Molecular and biochemical characterization of a fructose-6-phosphate-forming and ATP-dependent fructokinase of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus litoralis</Emphasis>

Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases. We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli. We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties. At its optimal temperature of 80°C, the apparent K_m and V_max values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration. The enzyme did not lose activity at 80°C for 4 h. Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa. Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.     

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K _i = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.     

Homo- and heterologous reporter proteins for evaluation of promoter activity in <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

A number of vectors were constructed based on the plasmid from the broad range of pMHA200 hosts. Also, the expression of some key genes of the haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z was studied. The activities of the promoter regions of genes for hexulose phosphate synthase, glutamine synthetase, and glucokinase, as well as the promoter of the ectABC-ask operon, which encodes enzymes for osmoprotectant ectoine biosynthesis, were evaluated with the use of the gfp gene; the evaluation was proven to be ineffective. Conversely, glucokinase and a heterologous enzyme of chloramphenicol acetyltransferase were useful for the evaluation of promoter activity. In M. alcaliphilum 20Z cells, the expression level of chloramphenicol acetyltransferase transcribed from the methanol dehydrogenase promoter was higher as compared with that of glucokinase. This seems to be due to a regulatory mechanism for homologous protein expression. The introduction of a synthetic nucleotide sequence forming the secondary structure in the 5′ untranslated region of the glucokinase mRNA resulted in an increase of this enzyme level. This is the first attempt to use M. alcaliphilum 20Z for homo- and heterologous protein expression.     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Transport of glucose,fructose and sucrose by Streptanthus tortuosus suspension cells

Streptanthus tortuosus Kell. suspension cells will grow in a medium with sucrose as carbohydrate source. It was investigated whether the cells are able to take up sucrose or whether sucrose has to be hydrolyzed to glucose and fructose which eventually are taken up. The detailed quantitative analysis of sugar-uptake rates in the low concentration range up to 1 mM showed the following features: (i) There is definitely no sucrose-uptake system working in the low concentration range; any uptake of radioactivity from labelled sucrose proceeds via hydrolysis of sucrose by cell-wallbound invertase. (ii) Hexoses are taken up by two systems, a glucose-specific system with a K _m of 45 M and a high V _max for glucose and a K _m of 6 mM and a low V _max for fructose, and a fructosespecific system with a K _m of 500 M and high a V _max for fructose and a K _m of 650 M and a low V _max for glucose. (iii) There is a more than tenfold preference for uptake of the fructose derived from sucrose versus uptake of free fructose, with the result that the kinetic disadvantage of the fructoseuptake system compared to the glucose-uptake system is diminished if sucrose is supplied as the carbon source. It is speculated that invertase might work as an enzyme aiding in fructose transport.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - FW fresh weight     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

Hexose kinases from the plant cytosolic fraction of soybean nodules

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K_m 0.075 millimolar) than fructose (K_m 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K⁺ ions for maximum activity. The enzyme was specific for fructose (apparent K_m 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 ± 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.     

Kinetics of Sugar Transport and Phosphorylation Influence Glucose and Fructose Cometabolism by Zymomonas mobilis

The competitive inhibition of fructokinase by glucose has been proposed as the mechanism by which Zymomonas mobilis preferentially consumes glucose from mixtures of glucose and fructose and accumulates fructose when growing on sucrose. In this study, incorporation of radioactive fructose into biomass was used as a measure of fructose catabolism. It was determined that the rate of fructose incorporation by Z. mobilis CP4 was somewhat lower in the presence of an equimolar concentration of glucose but that the inhibition of fructokinase by glucose was not nearly as severe in vivo as was predicted from in vitro studies. Interestingly, addition of glucose to a culture of Z. mobilis CP4-M2, a glucokinaseless mutant, resulted in an immediate and nearly complete inhibition of fructose incorporation. Furthermore, addition of nonmetabolizeable glucose analogs had a similar effect on fructose catabolism by the wild-type Z. mobilis CP4, and fructose uptake by Z. mobilis CP4-M2 was shown to be severely inhibited by equimolar amounts of glucose. These results suggest that competition for fructose transport plays an important role in preferential catabolism of glucose from sugar mixtures. Indeed, the apparent K(infm) values for sugar uptake by Z. mobilis CP4 were approximately 200 mM for fructose and 13 mM for glucose. Other experiments supported the conclusion that a single facilitated diffusion transport system, encoded by the glf gene, is solely responsible for the uptake of both glucose and fructose. The results are discussed with regard to the hypothesis that the kinetics of sugar transport and phosphorylation allow the preferential consumption of glucose and accumulation of fructose, making the fructose available for the enzyme glucose-fructose oxidoreductase, which forms sorbitol, an important osmoprotectant for Z. mobilis when growing in the presence of high sugar concentrations.     

Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent K_m for glucose of approximately 1.1 - 2.9 mM, consistent with the function of Gif as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z mobilis glucokinase gene, in addition to glf, did not affect the K_m of Glf for glucose in recombinant E. coli The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Gif for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.     

Phosphorolytic cleavage of sucrose by sucrose-grown ruminal bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain k3

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90 % sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase (EC 2.4.1.7) of molar mass ≈54 kDa and maximum activity at pH 6.0 and 45 °C. The calculated Michaelis constant (K _m) for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction (v _lim) were 1.19 and 0.64 μmol/L per mg protein per min, respectively.     

Purification of rabbit liver phosphofructokinase and its properties under simulatingin vivo conditions

Phosphofructokinase (EC 2.7.1.11) from rabbit liver was purified to homogeneity. Preincubation of enzyme results in nonlinearity of enzyme activity with enzyme concentration. Therefore K_0.5 of enzyme for fructose 6 phosphate in the absence or presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both was determined at physiological concentrations of its various effectors by taking the initial rate obtained by adding the enzyme last. They decrease the K_0.5 value from 4.1 mM to about 0.2mM. The K_0.5 of enzyme for fructose 2,6 bisphosphate was also determined under the above conditions. It is about 4.3ΜM. Transient kinetics of phosphofructokinase at varying concentrations of enzyme in the presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both were studied. It was found that although they decrease t_1/2 i.e. the time to reach half the maximal steady rate by about 5–8 fold, it was about constant at varying concentrations of the enzyme. These results indicate that fructose 2,6 bisphosphate and polyethylene glycol decrease K_0.5 of the enzyme for fructose 6 phosphate not by associating the enzyme to higher aggregates, but by a different mechanism.     

Characterization and compartmentation,in green leaves,of hexokinases with different specificities for glucose,fructose, and mannose and for nucleoside triphosphates

When green leaves of spinach (Spinacia oleracea L.) were surveyed for the presence of hexokinases which utilize glucose, fructose and-or mannose as a substrate, four kinases could be distinguished by their order of elution during chromatography on diethylaminoethyl (DEAE)-cellulose: (i) a hexokinase I with a specificity for fructose, glucose, and mannose, (ii) a fructokinase I with a specificity for fructose, (iii) a hexokinase II with a specificity for glucose, fructose and mannose, and (iv) a fructokinase II with a specificity for fructose. Hexokinases I and II had high apparent K_m values for fructose (8 and 15 mM, respectively) and medium or low apparent K_m values for glucose (150 and 18 μM, respectively) and mannose (18 and 15 μM, respectively). Maximal velocities were highest with fructose, medium with glucose and lowest with mannose. That hexokinases I and II used several sugars as substrate was concluded (i) from their identical elution profiles during enzyme separation and (ii) because their activities with two or three sugars at a time was always lower than the sum of activities with one substrate, indicating competition of the sugars for the reaction with the enzymes. Fructokinases I and II were very specific for fructose (85 and 140 μM, respectively) and had only little, if any, activity with glucose or mannose. All kinases showed varying degrees of activity with nucleoside triphosphates other than ATP. In the presence of all three sugars, hexokinases I and II were considerably more active with ATP than with uridine-, cytidine-, and guanosine 5'-triphosphate (UTP, CTP, GTP) except that, in the presence of glucose, hexokinase I was almost as active with UTP as with ATP. In the presence of fructose, fructokinase I exhibited highest activity with GTP and a gradually decreasing level of activity with CTP, UTP, and ATP. The activities in the presence of the other two sugars were highest with ATP. Fructokinase II was most active with ATP and fructose and progressively less active with GTP, UTP, and CTP. Cell fractionation by isopycnic density-gradient centrifugation or differential centrifugation indicated that fructokinase II was associated with chloroplasts, hexokinase II with mitochondria, and the other two kinases with the non-particulate cell fraction. In green leaves of pea (Pisum sativum L.), only a hexokinase (II) and fructokinase (II) were present. Corn (Zea mays L.) leaves exhibited only very low hexokinase activity. Dedicated to Prof. Dr. Hans Mohr on the occasion of his 60th birthday     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

Characterization of Sucrolysis via the Uridine Diphosphate and Pyrophosphate-Dependent Sucrose Synthase Pathway

The breakdown of sucrose to feed both hexoses into glycolytic carbon flow can occur by the sucrose synthase pathway. This uridine diphosphate (UDP) and pyrophosphate (PPi)-dependent pathway was biochemically characterized using soluble extracts from several plants. The sucrolysis process required the simultaneous presence of sucrose, UDP, and PPi with their respective K_m values being about 40 millimolar, 23 micromolar, and 29 micromolar. UDP was the only active nucleotide diphosphate. Slightly alkaline pH optima were observed for sucrose breakdown either to glucose 1-phosphate or to triose phosphate. Sucrolysis incrased with increasing temperature to near 50°C and then a sharp drop occurred between 55 and 60°C. The breakdown of sucrose to triose-P was activated by fructose 2,6-P₂ which had a K_m value near 0.2 micromolar. The cytoplasmic phosphofructokinase and fructokinase in plants were fairly nonselective for nucleotide triphosphates (NTP) but glucokinase definitely favored ATP. A predicted stoichiometric relationship of unity for UDP and PPi was measured when one also measured competing UDPase and pyrophosphatase activity. The cycling of uridylates, UDP to UTP to UDP, was demonstrated both with phosphofructokinase and with fructokinase. Enzyme activity measurements indicated that the sucrose synthase pathway has a major role in plant sucrose sink tissues. In the cytoplasmic sucrose synthase breakdown pathway, a role for the PPi-phosphofructokinase was to produce PPi while a role for the NTP-phosphofructokinase and for the fructokinase was to produce UDP.     

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K_m for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective `PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Subcellular localization of hexose kinases in pea stems: mitochondrial hexokinase

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K_m 76 micromolar) and mannose (K_m 71 micromolar) and a relatively low affinity for fructose (K_m 15.7 millimolar). The K_m for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al³⁺ was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.