Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.     

Induction of Ears from Maize Seeds in Vitro and Plant Regeneration from Ovaries of Unfertilized Ears

A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.     

A new colorimetric method for determining the isomerization activity of sucrose isomerase

A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.     

Active-site-directed inhibition of sucrose-phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin

Sucrose-phosphate synthase (SPS, E.C. 2.4.1.14) from spinach (Spinacia oleracea L.) was partially purified and the inhibition of the enzyme reaction by 1-deoxynojirimycin and Cibacron blue F3G-A analyzed. Cibacron blue was a high-affinity competitive inhibitor with respect to the substrate UDPglucose (K_i = 80 nM) and a mixed-type inhibitor with respect to fructose-6-phosphate. 1-Deoxynojirimycin was a mixed-type inhibitor of SPS with respect to UDPglucose [K_i(EI) = 5.8 mM] and a uncompetitive inhibitor with respect to fructose 6-phosphate. These results are discussed in relation to the mechanism of the reaction catalysed by SPS and the secondary structure of the enzyme.Abbreviations DN 1-deoxynojirimycin - Glc6P glucose-6-phosphate - Fru6P fructose-6-phosphate - SPS sucrose-phosphate synthase - UDPG1c UDPglucose We are grateful to M. Stitt (University of Heidelberg, Germany) for many helpful discussions and J. Harr and P. Bocion (both SANDOZ AGRO, Switzerland) for supporting the work.     

ATP Regulates Sodium Channel Kinetics in Pancreatic Islet Beta Cells

Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP]_i) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP]_i remains unclear. Here, we demonstrated that increasing [ATP]_i within a certain range of concentrations (2–8 mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2 mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12 mM). Furthermore, elevated ATP modulated the sodium channel kinetics in a dose-dependent manner. Increased [ATP]_i shifted both the current–voltage curve and the voltage-dependent inactivation curve of sodium channel to the right. Finally, the sodium channel recovery from inactivation was significantly faster when the intracellular ATP level was increased, especially in 8 mM [ATP]_i, which is an attainable concentration by the high glucose stimulation. In summary, our data suggested that elevated cytosolic ATP enhanced the activity of Na⁺ channels, which may play essential roles in modulating β cell excitability and insulin release when blood glucose concentration increases.     

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG₁, SG_?1, SG_?2, SG_?3, SG_?4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG₁ and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1–4Glcα1–4Glcα1–4Glcα1–4Glcα1–4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc _m -AltNAc-Glc _n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG₁ –SG_?4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V _derivative) with that toward PA-0 (V _PA-0), we found that the value of V _derivative/V _PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.     

Salt tolerance of a cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous chemical treatments

Suaeda fruticosa Forssk is a leaf succulent obligate halophyte that produces numerous seeds under saline conditions. Seeds are a good source of high quality edible oil and leaves are capable of removing substantial amount of salt from the saline soil besides many other economic usages. Little is known about the biochemical basis of salt tolerance in this species. We studied some biochemical responses of S. fruticosa to different exogenous treatments under non-saline (0 mM), moderate (300 mM) or high (600 mM) NaCl levels. Eight-week-old seedlings were sprayed twice a week with distilled water, hydrogen peroxide (H₂O₂, 100 μM), glycine betaine (GB, 10 mM), or ascorbic acid (AsA, 20 mM) for 30 days. At moderate (300 mM) NaCl, leaf Na⁺, Ca²⁺ and osmolality increased, along with unchanged ROS and antioxidant enzyme activities, possibly causing a better plant growth. Plants grew slowly at 600 mM NaCl to avoid leaf Na⁺ buildup relative to those at 300 mM NaCl. Exogenous application of distilled water and H₂O₂ improved ROS scavenging mechanisms, although growth was unaffected. ASA and GB alleviated salt-induced growth inhibition at 600 mM NaCl through enhancing the antioxidant defense system and osmotic and ion homeostasis, respectively.     

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.     

Regulation of glycolysis during acclimation of scallops (Patinopecten yessoensis Jay) to anaerobiosis

Some glycolytic metabolites in the adductor muscle were measured after transfer of scallops from aerobic to anaerobic saltwater for 12 h. The level of octopine increased gradually during the initial 3 h incubation, and thereafter the level increased rapidly up to 12 h. The ATP level also did not show any significant change for the initial 3 h, and then decreased rapidly. The fructose 2,6-biphosphate (Fru 2,6-BP) level increased drastically during the initial 3 h incubation, but thereafter the level did not show any significant change up to 12 h. In the short-term effects of anaerobiosis for 90 min, the level of fructose 6-phosphate (Fru 6-P) increased just after transfer to anaerobiosis, and then its level decreased. In contrast, the fructose 1,6-biphosphate (Fru 1,6-BP) level increased greatly, at the time when both glucose 6-phosphate (Glc 6-P) and Fru 6-P decreased. The Fru 2,6-BP level did not any significant change during the initial 15 min incubation, but thereafter the level increased gradually up to 90 min. Scallop 6-phosphofructo 1-kinase (EC 2.7.1.11) (PFK1) was strongly activated by 1 microM Fru 2,6-BP when 0.2 mM Fru 6-P was used as a substrate, but the activity was not affected at 5 mM Fru 6-P. In view of these results, the regulation mechanism of glycolysis is discussed.     

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h = 1.70 and S _0.5 = 6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K⁺ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h = 1.16) and GLK affinity for substrate was reduced (S _0.5 = 23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h = 1.60, S _0.5 = 9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h = 1.20, S _0.5 = 45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.     

Physiological response of the facultative halophyte,Aeluropus littoralis,to different salt types and levels

The present study investigated the effects of NaCl, KCl and Na₂SO₄ salts on the C₄ excreting halophyte Aeluropus littoralis in relation to growth, mineral status and photosynthesis in greenhouse conditions. Plantlets were subjected to five salinity levels: 0, 200, 400, 600 and 800 mM for 30 days. Growth decreased progressively with salinity increase, its reduction might be correlated with the high sodium (and/or chloride) accumulation in plant tissues, the decrease of leaf water status and the decline of the net photosynthetic rate and the intrinsic water use efficiency. Na₂SO₄ appeared more toxic than KCl and NaCl, especially at 200 mM. At 200 mM, Na₂SO₄ reduced plant growth by 61% while for other salt forms, the reductions were less than 20%. At this salt level, stomatal conductance showed a consistent pattern with plant growth and could adequately explain the variations between the effects of the three salt types.     

Metabolite pools during starch synthesis and carbohydrate oxidation in amyloplasts isolated from wheat endosperm

Starch synthesis and CO₂ evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with ¹⁴C-labelled hexose-phosphates. Amyloplasts converted [U-¹⁴C]glucose 1-phosphate (Glc1P) but not [U-¹⁴C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-¹⁴C]Glc1P and [U-¹⁴C]Glc6P were metabolized to CO₂, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium. Received: 29 March 1997 / Accepted: 5 June 1997     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves

The effects of two sodium salts on growth, fatty acids, and essential oil compositions were investigated in a medicinal and aromatic plant, Ocimum basilicum cultivated in hydroponic medium. Plants were subjected to an equimolar concentration of Na₂SO₄ (25 mM) and NaCl (50 mM) for 15 days. Our results showed that leaf growth rate was more depressed by 25 mM Na₂SO₄ than by 50 mM NaCl. The total fatty acid contents did not show any change in plants. α-Linolenic, palmitic, and linoleic acids were the major fatty acids. The identification of basil leaf fatty acids has not been previously studied and this work revealed the predominance of polyunsaturated fatty acids. Under both salts, leaf fatty acid composition remained unchanged. Regarding the essential oil yield, it decreased significantly by 28 % under 25 mM Na₂SO₄ and showed an increase by 27 % under 50 mM NaCl. The major volatile compound in leaves was linalool with 34.3 % of total essential oil constituents, followed by eugenol (19.8 %), 1.8-cineole (14.4 %) and methyl eugenol (5.2 %). Further, levels of eugenol and methyl eugenol were most modulated by salt, and the negative correlation between these two compounds reflects the stimulation of O-methyltransferase activity under both salts.     

Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor

NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased significantly compared with that under 200 mM NaCl treatment. Na⁺ secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was significantly higher than that under 200 mM NaCl without SNP. However, there was no significant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no significant difference in salt-secretion rate of individual glands, Na⁺ concentration in the leaves treated with 200 mM NaCl solution containing SNP was significantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na⁺ concentration in salt glands. Obviously, the efficiency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efficiency of the secretion process per gland.     

A dual-mode optical assay for iron(II) and gallic acid based on Fenton reaction

The hydroxyl radicals ( · OH) produced by the Fenton reaction of iron(II) and hydrogen peroxide (H₂O₂) can oxidize the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (Ox-TMB), resulting in a decrease in the fluorescence intensity of the reaction system and an increase in ultraviolet absorption. Ox-TMB had a visible absorption peak at 625 nm and a fluorescence peak around 420 nm. When gallic acid (GA) was added to the system, Ox-TMB was reduced to TMB, which made the color of the system disappear and the fluorescence recover. The linear ranges for determination of iron(II) were 0.5–10 μM (fluorometric) and 0.5–20 μM (colorimetric), and the detection limits were 0.25 μM (fluorometric) and 0.28 μM (colorimetric). The linear ranges for determination of GA were 0–80 μM (fluorometric) and 0–60 μM (colorimetric), and the detection limits were 0.31 μM (fluorometric) and 0.8 μM (colorimetric). The results of anti-interference experiments shew that this dual-mode assay had very good selectivity for the determination of iron(II) and GA.     

Effect of sodium chloride-induced stress on growth, proline, glycine betaine accumulation, antioxidative defence and bacoside A content in in vitro regenerated shoots of Bacopa monnieri (L.) Pennell

Growth, osmotic adjustment, antioxidant enzyme defense and the principle medicinal component bacoside A were studied in the in vitro raised shoot cultures of Bacopa monnieri, a known medicinal plant, under different concentrations of NaCl [0.0 (control), 50, 100, 150 or 200 mM]. A sharp increase in Na⁺ content was observed at 50 mM NaCl level and it was about 6.4-fold higher when compared with control. While Na⁺ content increased in the shoots with increasing levels of NaCl in the medium, both K⁺ and Ca²⁺ concentrations decreased. Significant reduction was observed in shoot number per culture; shoot length, fresh weight (FW), dry weight (DW) and tissue water content (TWC) when shoots were exposed to increasing NaCl concentrations (50–200 mM) as compared with the control. Decrease in TWC was not significant at higher NaCl level (150 and 200 mM). At 200 mM NaCl, growth of shoots was adversely affected and microshoots died under prolonged stress. Minimum damage to the membrane as assessed by malondialdehyde (MDA) content was noticed in the controls in contrast to sharp increase of it in NaCl-stressed shoots. Higher amounts of free proline, glycinebetaine and total soluble sugars (TSS) accumulated in NaCl-stressed shoots indicating that it is a glycinebetaine accumulator. About 2.11-fold higher H₂O₂ content was observed at 50 mM NaCl as compared with control and it reached up to 7.1-folds more at 200 mM NaCl. Antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase) also increased with a rise in NaCl level. Increase in bacoside A, a triterpene saponin content was observed only up to 100 mM NaCl level. Higher salt concentrations inhibited the accumulation of bacoside A. It appears from the data that accumulation of osmolytes, ions and elevated activities of antioxidant enzymes play an important role in osmotic adjustment in shoot cultures of Bacopa under salt stress.     

Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca

Glucose isomerase (GIase) catalyzes the isomerization of d-glucose to d-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5–10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K _m and K _cat values of the enzyme are 197 mM and 1,688 min^?1, respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.