Kinetics of formation of maltose and isomaltose through condensation of glucose by glucoamylase

A kinetic model was devised for the hydrolysis and synthesis of maltose and isomaltose by two glucoamylases from Rhizopus niveus and Aspergillus niger, and the validity of the model was verified experimentally at 313 K and pH 5.0. For both enzymes, the formations of maltose and isomaltose from glucose were parallel reversible reactions, and glucosyl transfer between maltose and isomaltose was not observed. The enzymes catalyzed rapid hydrolysis and synthesis of maltose. Isomaltose was hydrolyzed and synthesized more slowly, but the level produced from glucose was much higher than that of maltose. These hydrolysis and condensation reactions were expressed well by the model.     

Kinetics of condensation of glucose into maltose and isomaltose in hydrolysis of starch by glucoamylase

Kinetics of the condensation of glucose into maltose and isomaltose in the hydrolysis of starch by two types of glucoamylase (from Aspergillus niger and Rhizopus niveus) was studied both experimentally and theoretically. A kinetic model for the hydrolysis of starch by glucoamylase from A. niger was proposed. In this model the reversible hydrolysis of maltose and isomaltose and the kinetic parameters change were taken into consideration. Calculated values agreed approximately with the experimental results, and this simple kinetic model was found to have practical use. The rate of condensation of glucose into isomaltose by enzyme from A. niger was about three times larger than that by enzyme from R. niveus. At a higher initial concentration of starch a large amount of isomaltose was reversed, and the glucose yield was reduced significantly after very long reaction times.     

A kinetic model for the hydrolysis and synthesis of maltose, isomaltose, and maltotriose by glucoamylase

Kinetic results on the glucomylase-catalysed hydrolysis of maltose and maltotriose, and glucose polymerization into maltose and isomaltose up to 450 g/L total sugar concentration are presented. Whereas the enzyme has a faster hydrolytic and synthetic activity on alpha-(1-->4) than on alpha-(1-->6) linkages, at equilibrium, on the contrary, the isomaltose level which represents 15% (w/w) of the total sugar concentration at the highest investigated concentrations is much higher than the corresponding maltose level. Under a wide range of initial conditions, experimental results are adequately described by a new kinetic model with simple first- and second-order, or Michaelian-type, rate expressions for the reversible hydrolysis of maltotriose, maltose, and isomaltose. The model also accounts for the inhibition of hydrolysis by glucose, but does not consider the concentration of water which, under the present conditions, was not found kinetically limiting.     

Inhibition by maltose, isomaltose, and nigerose of the synthesis of high-molecular-weight D-glucans by the D-glucosyltransferases of Streptococcus sobrinus

Two D-glucosyltransferases are produced by Streptococcus sobrinus C211. One (GTF-S) catalyzes the conversion of sucrose into soluble alpha-(1----6)-linked alpha-(1----3)-branched D-glucans, and the other (GTF-I), of sucrose into alpha-(1----3)-linked alpha-(1----6)-branched D-glucans. These enzymes were studied by using maltose, isomaltose, and nigerose as inhibitors. Maltose and isomaltose were found to be competitive inhibitors of GTF-S, whereas nigerose has no effect on GTF-S activity. The Ki values for maltose and isomaltose were determined to be 11 and 15mM, respectively. Maltose, isomaltose, and nigerose competitively inhibit GTF-I. The Ki values for these inhibitors were found to be approximately 0.8, 2.5, and 15mM, respectively. The inhibitory properties of each disaccharide are interpreted in terms of conformational comparisons with sucrose.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil

The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V_max of the ATPase 2.7-fold but had no effect on the K_m of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN₃, as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.     

Oxidation of exogenous glucose, sucrose, and maltose during prolonged cycling exercise.

The purpose of the present study was to investigate whether combined ingestion of two carbohydrates (CHO) that are absorbed by different intestinal transport mechanisms would lead to exogenous CHO oxidation rates of >1.0 g/min. Nine trained male cyclists (maximal O(2) consumption: 64 +/- 2 ml x kg body wt(-1) x min(-1)) performed four exercise trials, which were randomly assigned and separated by at least 1 wk. Each trial consisted of 150 min of cycling at 50% of maximal power output (60 +/- 1% maximal O(2) consumption), while subjects received a solution providing either 1.8 g/min of glucose (Glu), 1.2 g/min of glucose + 0.6 g/min of sucrose (Glu+Suc), 1.2 g/min of glucose + 0.6 g/min of maltose (Glu+Mal), or water. Peak exogenous CHO oxidation rates were significantly higher (P < 0.05) in the Glu+Suc trial (1.25 +/- 0.07 g/min) compared with the Glu and Glu+Mal trials (1.06 +/- 0.08 and 1.06 +/- 0.06 g/min, respectively). No difference was found in (peak) exogenous CHO oxidation rates between Glu and Glu+Mal. These results demonstrate that, when a mixture of glucose and sucrose is ingested at high rates (1.8 g/min) during cycling exercise, exogenous CHO oxidation rates reach peak values of approximately 1.25 g/min.     

beta-Maltose is the metabolically active anomer of maltose during transitory starch degradation

Maltose is the major form of carbon exported from the chloroplast at night as a result of transitory starch breakdown. Maltose exists as an alpha- or beta-anomer. We developed an enzymatic technique for distinguishing between the two anomers of maltose and tested the accuracy and specificity of this technique using beta-maltose liberated from maltoheptose by beta-amylase. This technique was used to investigate which form of maltose is present during transitory starch degradation in bean (Phaseolus vulgaris), wild-type Arabidopsis (Arabidopsis thaliana), two starch deficient Arabidopsis lines, and one starch-excess mutant of Arabidopsis. In Phaseolus and wild-type Arabidopsis, beta-maltose levels were low during the day but were much higher at night. In Arabidopsis plants unable to metabolize maltose due to a T-DNA insertion in the gene for the cytosolic amylomaltase, (Y. Lu, T.D. Sharkey [2004] Planta 218: 466-473) levels of alpha- and beta-maltose were high during both the day and night. In starchless mutants of Arabidopsis, total maltose levels were low and almost completely in the alpha-form. We also found that the subcellular concentration of beta-maltose at night was greater in the chloroplast than in the cytosol by 278 microm. We conclude that beta-maltose is the metabolically active anomer of maltose and that a sufficient gradient of beta-maltose exists between the chloroplast and cytosol to allow for passive transport of maltose out of chloroplasts at night.     

Oxidation of dibenzofuran by Pseudomonas strains harboring plasmids of naphthalene degradation]

Pseudomonas strains harboring plasmids pBS3, pBS4, NAH7 were shown to carry out initial transformation of dibenzofurane to 4-[2'-(3'-hydroxy)-benzofuranyl]-2-keto-3-butenic acid due to broad substrate specificity of the enzymes of naphthalene catabolism nahA, nahB, nahC and nahD. These strains did not grow on dibenzofurane because of the inability of the enzyme nahE to split pyruvate of 4-[2'-(3' hydroxy)-benzofuranyl]-2-keto-3-butenic acid, which leads to accumulation of the latter. The strains harboring plasmids pBS2 and NPL-1 are not capable of any transformation of dibenzofurane.     

Crypticity of Myrothecium verrucaria spores to maltose and induction of transport by maltulose, a common maltose contaminant

Spores of the fungus Myrothecium verrucaria are cryptic to maltose and isomaltose. Induction of a transport system can be effected by several sugars whose order of effectiveness is: turanose > maltulose > sucrose > d-arabinose, d-fructose, nigerose, maltotriulose, kestose > melezitose, raffinose, nystose, and stachyose. The transport system is not specific to maltose and isomaltose, and it is apparently identical to an induced trehalose permease described previously. Induction of the permease is markedly influenced by spore age-older spores being more responsive. Pure maltose is not absorbed by spores. Absorption of commercial reagent-grade maltose is due to permease induction by maltulose as an impurity. Maltulose contamination of maltose was demonstrated by charcoal column chromatography and comparison of its physical, chemical, and permease-inductive properties with those of authentic maltulose. Maltose accumulates temporarily in spores after absorption and then decreases, although no conversion to glucose can be detected. Although spores contain small quantities of maltase, metabolism of maltose may be via some nonhydrolytic pathway.     

The enzymatic reaction for the production of panose and isomaltose by glucosyltransferase from Aureobasidium

Glucosyltransferase from Aureobasidium produced 212 mg ml^-1 of glucosyl-oligosaccharides (panose: Glcα1→6Glcα1→4Glc 189 mg ml^-1 and isomaltose: Glcα1→6Glc 23 mg ml^-1) from maltose: Glcα1→4Glc at a high concentration (500 mg ml^-1) and the efficiency of production was 42-4%. The enzymatic reaction from maltose to panose is reversible but that from panose to isomaltose is not.