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.     

The specificity of the synthetic reaction of two yeast alpha-glucosidases.

The specificity of the hydrolytic reaction has been compared to that of the synthetic reaction for maltase and isomaltase (alpha-methyl-D-glucosidase) from Saccharomyces oviformis. Maltase which hydrolyzes the alpha-1,4-disaccharide, maltose, and the alpha-1,6-disaccharide, isomaltose, catalyzes the formation of both maltose and isomaltose from free glucose. Isomaltase, which hydrolyzes isomaltose but not maltose, catalyzes the formation only of isomaltose from glucose. Both enzymes hydrolyze p-nitrophenyl-alpha-D-glucoside releasing the alpha-anomer of glucose. The enzymes utilize the alpha-anomer but not the beta-anomer for the synthesis of the disaccharides. These results are consistent with the double displacement mechanism for glycosidases and with the proposal that the glucosyl-enzyme complex is an intermediate in the reaction. The competitive inhibition by D-glucose is independent of its anomeric form for both enzymes.     

Effects of alpha-D-glucosylglycerol on the in vitro digestion of disaccharides by rat intestinal enzymes

Alpha-D-glucosylglycerol (GG) is a mixture of 2-O-alpha-D-glucosylglycerol (GG-II), (2R)-1-O-alpha-D-glucosylglycerol (R-GG-I) and (2S)-1-O-alpha-D-glucosylglycerol (S-GG-I). GG has been found to be slightly hydrolyzed in vitro only by rat intestinal enzymes, but hardly at all by other digestive juices. GG suppressed the hydrolysis of maltose, sucrose and isomaltose by rat intestinal enzymes because the amount of glucose in the digestion of a mixture of GG and disaccharide was less than the sum of that in each individual digestion. The consumption of GG was suppressed by isomaltose, but promoted by maltose, with the hydrolysis of GG being suppressed. Sucrose appeared to suppress only the consumption of S-GG-I, suggesting that S-GG-I was hydrolyzed by the active site of sucrase in a sucrase-isomaltase complex. Transglucosylation seems to have occurred more frequently in the individual digestion of maltose and isomaltose than in that of GG and sucrose. GG seemed to promote transglucosylation in the presence of maltose, to suppress it with sucrose, and to delay it with isomaltose.     

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.     

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.     

Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.

The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.     

Two Forms of Glucoamylase from Mucor rouxianus

Both of the two forms of glucoamylase (glucoamylases I and II) from the wheat bran culture of Mucor rouxianus hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but did not act on isomaltose and isomaltotriose. Phenyl α-maltoside was hydrolyzed into glucose and phenyl α-glucoside by both glucoamylases. Maltose was hydrolyzed about one-fifth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen, soluble starch in the yields of almost complete hydrolysis. They hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylase II hydrolyzed raw starch at 3-fold higher rate than glucoamylase I. The former hydrolyzed rice starch almost completely into glucose, whereas the latter hydrolyzed it incompletely (nearly 50%).     

Gluco-oligosaccharides synthesized by glucosyltransferases from constitutive mutants of Leuconostoc mesenteroides strain Lm 28

Aims:  To find different types of glucosyltransferases (GTFs) produced by Leuconostoc mesenteroides strain Lm 28 and its mutant forms, and to check the effectiveness of gluco-oligosaccharide synthesis using maltose as the acceptor.
Methods and Results:  Constitutive mutants were obtained after chemical mutagenesis by ethyl methane sulfonate. Lm M281 produced more active GTFs than that obtained by the parental strain cultivated on sucrose. GTF from Lm M286 produced a resistant glucan, based on endo-dextranase and amyloglucosidase hydrolysis. The extracellular enzymes from Lm M286 catalyse acceptor reactions and transfer the glucose unit from sucrose to maltose to produce gluco-oligosaccharides (GOS). By increasing the sucrose/maltose ratio, it was possible to catalyse the synthesis of oligosaccharides of increasing degree of polymerization (DP).
Conclusions:  Different types of GTFs (dextransucrase, alternansucrase and levansucrase) were produced from new constitutive mutants of Leuc. mesenteroides . GTFs from Lm M286 can catalyse the acceptor reaction in the presence of maltose, leading to the synthesis of branched oligosaccharides.
Significance and Impact of the Study:  Conditions were optimized to synthesize GOS by using GTFs from Lm M286, with the aim of producing maximum quantities of branched-chain oligosaccharides with DP 3–5. This would allow the use of the latter as prebiotics.     

Heterooligosaccharide synthesis catalyzed by α-glucosidase from Bacillus stearothermophilus

α-Glucosidase from Bacillus stearothermophilus was used as a catalyst for oligosaccharide synthesis by reversed hydrolysis. The yield of disaccharides and trisaccharides depended strongly on the units of enzyme activity added, and on the stability of the enzyme under reaction conditions. When glucose was the only saccharide present in the reaction mixture with α-glucosidase, isomaltose (51%), nigerose (25%), maltose (14%) and kojibiose (10%) were formed. In 50% glucose solution, disaccharide concentrations reached up to 400 mmol/l and trisaccharides were also produced. When other saccharides (mannose or xylose), in addition to glucose, were present in the reaction mixture, both homodisaccharides and heterodisaccharides were formed, their quantity being dependent on the glucose/saccharide acceptor ratios. The highest yields of oligosaccharides were observed with glucose alone, consistent with the observation that the enzyme stability was highest with glucose as the sole saccharide.     

Effects of castanospermine on purified lysosomal alpha-1,4-glucosidase

The indolizidine alkaloid castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) inhibits hydrolysis of maltose, glycogen and isomaltose by purified lysosomal alpha-glucosidase yielding Ki values of 0.095, 0.10 and 0.30 mumol/l, respectively. Castanospermine exhibited high affinity for both the maltose and isomaltose sites. In distinct contrast, the alkaloid exhibited little or no affinity for the site catalyzing hydrolysis of glycogen as indicated by a noncompetitive mode of inhibition. Kinetic data presented in this report indicate castanospermine to be a very potent inhibitor of lysosomal alpha-glucosidase.     

The structure of an α-D-glucan from Cyttaria harioti Fischer

A homogeneous glucan has been isolated from the fruiting bodies of Cyttaria harioti Fischer. Partial acid hydrolysis produced major amounts of isomaltose, whereas acetolysis gave maltose and maltotriose. Enzymic hydrolysis with amylo-glucosidase and pullulanase indicated a structure based on maltotriose residues connected by (1→6)-α-D linkages. This conclusion was supported by periodate-oxidation data which also showed that 3–7% of the glucose resisted oxidation. Methylation analysis confirmed the presence of (1→6) and (1→4) linkages in the ratio 1:2.4.     

黑曲霉突变株葡萄糖淀粉酶的底物特异性

黑曲霉(Aspergillus niger)突变株T-21葡萄糖淀粉酶(GAI)仅能水解多种淀粉及麦芽低聚糖生成唯一产物β-葡萄糖,其水解麦芽糖及麦芽三糖的速度分别为200和570mg葡萄糖·h~(-1)·mg~(-1).GAI水解α-1,4键的速度比水解α-1.6键快100多倍.除了马铃薯淀粉外,对其它淀粉及麦芽低聚糖几乎都能100%地水解,但不能水解环状糊精,其水解各麦芽低聚糖的最先产物都比原底物少一个葡萄糖单位,说明GAI为一外切型淀粉酶.GAI对麦芽糖、麦芽三糖、可溶性淀粉、糯米淀粉、糊精及糖原的Km值分别1.92mmol/L、0.38mmol/L、0.053%、0.045%、0.059%、及0.076%,V_(max)分别为590、1370、1270、1520、1120和1220mg葡萄糖·h~(-1)·mg~(-1).D-葡萄糖酸-δ-内酯及麦芽糖醇对此酶分别具有反竞争性抑制和混合性抑制.     

Resistance to quaternary ammonium compounds in Staphylococcus spp. isolated from the food industry and nucleotide sequence of the resistance plasmid pST827

K.A. ALBASHERI AND W.J. MITCHELL. 1995. Maltose metabolism in the obligate anaerobe Clostridium acetobutylicum was studied. The sugar is accumulated via an energy-dependent transport process which is not a phosphotransferase. Cell extracts were incapable of phosphorylating maltose in the presence or absence of phosphoenolpyruvate or ATP, but exhibited hydrolytic activity against a range of glucoside substrates. The activity was predominantly in the soluble fraction of cell extracts, indicating a cytoplasmic location in the cell. Gel filtration on Sephadex G100 indicated the presence of at least two α-glucosidases. One enzyme (maltase) was active with maltose and maltotriose, while the other (pNPGase) hydrolysed isomaltose and several glucoside analogues, but neither showed activity against starch. Both glucosidases were induced by isomaltose, maltose, glucose and starch, but not by xylose, sucrose or cellobiose. In the presence of both glucose and maltose, growing cells showed a preference for glucose, apparently due to regulation of maltose transport, which did not occur in glucose-grown cells.     

Some Catalytic Properties of an Exo-l,6-α-glucosidase (Glucodextranase) from Arthrobacter globiformis I42

An exo-l,6-α-glucosidase (EC 3.2.1.70) (glucodextranase) produced extraceUularly by Arthrobacter globiformis I42 was found to invert the configuration of glucose released from dextran, and to require calcium for protection against warming. Among isomaltodextrins used as substrates for this enzyme, the rate of hydrolysis for isomaltose was the lowest and increased with the degree of polymerization (d. p.) of the saccharides up to d. p. 7. The minor activities accompanying purified glucodextranase preparations (release of glucose from starch, splitting of maltose, nigerose and kojibiose) were ascribed to the glucodextranase itself. Fourteen native dextrans and soluble potato starch were subjected to digestion by this glucodextranase and the rate, process and extent of hydrolysis of these substrates were studied relative to the composition of non-l,6-α-linkages of these polysaccharides.     

A transglucosylase of Streptococcus bovis

1. A transglucosylase has been separated from the α-amylase of Streptococcus bovis by chromatography of the cell extract on DEAE-cellulose. 2. The transglucosylase can synthesize higher maltodextrins from maltotriose, but maltose, isomaltose and panose do not function as donors. 3. Iodine-staining polysaccharide may be synthesized from maltotriose provided that glucose is removed. Synthesis from maltohexaose results in dextrins of sufficient chain length to stain with iodine, but again maltodextrins of longer chain length are formed when glucose is removed from the system. 4. The transglucosylase degrades amylose in the presence of a suitable acceptor, transferring one or more glucosyl residues from the non-reducing end of the donor to the non-reducing end of the acceptor. With [¹⁴C]glucose as acceptor the maltodextrins produced were labelled in the reducing glucose unit only. 5. The acceptor activities of 25 sugars have been compared with that of glucose. Maltose has 50%, methyl α-glucoside has 15%, isomaltose and panose each has 8% and sucrose has 6% of the accepting efficiency of glucose. Mannose and sorbose also had detectable activity. With the exception of maltose all these sugars produced a different series of dextrins from that obtained with glucose. 6. It was concluded that S. bovis transglucosylase transfers α-(1→4)-glucosidic linkages in the same manner as D-enzyme, but some differences in specificity distinguish the two enzymes. Unlike D-enzyme, S. bovis transglucosylase can transfer glucosyl units, producing appreciable amounts of maltose both during synthesis from maltotriose and during transfer from amylose to glucose. 7. No evidence was found that the transglucosylase was extracellular. The enzyme is cell-bound, and is released by treatment of the cells with lysozyme and by suspension of the spheroplasts in dilute buffer. 8. The transglucosylase may be responsible for the storage of intracellular iodophilic polysaccharide that occurs when the cells are grown in the presence of suitable carbohydrate sources.     

Certain Properties of α-Glucosidase from Mucor racemosus

The substrate and inhibitor specificities, and α-glucosyltransfer products of the purified α-glucosidase from the mycelia of Mucor racemosus were investigated. The enzyme hydrolyzed maltose, maltotriose, phenyl α-maltoside, isomaltose, soluble starch, and amylose liberating glucose, but did not act on sucrose. The enzyme hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. Maltotriose was the main a-glucosyltransfer product formed from maltose, and isomaltose was that from soluble starch. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. The enzyme hydrolyzed amylose liberating a-glucose. The enzyme was a glycoprotein containing 4.1% of neutral sugar. The neutral sugar was identified as mannose in the acid hydrolyzate of the enzyme.     

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.     

Misregulation of maltose uptake in a glucose repression defective mutant of Saccharomyces cerevisiae leads to glucose poisoning

In hex2 mutants of Saccharomyces cerevisiae, which are defective in glucose repression of several enzymes, growth is inhibited if maltose is present in the medium. After adding [14C]maltose to cultures growing with ethanol, maltose metabolism was followed in both hex2 mutant and wild-type cells. The amount of radioactivity incorporated was much higher in hex2 than in wild-type cells. Most of the radioactivity in hex2 cells was located in the low molecular mass fraction. Pulse-chase experiments showed that 2 h after addition of maltose, hex2 cells hydrolysed maltose to glucose, which was partially excreted into the medium. 31P-NMR studies gave evidence that turnover of sugar phosphates was completely abolished in hex2 cells after 2 h incubation with maltose. 13C-NMR spectra confirmed these results: unlike those for the wild-type, no resonances corresponding to fermentation products (ethanol, glycerol) were found for hex2 cells, whereas there were resonances corresponding to glucose. Although maltose is taken up by proton symport, the internal pH in the hex2 mutant did not change markedly during the 5 h after adding maltose. The intracellular accumulation of glucose seems to explain the inhibition of growth by maltose, probably by means of osmotic damage and/or unspecific O-glycosylation of proteins. Neither maltose permease nor maltase was over-expressed, and so these enzymes were not the cause of glucose accumulation. Hence, the coordination of maltose uptake, hydrolysis to glucose and glycolysis of glucose is not regulated simply by the specific activity of the catabolic enzymes involved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amylosucrase from Neisseria polysaccharea: novel catalytic properties

Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.     

Kinetics, equilibria, and modeling of the formation of oligosaccharides from D-glucose with Aspergillus niger glucoamylases I and II

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were incubated with D-glucose at a number of temperatures and pH values. Kinetics and equilibria of the formation of alpha,beta-trehalose, kojibiose, nigerose, maltose, isomaltose, panose, and isomaltotriose, which with isomaltotetraose were the only products formed, were determined. There was no difference in the abilities of GA I and GA II to form these products. Activation energies for the formation of maltose and panose were lower than those of the other Oligosaccharides. Relative rates of oligosaccharide production based on glucoamylase hydrolytic activity did not vary significantly between pH 3.5 and 4.5 but were lower at pH 5.5. Maltose was formed much faster than any other product. Equilibrium concentrations at higher dissolved solids concentrations decreased in the order isomaltose, isomaltotriose, kojibiose, nigerose, maltose, alpha, beta-Mrehalose, panose, and isomaltotetraose. They were not appreciably affected by changes in temperature or pH. A kinetic model based on adsorption of D-glucose and the seven di- and trisaccharides by the first three glucoamylase subsites was formulated. Oligosaccharide formation was simulated with the model, using equilibrium data gathered for this article and subsite binding energies and kinetic parameters for oligosaccharide hydrolysis measured earlier. Agreement of simulated and actual oligosaccharide formation data through the course of the reaction was excellent except at very high solid concentrations.