Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from allium cepa

Sucrose: sucrose fructosyltransferase and fructan:fructan fructosyltransferase were isolated from the inner leaf bases of bulbing onion plants (Allium cepa) and separated by gel filtration on Bio-Gel P-150. Sucrose:sucrose fructosyltransferase produced only one trisaccharide, 1^F-fructosylsucrose, from sucrose. Fructan:fructan fructosyltransferase produced tetrasaccharide and higher polymers from trisaccharide. The trisaccharide found in the greatest concentration in onion, 6^G-fructosylsucrose, was produced from 1^F-fructosylsucrose by fructan:fructan fructosyltransferase and was not a product of sucrose:sucrose fructosyltransferase.     

Acceptor reactions of a novel transfructosylating enzyme from Bacillus sp.

Many different oligosaccharides were produced by transferring the fructose residue of sucrose to maltose, cellobiose, lactose and sucrose (self-transfer), where their yields of fructosylated acceptor products accounted for 26–30% (w/w). The maximum conversion yield (30%) was obtained in fructosyl cellobioside formation with 500 g sucrose l^–1 (substrate) and 200 g cellobiose l^–1 (acceptor). These four acceptors gave various products having DP (degree of polymerization) 2–7 by successive transfer reactions.     

Enzymatic Synthesis and Characterization of Fructooligosaccharides and Novel Maltosylfructosides by Inulosucrase from Lactobacillus gasseri DSM 20604

The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucrose and sucrose-maltose mixtures was investigated after optimization of synthesis conditions, including enzyme concentration, temperature, pH, and reaction time. The maximum formation of FOS, which consist of β-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained after 24 h of reaction at 55°C in the presence of sucrose (300 g liter⁻¹) and 1.6 U ml⁻¹ of IS–25 mM sodium acetate buffer–1 mM CaCl₂ (pH 5.2). The production of MFOS was also studied as a function of the initial ratios of sucrose to maltose (10:50, 20:40, 30:30, and 40:20, expressed in g 100 ml⁻¹). The highest yield in total MFOS was attained after 24 to 32 h of reaction time and ranged from 13% (10:50 sucrose/maltose) to 52% (30:30 sucrose/maltose) in weight with respect to the initial amount of maltose. Nuclear magnetic resonance (NMR) structural characterization indicated that IS from L. gasseri specifically transferred fructose moieties of sucrose to either C-1 of the reducing end or C-6 of the nonreducing end of maltose. Thus, the trisaccharide erlose [α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside] was the main synthesized MFOS followed by neo-erlose [β-d-fructofuranosyl-(2→6)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranose]. The formation of MFOS with a higher degree of polymerization was also demonstrated by the transfer of additional fructose residues to C-1 of either the β-2,1-linked fructose or the β-2,6-linked fructose to maltose, revealing the capacity of MFOS to serve as acceptors.     

The Metabolism of Fructose Polymers in Plants: VI. TRANSFRUCTOSYLATION IN LIVING TISSUE 3HELIANTHUS TUBEROSUS L.

Tracer ¹⁴CO₂ was supplied to the leaves of Jerusalem artichokeplants in the light and tissue was removed from an attachedtuber at intervals. The first compound to contain ¹⁴C in thetuber was sucrose; label next appeared in the trisaccharideI^F-fructosylsucrose, and more slowly in the higher oligo-andpolysaccharides. After 50 hours over 40 per cent. of the tracerpresent in the tuber was found in the ‘inulin’ fraction(DP>25). Degradation of the polymers showed that in the earlystages only the sucrosyl groups of the oilgosaccharides werelabelled. Later the labelling in the fructosyl residues of the‘tails’ of the oilgosaccharides increased to thelevel of that in the sucrosyl groups. The pattern in the fractionof DP> 20 did not correspond to that of the lower polymers,as the ‘tail’ appeared to become labelled first.Transfer of terminal fructosyl residues by known transfructosylaseactivity can explain the labelling of the sucrosyl residuesof the fructosan series, thus implicating this enxyme in thedistribution of fructose within the series, but the mechanismleading to ‘tail’ labelling is still obscure.     

Purification and Subsite Affinities of exo-lnulinase from Penicillium trzebinskii

An exo-inulinase was highly purified from the culture broth of Penicillium trzebinskii by anion exchange, hydrophobic, and gel filtration chromatographies. The enzyme was homogeneous by disc electrophoresis. The molecular weight was 8.7 × 10⁴, and the isoelectric point was pH 4.3. The enzyme hydrolyzed not only inulin and sucrose but also inulooligosaccharides [1^F(1-β-D-fructofuranosyl)_n-1fructose, F_n (n= 2—5)] and fructooligosaccharides [1^F(1-β-D-fructofuranosyl)_n-1 sucrose, GF_n, (n = 2—8)] liberating the nonreducing terminal fructose of the substrates. The substrate specificity was investigated. The K_m (mM) and k_o (sec^?1were: inulin, 0.042 and 159; sucrose, 6.5 and 169; F₂, 2.1 and 62.8; F₃, 0.40 and 126; F₄, 0.47 and 171; and F₅, 0.47 and 131, respectively. Dependence of K_m and k_o values on the degree of polymerization of substrates was observed. The subsite affinities in the active site were 1.05, 4.57, 1.45, 0.09, and — 0.16kcal/mol for subsite 1, 2, 3, 4, and 5, respectively.     

Purification and Characterization of Wheat beta(2-->1) Fructan:Fructan Fructosyl Transferase Activity

Fructans are the major storage carbohydrate in vegetative tissues of wheat (Triticum aestivum L.). Fructan:fructan fructosyl transferase (FFT) catalyzes fructosyl transfer between fructan molecules to elongate the fructan chain. The objective of this research was to isolate this activity in wheat. Wheat (cv Caldwell) plants grown at 25°C for 3 weeks were transferred to 10°C to induce fructan synthesis. From the leaf blades kept at 10°C for 4 days, fructosyl transferase activity was purified using salt precipitation and a series of chromatographic procedures including size exclusion, anion-exchange, and affinity chromatography. The transferase activity was free from invertase and other fructan-metabolizing activities. Fructosyl transferase had a broad pH spectrum with a peak activity at 6.5. The temperature optimum was 30°C. The activity was specific for fructosyl transfer from β(2→1)-linked 1-kestose or fructan to sucrose and β(2→1) fructosyl transfer to other fructans (1-FFT). Fructosyl transfer from oligofructans to sucrose was most efficient when 1-kestose was used as donor molecule and declined as the degree of polymerization of the donor increased from 3 to 5. 1-FFT catalyzed the in vitro synthesis of inulin tetra- and penta-saccharides from 1-kestose; however, formation of the tetrasaccharide was greatly reduced at high sucrose concentration. 6-Kestose could not act as donor molecule, but could accept a fructosyl moiety from 1-kestose to produce bifurcose and a tetrasaccharide having a β(2→1) fructose attached to the terminal fructose of 6-kestose. The role of this FFT activity in the synthesis of fructan in wheat is discussed.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “Protaminobacter rubrum”

“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a ³²⁵RLDRD³²⁹ motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg³²⁵ and Arg³²⁸ in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R³²⁵Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter⁻¹ and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter⁻¹) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g⁻¹ with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.     

Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10⁷) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Enzymology of Fructan Synthesis in Grasses: Properties of Sucrose-Sucrose-Fructosyltransferase in Barley Leaves (Hordeum vulgare L. cv Gerbel)

Fructan synthesis was induced in excised primary leaf blades of Hordeum vulgare L. cv Gerbel by illumination in 30 millimolar fructose. This treatment induced a 26-fold increase of sucrose-sucrose-fructosyltransferase (SST, EC 2.4.1.99) activity within 24 hours. Acid invertase (EC 3.2.1.26) activity remained about constant. By preparing protoplasts from induced leaves, approximately 80% of the invertase activity was removed with the cell walls while SST was retained. The protoplast homogenate was used to partially purify and characterize SST. Acid precipitation (pH 4.75) and anion exchange chromatography (fast protein liquid chromatography on Mono `Q') resulted in a recovery of about 80% of total SST activity. The principal activity (SST 1), accounting for 85% of the activity recovered, was purified about 200-fold. It was essentially free of invertase activity and catalyzed the synthesis of a trisaccharide which co-chromatographed with isokestose (1F-β-fructosylsucrose). The remaining 15% of SST activity (SST 2) was purified about 35-fold. It retained substantial invertase activity and catalyzed the synthesis of only one trisaccharide which co-chromatographed with kestose (6F-β-fructosylsucrose). It is concluded that barley leaves which store mainly fructan of the phlein type (β-2-6 polyfructosylsucrose), nevertheless contain sucrose-sucrose 1F-β-d-fructosyltransferase as the key enzyme of fructan synthesis.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Glucofructosan metabolism in Cichorium intybus roots

A 10-fold purification of sucrose sucrose fructosyl transferase from Cichorium intybus roots was achieved by ammonium sulphate fractionation and DEAE-cellulose column chromatography. The energy of activation for this enzyme was ca 48 kJ/mol sucrose. Sucrose sucrose fructosyl transferase and invertase were prominent during early months of growth. Evidence obtained from: (1) the changes in carbohydrate composition at monthly intervals; (2) comparative studies on fructosyl transferase and invertase at different stages of root growth; and (3) incubation studies with [¹⁴C]glucose, [¹⁴C]fructose and [¹⁴C]sucrose revealed that, during the later stages of root growth, fructosan hydrolase is responsible for fructosan hydrolysis. No evidence for the direct transfer of fructose from sucrose to high M_r glucofructosans was obtained.     

Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae

A high-density-cell fermentation process for production of an exracellular alginat lyase from Klebseilla pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscoous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of algiante lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1^–1) was found both for the maximum final concentration of enzyme (1330 U 1^–1) and for the maximum volumetric rate of enzyme production (67 U 1^–1 h^–1). The enzyme production dependes of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fiveflod increase compare with previously reproted daata where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the othr hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)^–1 h^–1.     

Partial purification and properties of phleinase induced in stem base of orchardgrass after defoliation

Phleinase induced in stem base of orchardgrass (Dactylis glomerata L.) after defoliation was partially purified with ammonium sulfate precipitation, DEAE-Sephadex chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The molecular weight of phleinase was 57,000 as determined by gel chromatography. The enzyme showed normal Michaelis-Menten kinetics and its K_m value was 91 millimolar for phlein of mean degree of polymerization 60 as substrate. Reaction velocity of the enzyme was proportional to molarity of phlein irrespective of its chain length (mean degree of polymerization, 30 to 314). Phleinase attacked terminal fructosyl linkage of phlein by multi-chain mechanism. Phleinase cleaved β-2,6 linkage, β-2,6 linkage branched with β-2,1 linkage, and β-2,1 linkage of fructan in order of affinity, but not sucrose. Phleinase exhibited an optimum activity at pH 5.5 at 40°C. Its complete inactivation occurred at 60 and 70°C without and with phlein, respectively. Heat inactivation of the enzyme was enhanced by p-chloromercuribenzoate and protected partially by l-cysteine. The enzyme was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and Hg²⁺. The modes of action of phleinase were compared with those of the related enzymes.     

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2°C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4°C. During storage at 8°C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8°C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8°C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4°C but remained the same throughout storage at 8°C. After exposure to 2°C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4°C but decreased slightly at 8°C. All observed changes became annulled after transfer of cold stored tubers to 18°C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.     

The production of ethanol plus fructose sweetener using fructose utilization negative mutants of Zymomonas mobilis

Summary Two mutants, unable to utilize fructose (Fru^–) as a sole source of carbon and energy, were isolated fromZymomonas mobilis following ethyl methane sulfonate (EMS) mutagenesis. The frequency of stable Fru^– mutants among survivors of mutagenesis was 1 in 10⁴. The two Fru^– mutants were able to cleave sucrose to glucose and fructose, and then ferment only the glucose to ethanol while accumulating fructose close to the theoretical value. Under controlled fermentation conditions, sucrose was converted to ethanol plus 80% or higher purity fructose syrup in a single-stage batch fermentation process, improving the Sucrotech Process significantly.     

Purification of membrane-bound dopamine beta-monooxygenase from chromaffin granules: relation to soluble dopamine beta-monooxygenase

Previous studies have indicated that α-d-1-fluoroglucose is a glycosyl donor for glucosyl transferases (5, 6) including dextransucrases formed by Leuconostoc and Streptococcus mutans. The present report confirms these observations with dextransucrase isolated from S. sanguis and conclusively establishes the details of this reaction as well as proving that mechanism of fluoroglucose transfer is comparable to that glucosyl transfer from sucrose. A new procedure for monitoring the reaction is reported, and is based on the measurement of proton formation using the pH indicator, bromcresol purple. Production of F^? was found to be stoichiometric with proton production. Rate studies with the substrate indicate that α-1-fluoroglucose undergoes spontaneous hydrolysis, which is greatly increased in the presence of nucleophilic buffers. When [¹⁴C]maltose and α-1-fluoroglucose or [¹⁴C]α-1-fluoroglucose and maltose were incubated with dextransucrase, a series of oligosaccharide products was observed. The results indicate that the glucosyl moiety of α-1-fluoroglucose transferred to the acceptor. The nature of formation of the products are consistent with a series of precursor-product reactions. Product analysis of the saccharides by borohydride reduction analysis demonstrated that the glucosyl unit was added to the nonreducing end of maltose. When either [¹⁴C]fructose or [¹⁴C]-α-1-fluoroglucose were incubated with enzyme, a reaction was observed which was analogous to the isotopic-exchange reaction catalyzed by the enzyme in the presence of [¹⁴C]fructose and sucrose.     

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)₈-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp²⁴¹, Glu²⁹⁵, Asp³⁶⁹, His¹⁴⁵, and His³⁶⁸) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a K_m of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe³⁺ and Hg²⁺ and was enhanced by Mn²⁺ and Mg²⁺. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu⁴⁹⁸ and Arg³¹⁰ with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.