Hydrolysis Of Maltooligosaccharides Research Articles

Amyrel, a novel glucose-forming α-amylase from Drosophila with 4-α-glucanotransferase activity by disproportionation and hydrolysis of maltooligosaccharides.

The α-amylase paralogue Amyrel present in true flies (Diptera Muscomorpha) has been classified as a glycoside hydrolase in CAZy family GH13 on the basis of its primary structure. Here, we report that, in fact, Amyrel is currently unique among animals as it possesses both the hydrolytic α-amylase activity (EC 3.2.1.1) and a 4-α-glucanotransferase (EC 2.4.1.25) transglycosylation activity. Amyrel reacts specifically on α-(1-4) glycosidic bonds of starch and related polymers but produces a complex mixture of maltooligosaccharides, which is in sharp contrast with canonical animal α-amylases. With model maltooligosaccharides G2 (maltose) to G7, the Amyrel reaction starts by a disproportionation leading to Gn - 1 and Gn+1 products, which by themselves become substrates for new disproportionation cycles. As a result, all detectable odd- and even-numbered maltooligosaccharides, at least up to G12, were observed. However, hydrolysis of these products proceeds simultaneously, as shown by p-nitrophenyl-tagged oligosaccharides and microcalorimetry, and upon prolonged reaction, glucose is the major end-product followed by maltose. The main structural determinant of these atypical activities was found to be a Gly-His-Gly-Ala deletion in the so-called flexible loop bordering the active site. Indeed, engineering this deletion in porcine pancreatic and Drosophila melanogaster α-amylases results in reaction patterns similar to those of Amyrel. It is proposed that this deletion provides more freedom to the substrate for subsites occupancy and allows a less-constrained action pattern resulting in versatile activities at the active site.

Structural Advantage of Sugar Beet α-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate

The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower Km and higher kcat/Km for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log Ki of the acarviosyl-maltooligosaccharides and log(Km/kcat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the Km for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.

Characterization and mechanism of action of Microbacterium imperiale glucan 1,4-α-maltotriohydrolase

In this study, glucan 1,4-α-maltotriohydrolase (AMTS) from Microbacterium imperiale was purified and characterized. Hydrolysis by AMTS was affected by starch structure (e.g., amylose versus amylopectin) and hydrolysis time. During the initial phase of hydrolysis of maltooligosaccharides (G4–G7), AMTS displayed a unique transfer specificity to the transfer of maltotriosyl units. After extensive hydrolysis, maltotriose became the major end product, followed by glucose and maltose. Maltotetraose (G4) was the smallest donor in transglycosylation reactions by AMTS. This is the first study that reports transglycosylation activity of AMTS on maltooligosaccharides. The results of this study suggest that high purity maltotriose can be produced by the hydrolytic action of AMTS on starch.

Capillary electrophoresis coupled to electrospray mass spectrometry through a coaxial sheath flow interface and semi‐permanent phospholipid coating for the determination of oligosaccharides labeled with 1‐aminopyrene‐3,6,8‐trisulfonic acid

For the first time, a semi-permanent phospholipid coating is utilized in capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). Although phospholipids in free solution are generally avoided in ESI, they did not interfere with the detection of linear and branched oligosaccharides using ESI operated in negative mode. The CE and ESI were coupled using a coaxial sheath flow interface. The separation was operated in reversed polarity and the electroosmotic flow was effectively suppressed by the phospholipid coating. The method was characterized with linear oligosaccharides and used to monitor the enzymatic hydrolysis of maltooligosaccharides with α-amyloglucosidase. Branched oligosaccharides were separated and detected with the system. The enzyme β1-4 galactosidase was used to distinguish branched isomeric oligosaccharides derived from asialofetuin.

Identification of the Essential Catalytic Residues and Selectivity-Related Residues of Maltooligosyltrehalose Trehalohydrolase from the Thermophilic Archaeon Sulfolobus solfataricus ATCC 35092

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75 degrees C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.

Kinetics of Maltooligosaccharide Hydrolysis in Subcritical Water

The kinetics of the hydrolysis of maltooligosaccharides with a degree of polymerization (DP) of 3-6 in subcritical water was studied using a tubular reactor at temperatures between 200 and 260 degrees C and at a constant pressure of 10 MPa. The maltooligosaccharide disappearance and product formation at residence times shorter than 50 s could be expressed by first-order kinetics. The rate constants for the hydrolysis of each maltooligosaccharide were evaluated. There was a tendency that the exosite glucosidic bond was hydrolyzed faster than the endo-site one irrespective of the DP of the maltooligosaccharide. The hydrolysis of the maltooligosaccharides was consecutively preceded, and the time dependence of the hydrolysis for maltooligosaccharides with different DPs could be calculated by simultaneously solving the mass balance equations for all the possible saccharides.

Amylolytic activity of IgG and sIgA immunoglobulins from human milk

Background: New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. Methods: The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6- O-ethylidene-α- d-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. Results: All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed α-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6- O-ethylidene-α- d-maltoheptaoside in the range of 10 −4 M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-α- d-glucopyranose. Conclusions: IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.

生体高分子 高圧力による酵素反応生成物の制御

To investigate the possibility that pressure can control the products for the enzyme reactions, pressure effects on the time course of the products' composition accompanying the hydrolysis of maltooligosaccharides [maltotetraose (G4), maltopentaose (G5), and maltohexaose (G6) ] and amylose catalyzed by porcine pancreatic α-amylase (PPA) were measured up to 300 MPa at 30°C. The composition of products for the hydrolysis of G5 substrates changed slightly by compression. But for G4, G6 and amylose substrate, pressure induced some changes in the composition of the products. These results tell us that pressure is one of the efficacious tools to control the products of enzyme reaction of α-amylase. The mechanism of an interesting pressure-induced reaction catalyzed by PPA is discussed in terms of the volume differences among enzyme-substrate (ES) complexes.

Ostrich intestinal glycohydrolases: distribution, purification and partial characterisation

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase–glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity chromatography, while jejunal sucrase–isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl–Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M r 145 000, 125 000 and 115 000. Although the N-terminal amino acid sequences bear no homology to SI, the M r 115 000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58°C, but were quickly denatured above 60°C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters ( K m, k cat and k cat/ K m) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.

Effect of pressure on the mechanism of hydrolysis of maltotetraose, maltopentaose, and maltohexose catalyzed by porcine pancreatic α-amylase

Pressure effects on the time course of the products' composition accompanying the hydrolysis of maltooligosaccharides [maltotetraose (G4) maltopentaose (G5), and maltohexaose (G6)] catalyzed by porcine pancreatic α-amylase (PPA) were measured up to 300 MPa at 30°C. The composition of products, glucose (G1), maltose (G2), and maltotriose (G3), for the hydrolysis of G4, and G5 substrates changed a little by compression. But for G6 substrate, pressure induced some changes in the composition of products, G2, G3, and G4, respectively. From the pressure dependence of the observed rate constants on PPA catalyzed hydrolysis of G6, the volume difference between two kinds of Michaelis complexes of α-amylase-G6 is about 5.4 cm 3/mol. The mechanism of an interesting pressure-induced reaction catalyzed by PPA is discussed in the terms of the reaction volumes.

Acarbose and 1-Deoxynojirimycin Inhibit Maltose and Maltooligosaccharide Hydrolysis of Human Small Intestinal Glucoamylase–Maltase in Two Different Substrate-Induced Modes

The inhibition of the glucoamylase–maltase-catalyzed maltose and maltooligosaccharide hydrolysis by acarbose and 1-deoxynojirimycin has been demonstrated. Acarbose and 1-deoxynojirimycin act as potent competitive inhibitors withKi= 0.8 μmfor the hydrolysis of maltose and withKivalues of 0.4 and 0.3 μm, respectively, for the hydrolysis of maltooligosaccharides. In a previous work (Güntheret al., Arch. Biochem. Biophys.327, 295–302, 1996) using maltitol and maltobionate as inhibitors we were able to discriminate two different binding modes for glucoamylase–maltase: a maltose and an oligosaccharide binding mode. Here we found that structurally quite different substances, namely, the pseudotetrasaccharide acarbose and the monomeric glucose analog 1-deoxynojirimycin, act as competitive inhibitors for maltose and maltooligosaccharide hydrolysis. TheKivalues for all used maltooligosaccharides are nearly equal, but for maltose hydrolysis theKivalues are significantly higher by a magnitude factor of two. The differences concerningKivalues can be explained by means of the two-binding-mode model.

Maltitol and Maltobionate Act Differently on Maltose- and Maltooligosaccharide Hydrolysis by Human Small Intestinal Glucoamylase-Maltase Indicating Two Different Enzyme Binding Modes

The hydrolysis of maltose and maltotriose at the same catalytic site of glucoamylase-maltase has been demonstrated. Maltitol acts as a competitive inhibitor,Ki= 69 (±10) mM, of the maltose hydrolysis and as a noncompetitive inhibitor of the hydrolysis of maltotriose,Ki= Kii= 29 (±4) mM, and maltotetraose,Ki= Kii= 30 (±3) mM. Maltobionate was not hydrolyzed by the enzyme and did not influence the maltose hydrolysis. In contrast, in hydrolysis of maltooligosaccharides it acts as an uncompetitive inhibitor. For the hydrolysis of maltotriose,Kii= 25 (±8) mM, and maltotetraose,Kii= 30 (±4) mMwas found. According to a characteristic of this rare inhibition pattern a simultaneous decrease of the apparentKmand the apparentVmaxof maltooligosaccharide hydrolysis with increasing maltobionate concentrations was observed. We were able to discriminate two different binding modes for glucoamylase-maltase. Maltitol binds to the free enzyme (maltose binding mode) as well as to the maltooligosaccharide–enzyme complex, whereas maltobionate binds only to the oligosaccharide–enzyme complex (oligosaccharide binding mode). This could be shown by the different inhibition behaviors of maltitol and maltobionate depending on the substrate: maltose or maltooligosaccharides.

Refined Crystal Structures of Glucoamylase from Aspergillus awamori var. X100

The refined crystal structure of a proteolytic fragment of glucoamylase from Aspergillus awamori var. X100 have been determined at pH 6 and 4 to a resolution of 2·2 Å and 2·4 Å, respectively. The models include the equivalent of residues 1 to 471 of glucoamylase from Aspergillus niger and a complete interpretation of the solvent structure. The R-factors of the pH 6 and 4 structure are 0·14 and 0·12, respectively, with root-mean-square deviations of 0·014 Å and 0·012 Å from expected bondlengths. The enzyme has the general shape of a doughnut. The "hole" of the doughnut consists of a barrier of hydrophobic residues at the center, which separates two water-filled voids, one of which serves as the active site. Three clusters of water molecules extend laterally from the active site. One of the lateral clusters connects the deepest recess of the active site to the surface of the enzyme. The most significant difference in the pH 4 and 6 structures is the thermal parameter of water 500, the putative nucleophile in the hydrolysis of maltooligosaccharides. Water 500 is associated more tightly with the enzyme at pH 4 (the pH of optimum catalysis) than at pH 6. In contrast to water 500, Glu179, the putative catalytic acid of glucoamylase, retains the same conformation in both structures and is in an environment that would favor the ionized, rather than the acid form of the side-chain. Glycosly chains of 5 and 8 sugar residues are linked to Asparagines 171 and 395, respectively. The conformations of the two glycosly chains are similar, being superimpossable on each other with a root-mean-square discrepancy of 1·9 Å. The N-glycosly chains hydrogen bond to the surface of the protein through their terminal sugars, but otherwise do not interact strongly with the enzyme. The structures have ten serine/threonine residues, to each of which is linked a single mannose sugar. The structure of the ten O -glycosylated residues taken together suggests a well-defined conformation for proteins that have extensive O-glycosylation of their polypeptide chain.

Refined structure for the complex of 1-deoxynojirimycin with glucoamylase from Aspergillus awamori var. X100 to 2.4-A resolution.

The three-dimensional structure of the complex of 1-deoxynojirimycin with glucoamylase II-(471) from Aspergillus awamori var. X100 has been determined to 2.4-A resolution. The model includes residues corresponding to residues 1-471 of glucoamylase I from Aspergillus niger, two molecules of bound 1-deoxynojirimycin and 605 sites for water molecules. The crystallographic R factor from refinement is 0.119, and the root-mean-squared deviation in bond distances is 0.012 A. The inhibitor complex confirms the location of the active site in the packing void of the alpha/alpha-barrel as proposed by Aleshin et al. [Aleshin, A., Golubev, A., Firsov, L., & Honzatko, R. B. (1992) J. Biol. Chem. 267, 19291-19298]. One inhibitor molecule is associated with strong electron density and represents the principal site of interaction of 1-deoxynojirimycin with the enzyme. The other 1-deoxynojirimycin molecule is associated with weak electron density and therefore, probably represents a binding site of low affinity. Interactions of 1-deoxynojirimycin with the enzyme at its principal site involve Arg 45, Asp 55, Arg 305, and carbonyl 177. In addition, a water molecule (water 500) hydrogen bonds to Glu 400 and the 6-hydroxyl of 1-deoxynojirimycin and is at an approximate distance of 3.3 A from the "anomeric" carbon of the inhibitor. The structural arrangement of functional groups near the inhibitor molecule suggests that Glu 179 is a catalytic acid, Glu 400 a catalytic base, and water 500 the attacking nucleophile in the hydrolysis of maltooligosaccharides. The relevance of the X-ray work to proposed mechanisms of enzymatic hydrolysis of oligosaccharides is discussed.

Kinetics of enzymic hydrolysis of malto-oligosaccharides: A comparison with acid hydrolysis

The hydrolysis of malto-oligosaccharides G3-G6 catalysed by porcine pancreatic alpha-amylase was investigated kinetically at 25°. Kinetic parameters corresponding to different positions of enzymic attack were determined and product inhibition was evaluated. The enzymic hydrolysis was compared in terms of reaction rate and pattern of action with hydrolysis in 0.1m H2SO4 at 70°. Mathematical models for the mechanism of hydrolysis were developed and a good rationalisation of the experimental results was achieved.

Fractionation of isozymes and determination of the subsite structure of glucoamylase from Rhizopus niveus.

Four isozymes of glucoamylase were isolated from a commercial preparation of the enzyme from Rhizopus niveus by ion-exchange chromatography on CM-Sephadex C-50 and/or the preparative isoelectric focusing method on Sephadex IEF.The kinetic parameters, Michaelis constant Km and molecular activity k0, for the hydrolysis of maltooligosaccharides (degree of polymerization DP from 2 to 7) were determined on the major component enzyme at pFl 4.5 and 25°C. It was found that Km decreases and k0 increases with increasing DP. From the DP-dependences of Km and k0, the subsite affinities of the seven subsites of the enzyme active site were evaluated on the basis of subsite theory. These values are very similar to those obtained for an unfractionated preparation of Rhizopus delemar glucoamylase [Hiromi et al, B.B.A., 302, 362 (1973)].

Studies on action pattern of amylase-catalized hydrolysis of amylose using TNS fluorescence as a probe.

A large fluorescence enhancement of 2-p-toluidinylnaphthalene-6-suIfonate (TNS) caused by amylose decreases as the substrate is degraded by amylases. This property was used to follow the enzymatic hydrolysis of amylose and analyze the action pattern of six kinds of amylases. This method can substitute the conventional blue value method, and is more sensitive for short chain amylodextrins. The advantage of the new method over the blue value method is that it is useful to monitor the hydrolysis of maltooligosaccharides to which the blue value method cannot be applicable, and that it enables continuous monitoring of the enzyme reactions.

Effect of photooxidation of bacterial liquefying α-amylase dependent on the degree of polymerization of linear substrates

The effect of pH on the rate of inactivation of liquefying α-amylase from Bacillus subtilis (α-1,4-glucan 4-glucanohydrolase (liquefying), EC 3.2.1.1) by photosensitized oxidation in the presence of methylene blue indicated that the modification of a histidyl residue causes loss of the enzyme activity for the hydrolysis of soluble starch catalyzed by this enzyme. The activity of the enzyme photooxidized at 25°C and pH 7.0 was measured by using maltooligosaccharides with the degree of polymerization n ranging from 3 to 12.6 as substrates. A remarkable difference in the effect of the modification was observed depending on the n value of the substrate; the rate of hydrolysis of maltooligosaccharides with n larger than 5 was crucially decreased, whereas that of smaller substrates was not appreciably affected. This result indicates that the affinity of a subsite which is quite distant from the catalytic site is decreased in consequence of the modification. The location of the damaged subsite was estimated by considering the predominant productive binding modes which can be predicted from the knowledge already obtained concerning the arrangement of subsite affinities and the action patterns of this enzyme.