No evidence for the occurrence of substrate inhibition of Arabidopsis thaliana sucrose synthase-1 (AtSUS1) by fructose and UDP-glucose

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and NDP into the corresponding nucleotide-sugars and fructose. The Arabidopsis genome possesses six SUS genes (AtSUS1–6) that code for proteins with SuSy activity. As a first step to investigate optimum fructose and UDP-glucose (UDPG) concentrations necessary to measure maximum sucrose-producing SuSy activity in crude extracts of Arabidopsis, in this work we performed kinetic analyses of recombinant AtSUS1 in two steps: (1) SuSy reaction at pH 7.5, and (2) chromatographic measurement of sucrose produced in step 1. These analyses revealed a typical Michaelis-Menten behavior with respect to both UDPG and fructose, with Km values of 50 μM and 25 mM, respectively. Unlike earlier studies showing the occurrence of substrate inhibition of UDP-producing AtSUS1 by fructose and UDP-glucose, these analyses also revealed no substrate inhibition of AtSUS1 at any UDPG and fructose concentration. By including 200 mM fructose and 1 mM UDPG in the SuSy reaction assay mixture, we found that sucrose-producing SuSy activity in leaves and stems of Arabidopsis were exceedingly higher than previously reported activities. Furthermore, we found that SuSy activities in organs of the sus1/sus2/sus3/sus4 mutant were ca. 80–90% of those found in WT plants.     

Sucrose synthase isoforms in cultured tobacco cells.

The plant enzyme sucrose synthase (SuSy; EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose (UDP-Glc) and fructose. The enzyme exists in different isoforms and is both located in the cytosol, membrane-bound and associated to the actin cytoskeleton. We here investigate sucrose synthase from tobacco (Nicotiana tabacum L.) BY-2 heterotrophic cell suspensions. Two different isoforms of sucrose synthase SuSy1 and SuSy2, could be purified from cytosolic extracts of these cells using a combination of poly(ethylene glycol) (PEG) precipitation, gel filtration, ion-exchange chromatography and affinity chromatography. They were clearly distinct, both with regard to the binding to the ion-exchange column and with regard to their kinetic and regulatory properties. SuSy1, the more abundant species, showed lower V(max) and K(m) for sucrose and UDP compared to the less abundant SuSy2. The activity of SuSy2 in the breakdown direction was stimulated by 60% by actin, in contrast to that of SuSy1, which showed a 17% inhibition. An indication of interaction between SuSy1 and actin was obtained by partitioning in aqueous Dextran-PEG two-phase systems. Furthermore, fructose 2,6-bisphosphate (F26BP) at micromolar concentrations stimulated SuSy2 in the presence of actin while SuSy1 was strongly inhibited by fructose. Possible roles of these two isoforms in the sucrose turnover in BY-2 cells are discussed.     

Evidence for a tonoplast-associated form of sucrose synthase and its potential involvement in sucrose mobilization from the vacuole

The following work presents new evidence for a tonoplast localization of sucrose synthase and its functional role during the mobilization of vacuolar sucrose. Highly purified tonoplast vesicles were associated with significant SuSy levels as determined by immuno-recognition, enzymatic activity, and by-product measurements. Total tonoplast-bound SuSy was estimated to be approximately 7% of the total tissue activity. SuSy affinity to the tonoplast was confirmed by the lack of SuSy displacement by ionic washes and also by the tonoplast ability to bind to exogenously added SuSy as compared to the cytosolic marker alcohol dehydrogenase. UL-[(14)C]sucrose-loaded vesicles incubated with ATP and UDP produced [(14)C]UDP-Glc as determined by UDP-Glc dehydrogenase and by the ability of the product to bind to DEAE-cellulose and to co-migrate with authentic UDP-Glc on TLC. ATP alone induced sucrose efflux but not the production of [(14)C]ADP-Glc. Kinetic analysis of [(14)C]UDPG formation under conditions of low sucrose availability suggests sucrose channelling between the ATP-dependent sucrose transporter and SuSy, thus corroborating the association of SuSy with the tonoplast and its involvement in sucrose mobilization from the vacuole.     

A kinetic study of sugarcane sucrose synthase.

The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, +/- SE) were, for sucrose, 35.9 +/- 2.3; for UDP, 0.00191 +/- 0.00019; for UDP-glucose, 0.234 +/- 0.025 and for fructose, 6.49 +/- 0.61. values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.     

Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance.

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.     

Biosynthesis of type 3 capsular polysaccharide in Streptococcus pneumoniae. Enzymatic chain release by an abortive translocation process

The type 3 polysaccharide synthase from Streptococcus pneumoniae catalyzes sugar transfer from UDP-Glc and UDP-glucuronic acid (GlcUA) to a polymer with the repeating disaccharide unit of [3)-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->]. Evidence is presented that release of the polysaccharide chains from S. pneumoniae membranes is time-, temperature-, and pH-dependent and saturable with respect to specific catalytic metabolites. In these studies, the membrane-bound synthase was shown to catalyze a rapid release of enzyme-bound polysaccharide when either UDP-Glc or UDP-GlcUA alone was present in the reaction. Only a slow release of polysaccharide occurred when both UDP sugars were present or when both UDP sugars were absent. Chain size was not a specific determinant in polymer release. The release reaction was saturable with increasing concentrations of UDP-Glc or UDP-GlcUA, with respective apparent K(m) values of 880 and 0.004 micrometer. The apparent V(max) was 48-fold greater with UDP-Glc compared with UDP-GlcUA. The UDP-Glc-actuated reaction was inhibited by UDP-GlcUA with an approximate K(i) of 2 micrometer, and UDP-GlcUA-actuated release was inhibited by UDP-Glc with an approximate K(i) of 5 micrometer. In conjunction with kinetic data regarding the polymerization reaction, these data indicate that UDP-Glc and UDP-GlcUA bind to the same synthase sites in both the biosynthetic reaction and the chain release reaction and that polymer release is catalyzed when one binding site is filled and the concentration of the conjugate UDP-precursor is insufficient to fill the other binding site. The approximate energy of activation values of the biosynthetic and release reactions indicate that release of the polysaccharide occurs by an abortive translocation process. These results are the first to demonstrate a specific enzymatic mechanism for the termination and release of a polysaccharide.     

The structure of sucrose phosphate synthase from Halothermothrix orenii reveals its mechanism of action and binding mode

Sucrose phosphate synthase (SPS) catalyzes the transfer of a glycosyl group from an activated donor sugar, such as uridine diphosphate glucose (UDP-Glc), to a saccharide acceptor D-fructose 6-phosphate (F6P), resulting in the formation of UDP and D-sucrose-6'-phosphate (S6P). This is a central regulatory process in the production of sucrose in plants, cyanobacteria, and proteobacteria. Here, we report the crystal structure of SPS from the nonphotosynthetic bacterium Halothermothrix orenii and its complexes with the substrate F6P and the product S6P. SPS has two distinct Rossmann-fold domains with a large substrate binding cleft at the interdomain interface. Structures of two complexes show that both the substrate F6P and the product S6P bind to the A-domain of SPS. Based on comparative analysis of the SPS structure with other related enzymes, the donor substrate, nucleotide diphosphate glucose, binds to the B-domain of SPS. Furthermore, we propose a mechanism of catalysis by H. orenii SPS. Our findings indicate that SPS from H. orenii may represent a valid model for the catalytic domain of plant SPSs and thus may provide useful insight into the reaction mechanism of the plant enzyme.     

Examination of the mechanism of sucrose synthetase by positional isotope exchange

The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. [beta-18O2, alpha beta-18O]UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of [beta-18O2, alpha beta-18O]UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.     

Characterization of the purified hyaluronan synthase from Streptococcus equisimilis

Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg(2+) to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was approximately 40% more active in 25 mM, compared to 50 mM, PO(4) in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5-11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK(a) values at pH 6.6 and 11.8. As the pH was increased from approximately 6.5, both K(m) and V(max) increased until pH approximately 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Delta9) fatty acids. The apparent E(act) for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V(max) at 21, 6.5, and 3.5 mM, respectively. The apparent K(i) values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM.     

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.     

Non-Michaelian kinetics of rabbit muscle uridine diphosphoglucose pyrophosphorylase

The kinetic properties of rabbit muscle uridine diphosphoglucose (UDP-Glc) pyrophosphorylase have been studied, in both directions, with respect to substrate saturation, product inhibition, and cation requirement for activity. The results demonstrate that UDP-Glc pyrophosphorylase is a non-Michaelian enzyme: the synthetic reaction is characterized by a marked inhibition by glucose-1-phosphate (at concentrations higher than 0.3 mM) and by an hyperbolic saturation for UTP. In the reverse reaction, instead, the saturation function for UDP-Glc is hyperbolic and that for inorganic pyrophosphate is sigmoid, with a high Hill coefficient of (nH) 2.5. The study of the metal requirement indicates a distinctive ability of cations to stimulate the reactions of synthesis and degradation of the sugar nucleotide and a different stoichiometry of the metal chelates involved. The reaction mechanism is of the ordered-sequential type and the data of product inhibition allowed the identification of glucose-1-phosphate as the first substrate bound and UDP-Glc as the last product released. The inhibition pattern by UDP-Glc gives evidence for cooperativity also in the binding of this molecule.     

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.     

The mechanism of glycogen synthetase as determined by deuterium isotope effects and positional isotope exchange experiments

The reaction mechanism for glycogen synthetase from rabbit muscle was examined by alpha-secondary deuterium isotope effects and positional exchange experiments. Incubation of glycogen synthetase with [beta-18O2,alpha beta-18O]UDP-Glc did not result in any detectable positional isotope exchange from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. Glucono-1,5-lactone was found to be a noncompetitive inhibitor versus UDP-Glc. The kinetic constants, K(is) and K(ii), were found to be 91 +/- 4 microM and 0.70 +/- 0.09 mM, respectively. Deoxynojirimycin was a nonlinear inhibitor at pH 7.5. The alpha-secondary deuterium isotope effects were measured with [1-2H]UDP-Glc by the direct comparison method. The isotope effects on Vmax and Vmax/K were found to be 1.23 +/- 0.04 and 1.09 +/- 0.06, respectively. The inhibitory effects by glucono-lactone and deoxynojirimycon plus the large alpha-secondary isotope effect on Vmax have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. The lack of a detectable positional isotope exchange reaction in the absence of glycogen suggests the formation of a rigid tight ion pair between UDP and the oxocarbonium ion intermediate.     

β(1–3)Glucan synthase ofNeurospora crassa: Reaction sequence based on kinetic evidence

On the basis of the kinetic effects of substrate, activator, and inhibitors on (1–3) glucan synthase activity ofNeurospora crassa, we propose the following reaction sequence for glucan synthesis. First, enzyme binds laminaribiose (activator), forming an enzyme-laminaribiose complex. Substrate (UDP-Glc) binding follows. UDP-Glc is hydrolyzed, releasing UDP, while the glucose residue remains associated with glucan synthase. The resulting enzyme-activator-glucose complex binds another UDP-Glc. It is likely that linear competitive inhibitors act at this step. Initial polymerization occurs, forming a disaccharide (which remains bound to glucan synthase) and UDP, which is released. The resulting enzyme-activator-disaccharide binds another UDP-Glc, and Glc is covalently added; further polymerization occurs by addition of Glc (from UDP-Glc) to the growing glucan chain, which remains associated with glucan synthase. Uncompetitive inhibitors are likely to affect enzyme activity at this step.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

Kinetics of action of chymosin (rennin) on some peptide bonds of bovine beta-casein

The first steps of proteolysis of bovine beta-casein by chymosin were studied quantitatively by using reverse-phase high-performance liquid chromatography (RP-HPLC). Although chymosin has a broad specificity, it has been possible to selectively study the hydrolysis of two bonds (Ala-189-Phe-190 and Leu-192-Tyr-193) by choosing appropriate conditions. The disappearance of the substrate and the appearance of the reaction products as a function of time were followed at 220 nm by RP-HPLC. For concentrations where beta-casein was in a micellar form, the Michaelian parameters corresponding to the cleavage of bond 192-193 were determined by measuring initial rates of reaction at different substrate concentrations in a time period for which splitting of bond 189-190 was negligible. The following results were obtained; k1cat = 1.54 s-1, K1m = 0.075 mM, and k1cat/K1m = 20.6 mM-1 s-1. Under conditions where the protein was in a monomeric state, the following parameters were determined for the splitting of bond 192-193 by integrating the Michaelis equation: k2cat = 0.056 s-1, K2m = 0.007 mM, and k2cat/K2m = 79.7 mM-1 s-1. Under the latter conditions the four enzymic reactions involved in the cleavage of bonds 189-190 and 192-193 were first-order reactions. The four corresponding apparent rate constants were calculated by using a computer program. Excellent agreement was obtained between concentrations of four molecular species measured during the reaction period and those calculated by using the four apparent rate constants.     

Production of galactinol from sucrose by plant enzymes

Galactinol, 1-O-(alpha-D-galactopyranosyl)-myo-inositol, was produced from sucrose as a starting material. UDP-Glc was prepared with sucrose and UDP using sucrose synthase partially purified from sweet potato roots. Then, the UDP-Glc was converted to UDP-Gal using yeast UDP-Gal 4-epimerase from a commercial source. Finally, galactinol was produced from the UDP-Gal and myo-inositol using galactinol synthase partially purified from cucumber leaves. The product was identified as galactinol by the retention times of HPLC, alpha-galactosidase digestion, and NMR spectrometry.     

alpha-Lactalbumin (LA) stimulates milk beta-1,4-galactosyltransferase I (beta 4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of beta 4Gal-T1 x LA complex with UDP-Glc

beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K(m) for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K(m) for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1.LA complex with UDP-Glc x Mn(2+) and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.     

Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.