically increasing functionof concentration. An ANOVA for the sucrose ratings revealedno main effect for disease state, but there was a significantdisease state X concentration effect (P < 0.001). Futuretesting is necessary to determine if this change in preferenceis accompanied by changes in the magnitude ratings for sucrose.     

Differences in the taste quality of maltose and sucrose in rats: issues involving the generalization of conditioned taste aversions

The present study employed a conditioned taste aversion generalizationparadigm to test the hypothesis that maltose produces tastesensations in the rat which are qualitatively distinguishablefrom sucrose. Since stimulus generalization can occur in boththe quality and intensity domains, an intrachemical (acrossconcentration) generalization gradient was established to aidin the interpretation of the interchemical (across molecules)generalization gradient. Moreover, since the commonly used intaketest is vulnerable to nontaste post-ingestional influences,the present study measured immediate responses to 100 µlstimulus samples, thus increasing our confidence that the behaviorwas under orosensory control. In Experiment 1, naive water deprivedrats were trained in a specially designed gustometer to maintaindrinking-spout contact for intermittent water reinforcement.Following this, rats in the experimental group were given threeexposures to 0.1 M sucrose on separate days, with the firsttwo exposures immediately preceding an injection of LiCl. Acontrol group was treated identically but received distilledwater instead of sucrose. Rats were then tested in the gustometerfor their avoidance of three equimolar concentrations of sucroseand maltose. Rats received ten trials of each stimulus quasi-randomlypresented in two sessions. Results indicated that all sucroseconcentrations were avoided (in experimental group only), butonly the 0.3 M concentration of maltose was avoided. The lowestsucrose concentration was significantly less avoided than thehigher concentrations. Intensity generalization gradients aresuch that intensities weaker than the conditioned stimulus (CS)produce just as much or less of a conditioned response (CR)and intensities stronger than the CS produce just as much ora greater CR than that elicited by the CS itself. Therefore,based on the results of Experimental, it was predicted thatif 0.1 M maltose served as the CS, the order of avoidance shouldbe: 0.3 M sucrose 0.1 M sucrose 0.03 M sucrose 0.3 M maltose 0.1 M maltose 0.03 M maltose, if it were true that maltoseand sucrose produce identical sensations that differ only inintensity. Experiment 2 explicitly tested this prediction usingthe same procedure as Experiment 1 except that 0.1 M maltoseserved as the CS. The observed order of avoidance was 0.3 Mmaltose > 0.1 M maltose > 0.03 M maltose = 0.3 M sucrose= 0.1 M sucrose = 0.03 M sucrose. In both experiments the intrachemicalgeneralization gradient broadened and the interchemical generalizationgradient steepened upon retesting. In conclusion, qualitativedifferences between maltose and sucrose explain the outcomesof these experiments better than differences in the relativeintensity of these sugars at isomolar concentrations.     

Amylose synthesized in vitro by amylosucrase: morphology, structure, and properties

The recombinant amylosucrase from Neisseria polysaccharea was used to synthesize in vitro amylose from sucrose as unique substrate. The morphology and structure of the insoluble residue were shown to depend only on the initial sucrose concentration (100, 300, or 600 mM), which controlled both the chain length and concentration at the precipitation stage. The average degree of polymerization (DP) in the precipitated product varied from 58 for the lowest initial sucrose concentration (100 mM) to 45 and 35 for higher sucrose concentrations (300 and 600 mM, respectively). The shorter chains (DP 35 and 45), produced in high yields (54 and 24 g/L respectively), precipitated as polycrystalline aggregates with exceptional crystallinity, without optimization of the reaction medium for crystallization. The longer chains (DP 58), produced in lower amount (2.9 g/L), formed networks similar to those observed for amylose gels. All synthesized products displayed a B-type crystal structure. Their melting behavior was also studied, the thermostability being higher for the precipitate containing the longer chains. Further thermal treatments were shown to still improve the crystallinity and yield substrates usable as new standards for the determination of the relative crystallinity of starchy products. The kinetics of chain elongation and aggregation were thoroughly investigated in order to explain how the action of amylosucrase resulted in such different amylose structures. These results emphasize the potentiality of amylosucrase in the design of amylodextrins with controlled morphology, structure, and physicochemical properties.     

Inorganic phosphate self-sufficient whole-cell biocatalysts containing two co-expressed phosphorylases facilitate cellobiose production

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).     

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.