Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.     

Molecular cloning, nucleotide sequencing, and expression of genes encoding alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii and the mesophile Clostridium beijerinckii

Proteins play a pivotal role in thermophily. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed inE. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme.     

Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii

Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophile Clostridium beijerinckii (CBADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26 °C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed in Escherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.     

Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations

The present study demonstrates the comparative thermal, conformational and kinetic stabilities of the three closely related enzymes; the mesophilic yeast alcohol dehydrogenase (YADH), horse liver alcohol dehydrogenase (HLADH), and the extreme-thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). The mid-point unfolding temperatures for TBADH and HLADH were at least 10 °C and 6 °C higher, respectively, than that of YADH. When YADH was completely inactivated by thermal stress, the residual activities of HLADH and TBADH were 70% and 100%, respectively. The optimum temperature (T_opt) activities of HLADH and TBADH were at least 40 °C and 55 °C higher, respectively, than that of YADH. Due to the higher rigidity of HLADH and TBADH, the enzymatic activation energies of HLADH and TBADH were higher than that of YADH. Geometric X-ray analysis indicated a comparatively higher coil (turn and loop) percentage in TBADH and HLADH than in YADH. Pairwise alignment for TBADH/HLADH exhibited a similarity score approximately 2.5-fold greater than that of the TBADH/YADH pair. Multiple alignments made with ClustalW revealed a higher number of conserved proline residues in the two most stable enzymes (HLADH/TBADH). These extra prolines tend to occur in surface loops and are likely to be responsible for the increased stability of TBADH and HLADH, by loop rigidification.     

Bioaffinity purification of NADP(+)-dependent dehydrogenases: studies with alcohol dehydrogenase from Thermoanaerobacter brockii.

This study describes the development and application of a bioaffinity chromatographic system for the one-step purification of an NADP(+)-dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme-specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking-on tactic). Specifically, the following is described: Evaluation of 8'-azo-linked, C(8)-linked, N(1)-linked, and N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of TBADH; evaluation of 2', 5'-ADP as a stripping ligand for TBADH bioaffinity purifications using an 8'-azo-linked immobilized NADP(+) derivative in the locking-on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP(+) derivatives investigated, only the 8'-azo-linked immobilized NADP(+) derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking-on ligand. This is in contrast to other NADP(+)-dependent dehydrogenases where the immobilized N(6)-linked cofactor proved to be suitable. While the one-step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations.     

Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.     

Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii

Summary Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophileClostridium beijerinckii (CBADH) and the other from the extreme thermophileThermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26°C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed inEscherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.     

Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.     

Oligomeric integrity--the structural key to thermal stability in bacterial alcohol dehydrogenases.

Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30 degrees in their melting temperatures. The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 A, respectively. Comparison of these two very similar structures (RMS difference in Calpha = 0.8 A) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, "charged-neutral" hydrogen bonds, and prolines as well as improved stability of alpha-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.     

基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生

【目的】构建己糖激酶与葡萄糖-6-磷酸脱氢酶的大肠杆菌共表达体系,以葡萄糖为底物实现辅酶NADPH的高效再生。【方法】通过分子生物学方法,克隆己糖激酶HKgs、HKpp基因,并于Escherichia coli BL21(DE3)中表达,再将己糖激酶HKgs、HKpp分别与葡萄糖-6-磷酸脱氢酶Gpd PP共表达,实现NADPH的原位再生。比较两个共表达工程菌的辅酶再生效果,并针对催化活力较高的工程菌BL21(HKgs+Gpd PP)进行表达条件优化。【结果】NADPH再生活力达到856 U/L。该辅酶再生体系与醇脱氢酶Adh R联合催化,使不对称还原4-氯乙酰乙酸乙酯的催化活力提高至原始值的2.5倍。【结论】通过己糖激酶与葡萄糖-6-磷酸脱氢酶在大肠杆菌中的共表达,构建了一个新的NADPH高效再生体系,并用于醇脱氢酶催化的不对称还原反应。     

Use of a polymer-bound flavin derivative for the rapid regeneration of NAD(P)+ from NAD(P)H in dehydrogenase systems

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters (Km and kcat) revealed an overall catalytic efficiency (kcat/Km) 185-fold greater compared to riboflavin. Various factors affecting the chemical regeneration of NAD+ from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2-4 compared to the other buffers. The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300-fold NADH----NAD+ conversions for the system using yeast alcohol dehydrogenase and up to 1500-fold NADPH----NADP+ regenerations for the system using glucose-6-phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.     

Development of immunosensors for the analysis of 1-naphthol in organic media

Immunosensor systems have been developed for the rapid determination of 1-naphthol. In this work, the comparison of performance of immunosensors working in aqueous and organic media was done. Direct, indirect and capture formats were studied. Immunoreagents were immobilized on controlled pore glass (CPG), hidroxysuccinimide agarose gel or on azlactone Protein A/G supports. The Protein A/G-based sensor showed the best performance. In aqueous media, a LOD of 16.2 microg l(-1) and a DR of 33.7-586.6 microg l(-1) were achieved employing Tween 20 at a concentration ranging from 0.01 to 0.05% v/v. Maximum sensitivity was reached with 0.025% of surfactant. Binary mixtures of methanol or acetonitrile with aqueous buffer and ternary mixtures of methanol/isopropanol or ethyl acetate/methanol with the same buffer were studied as organic media. The mixture 50% MeOH-50% 20 mM sodium phosphate, pH 8, with 0.05% (v/v) Tween 20 resulted to be the best. A detection limit of 12.0 microg l(-1) and a dynamic range of 53.6-17,756.0 microg l(-1) were reached. The recycling of Protein A/G-based sensor working in this media was about 300 assays. Preconcentration factors around 250 were achieved using methanol as extracting solvent. It has been demonstrated that the technique can be successful in carrying out the analysis of low solubility in water analytes, such as 1-naphthol. The sensors developed can use higher concentrations of organic solvent (up to 50% methanol) compared to ELISA. On the other hand, the advantage of preconcentration can also be taken for the use of the same procedure as recommended for standard sample treatments.     

The kinetics of intact microsome glucose-6-phosphatase are sigmoid at physiologic glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) were studied with intact and detergent-disrupted microsomes from normal and diabetic rats. Glucose-6-P concentrations employed (12 microM to 1.0 mM) spanned the physiologic range. With the enzyme of intact microsomes from both groups, plots of v versus [glucose-6-P] were sigmoid. Hanes plots (i.e. [glucose-6-P]/v versus [glucose-6-P]) were biphasic (concave upwards). A Hill coefficient of 1.45 was determined with substrate concentrations between 12 and 133 microM. Disruption of microsomal integrity abolished these departures from classic kinetic behavior, indicating that sigmoidicity may result from cooperative interaction of glucose-6-P with the glucose-6-phosphatase system at the substrate translocase specific for glucose-6-P. With the enzyme from normal rats the [glucose-6-P] at which the enzyme was maximally sensitive to variations in [glucose-6-P] (which we term "Smax"), determined from plots of dv/d [glucose-6-P] versus [glucose-6-P], was in the physiologic range. The Smax of 0.13 mM corresponded well with the normal steady-state hepatic [glucose-6-P] of 0.16 mM, consistent with glucose-6-phosphatase's function as a regulatory enzyme. With the diabetic enzyme, in contrast, values were 0.30 and 0.07 mM for the Smax and steady-state level, respectively. We suggest that the decreasing sensitivity of glucose-6-phosphatase activity to progressively diminishing glucose-6-P concentration, inherent in its sigmoid kinetics, constitutes a mechanism for the preservation of a residual pool of glucose-6-P for other hepatic metabolic functions in the presence of elevated concentrations of glucose-6-phosphatase such as in diabetes.     

Novel Active Chrysanthemic Esters

The rates of recycling and turnover of glucose in 5-day- and 21-day-old lambs and adult sheep were measured by the method of single simultaneous injection of glucose-6-³H and glucose-6-¹⁴C. The ³H/¹⁴C ratio decreased linearly with time and was 0.58 and 0.60 in lambs of 5-day- and 21-day-old, respectively, and 0.82 in adult sheep at 120 min after injection of the labeled glucose. The pool size and turnover rate of glucose considerably decreased with age. The rate of glucose recycling was significantly higher in lambs of both ages (22.0 and 26.2%, respectively) than in adult sheep (11.1 %).     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase.

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Estimation of Waste Battery Generation and Analysis of the Waste Battery Recycling System in China

China produces and consumes a large amount of batteries annually, which leads to many waste batteries needing to be recycled. The collection and recycling system of primary, alkaline secondary, and lithium‐ion secondary batteries in China is particularly poor, and waste battery recycling enterprises generally sustain economic losses if they solely use waste batteries as raw materials. Increasing the profits of waste battery recycling systems is a key problem that needs to be considered. This article quantitatively analyzes waste battery generation in China by using annual sales data and probable lifetime distribution of various batteries. The results show that the rapid growth of battery usage has led to an increased generation of waste batteries and the percentage of different types of waste batteries is changing over time. In 2013, the total quantity of all waste batteries in the medium lifetime scenario reached 570 kilotons, of which primary, alkaline secondary, and lithium‐ion secondary waste batteries accounted for approximately 36%, 28%, and 35%, respectively. Based on a real‐world case study of a typical domestic waste battery recycling enterprise in China, material flow analysis and cost‐benefit analysis were conducted to study the development of the recycling process of comingled waste batteries. Through scenario analysis, we conclude that increasing the use of waste batteries as raw materials and the recycling of other materials that are less valuable reduces the profits of the waste battery recycling enterprise. Higher profits can be achieved by adding the production of high value‐added downstream products and government support. At the same time, the essential role of the government in developing a waste battery recycling system was identified. Finally, relevant suggestions are made for improvements in both the government and enterprise sectors.     

Structured bienzymatical models formed by sequential enzymes bound into artificial supports: Active glucose transport effect

Summary Two different artificial membrane systems bearing two built-in sequential enzymes are studied and compared in this communication.The first is a nonstructured membrane bearing two mixed enzymes: -galactosidase and glucose-oxidase. Its use enables a mathematical model to be formulated describing the heterogeneous phase kinetics of a bienzymatic system. The second is a multi-layer membrane system in which the structural dissymmetry involves a spatial orientation of the reacting metabolites, resulting in active glucose transport.The latter system consists of two active leaflets, the first phosphorylating glucose (hexokinase+ATP), the second dephosphorylating glucose-6 phosphate (phosphatase). On either side of this system, a perm-selective proteic layer allows the passage of glucose but not of glucose-6 phosphate. When positioned between two compartments containing glucose, such a membrane accumulates glucose on its phosphatase side, while degrading ATP.The accumulation of glucose as a function of the initial concentration shows the classical saturation of the transport system. Fructose competes with glucose transport.The chemical balance of these two reactions has the appearance of hydrolysis of ATP. Vectorial catalysis is a result of the dissymmetry in distribution of active sites and can be explained by an oscillatory concentration profile of glucose inside the membrane.The bienzymatic mechanism, a model of which is given here, is valid for any thickness of active layers and applicable to a system where both active sides are part of the same molecule as soon as it forms a uniformly oriented monolayer throughout the membrane structure.     

Re-evaluating the recycling hypothesis in the germinal centre

Mathematical models have been used to study different aspects of the germinal centre reaction, in particular, affinity maturation of antibodies and the hypothesis of recycling. So far, interpretation of several theoretical and experimental results has pointed to the existence of recycling. However, theoretical models have seldom been compared with experimental data from specific immune responses and the potential relevance of recycling in the germinal centre is still an open problem. In this article, we propose a model without recycling that takes into account selection mechanisms that were previously uncovered experimentally. We apply the model to several experimental systems that use different Ag and compare the results with experimental data of affinity maturation whenever available. The results obtained for a primary immune response to the hapten (4-hydroxy-3-nitrophenyl)-acetyl show that recycling is not a necessary mechanism to achieve the level of affinity maturation observed in germinal centre reactions. Similar levels of affinity maturation are obtained for other responses, although for antibodies involving several affinity-enhancing mutations the affinity maturation obtained with the model is much lower. Interpretation of these results and consequences towards the concept of recycling are discussed.     

Determination of a glucose-dependent futile recycling rate constant from an intraperitoneal glucose tolerance test

Increased glucose cycling between glucose and glucose-6-phosphate is characteristic of insulin resistance and hyperglycemia seen with Type II diabetes. Traditionally, glucose cycling is determined by the difference between hepatic glucose output measured with separate [2-3H]glucose and [6-3H]glucose infusions. We demonstrate a novel method for determining hepatic glucose recycling from an intraperitoneal glucose tolerance test (IPGTT). A single tracer, [1, 2-13C(2)]glucose (a M2 glucose isotopomer), was administered at 1mg/g body weight to 4-month-old C57BL/6 mice. Hepatic glucose recycling was monitored by the appearance of a plasma M1 isotopomer of glucose, which is produced by the action of the pentose cycle on the M2 glucose isotopomer in the liver. The initial M2 enrichment was 56% and decreased to 13% at the end of 3 h, and the M1 enrichment peaked at 2 h. The ratio of plasma M1/M2 glucose increased linearly with time to approximately 25%, and the regression of the M1/M2 ratio against time gives a slope, termed the in vivo glucose-dependent futile recycling rate constant k(HR). k(HR) estimates glucose/glucose-6-phosphate futile cycling, along with glucose recycling through the pentose cycle. These observations demonstrate complex substrate cycling during an IPGTT using a single stable isotope tracer.     

Glucose condensation by glucoamylase in organic solvents

Summary Oligosaccharides were synthesized through the enzymatic condensation of D-glucose by glucoamylase in water-organic mixtures with high concentrations of two of diethylene glycol diethyl ether or triethylene glycol dimethyl ether. The effect of water content on the yield of reaction was studied; maximum yield was obtained with 10% (v/v) of water in the two systems. Kinetics of synthesis and products composition were different with the two solvents. 37% of glucose were condensed by action of glucoamylase from a reaction medium containing 20 g/L of glucose and 90% (v/v) of diethylene glycol diethyl ether.