Series Of Maltooligosaccharides Research Articles

Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides

The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 → 4) and (α1 → 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 → 4)/(α1 → 6) alternating structure.

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121

Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1→4) also (α1→6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)-DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.

The Effect of Molecular Size on Molecular Mobility in Amorphous Oligosaccharides

The physical properties and especially the molecular mobility of amorphous carbohydrate matrixes directly influence the stability of foods, feeds, and pharmaceuticals and the dessication tolerance of animals and plants during anhydrobiosis. Phosphorescence of the sodium salt of erythrosin B was used to investigate the local molecular mobility in pure amorphous solids of a homologous series of malto-oligosaccharides (maltose, G2; maltotriose, G3; maltotetraose, G4; maltopentaose, G5; maltohexaose, G6; and maltoheptaose, G7); sucrose and maltodextrin DE18 (a hydrolytic fraction of starch) were investigated for comparison. Measurements of the temperature-dependence of the phosphorescence emission energy, an indicator of the extent of local dipolar relaxation, and the phosphorescence emission lifetime, an indicator of the rate of collisional quenching of the excited state by matrix molecules, demonstrate that the local matrix molecular mobility increases with molecular size, and thus, with an increase in T g, in these glucose oligosaccharides. Master curves of the spectroscopic measures of matrix mobility for each oligosaccharide, plotted against T–T g, were not superimposable, suggesting that local properties of the amorphous sugar matrixes, rather than T g per se, influence local matrix mobility. Indicators of spectral heterogeneity also varied with molecular size, indicating that dynamic site heterogeneity also increased with molecular size and thus, T g. These results emphasize the importance of additional research in developing appropriate “molecular rules” for designing amorphous matrix systems with better long-term stability for foods, feeds, or pharmaceuticals.

The unique glycoside hydrolase family 77 amylomaltase fromBorrelia burgdorferiwith only catalytic triad conserved

Glycoside hydrolase family 77 (GH77) contains prokaryotic amylomaltases and plant-disproportionating enzymes (both possessing the 4-alpha-glucanotransferase activity; EC 2.4.1.25). Together with GH13 and GH70, it forms the clan GH-H, known as the alpha-amylase family. Bioinformatics analysis revealed that the putative GH77 amylomaltase (MalQ) from the Lyme disease spirochaete Borrelia burgdorferi genome (BB0166) contains several amino acid substitutions in the positions that are important and conserved in all GH77 amylomaltases. The most important mutation concerned the functionally important arginine positioned two residues before the catalytic nucleophile that is replaced by lysine in B. burgdorferi MalQ. Similar remarkable substitutions were found in two other putative GH77 amylomaltases from related borreliae. In order to confirm the exclusive sequence features and to verify the eventual enzymatic activity, the malQ gene from B. burgdorferi was amplified using PCR. A c. 1.5-kb amplified DNA fragment was sequenced, cloned and expressed in Escherichia coli, and the resulting recombinant protein was preliminarily characterized for its activity towards glucose (G1) and a series of malto-oligosaccharides (G2-G7). This study confirmed that the remarkable substitution of the arginine really exists and the GH77 MalQ protein from B. burgdorferi is a functional amylomaltase because it is able to hydrolyse the malto-oligosaccharides as well as to form their longer transglycosylation products.

Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites

Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced alpha-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over beta-CD, calculated by comparing k(cat)/K(m) ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes.

Gene cloning and characterization of a novel α‐amylase from alkaliphilic Alkalimonas amylolytica

A gene encoding an extracellular alpha-amylase (AmyA) was cloned from the alkaliphilic bacterium Alkalimonas amylolytica by enzymatic activity screening in Escherichia coli DH5alpha. The gene amyA consists of 1764 base pairs and was predicted to encode a 587-amino acid protein encompassing a 31-amino acid signal peptide. In addition, a 459-amino acid catalytic domain and a 97-amino acid starch-binding domain (SBD) were found. The SBD showed little similarity to other known SBDs; instead, it contains conserved amino acids typically belonging to the carbohydrate-binding module (CBM) family 20. AmyA could act on both granular and gelatinized starch. The catalytic domain of the enzyme showed little similarity to other known alpha-amylases. Rather, AmyA contains four characteristic conserved regions of glycoside hydrolase family 13. The recombinant enzyme was a liquefying enzyme with the highest activity at 50 degrees C and pH 9.5. The enzyme displayed a unique endo-product profile and action pattern on soluble starch to yield a series of malto-oligosaccharides ranging from maltose to maltoheptaose. The activity of the enzyme was enhanced by Co(2+), but not affected by 5 mM EDTA. Taken together, AmyA from A. amylolytica has potential to be used in paper, textile, detergent and other industries where starch needs to be degraded in an alkaline environment.

Mapping of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley α-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3–12, revealed unfavorable binding energies at the internal subsites −3 and −5 and at subsites −8 and +3/+4 defining these subsites as binding barriers. Barley α-amylase 1 mutants Y105A and T212Y at subsite −6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (−2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in α-amylases.

The mechanism of action of α-amylase from Lactobacillus fermentum on maltooligosaccharides

The action pattern of Lactobacillus fermentum alpha-amylase (FERMENTA) was examined using a series of maltooligosaccharides (G2-G7) as substrates. Structurally, this enzyme has a molecular mass (106 kDa) almost twofold higher than alpha-amylases from mammalians and cereals. The product pattern was investigated through an analysis of products and substrates using HPAEC with pulsed amperometric detection. FERMENTA was consistent with an endo-type of amylase. The bond cleavage frequencies were studied using maltooligosaccharides of various chain lengths as substrate, i.e. maltose up to maltoheptaose and DP 4900-amylose catalyzed by FERMENTA. The catalytic efficiency (k(cat)/K(m)) increased with chain length from maltose (8.7 x 10(4) M(-1) s(-1)) up to amylose (1 x10(9) M(-1) s(-1)). These action pattern results revealed that FERMENTA can readily cleave the third linkage from the reducing end of the maltooligosaccharides (G5-G7).

CHARACTERISTICS OF ALPHA-AMYLASE IN BULB SCALES OF TULIP (TULIPA GESNERIANA L.)

We conducted biochemical and molecular analysis of tulip amylase. Enzyme extract was obtained from the scales of tulip bulbs after cold treatment for forcing cultivation, and some characteristics of the amylase were investigated. The amylase activity was measured by iodine-starch method. The degradation of soluble starch produced a series of maltooligosaccharides with different sizes. This indicates that the amylase in tulip bulb scale is endotype α -amylase. The optimum pH for amylase activity was about 5.6. The enzyme activity was stable at neutral and alkaline pH (pH 5-10) at 4°C for 24h, but the activity remarkably decreased in acidic pH (pH<4.5). The α -amylase requires Ca 2+ for its activity and stability. The optimum temperature for 5 min incubation was about 75°C in the presence of Ca 2+ . The addition of 2 mM or 20 mM of Cu 2+ , Hg 2+ and Zn 2+ inhibited the amylase activity, but Ca 2+ , Mg 2+ , Fe 2+ , Co 2+ and Ni 2+ did not affect. A cDNA of α -amylase was obtained by RT-PCR method using the primer designed from the database of the amino acid and DNA sequences of amylases in higher plants. The deduced amino acid sequence of the α -amylase from tulip bulb scales has high similarity (identity 60%) to those of the rice and maize.

Comparison of oligosaccharide compositions in male nuptial secretions of three cockroach species of the genus Blattella.

Male cockroaches of the genus Blattella secrete courtship pheromones from abdominal tergal glands. Conspecific females mount courting males and feed on their tergal exudates. In the German cockroach, B. germanica, a complex mixture of oligosaccharides was previously identified as the major phagostimulants in the male tergal secretion. We compared the oligosaccharide compositions of three Blattella species from different habitats, B. germanica (house), B. lituricollis (field), and B. nipponica (forest), by gas chromatographic (GC) analysis of their trifluoroacetylated derivatives. The three possessed a series of maltooligosaccharides and oligoglucosyl trehaloses with alpha1-->4 and alpha1-->1 linkages in common. However, B. germanica was conspicuous for its accumulation of a oligoglucosyl trehalose with a alpha1-->6 linkage [O-alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl alpha-D-glucopyranoside] as the most prominent component of the secretion. Such diversification of the male pheromonal compositions within the genus might be reflected by biosynthetic systems in males and chemosensory properties in females suited for their natural habitat.

Synthesis of Malto-Oligosaccharides Via the Acceptor Reaction Catalyzed by Cyclodextrin Glycosyltransferases

The disproportionation activity (intermolecular transglycosylation) of cyclomaltodextrin glycosyltransferases (CGTases) from Thermoanaerobacter sp. and Bacillus circulans strain 251 was studied. Using soluble starch as donor, the CGTase from Thermoanaerobacter sp. showed the highest transglycosylation activity with all the malto-oligosaccharides tested as acceptors. At ratios of starch: D-glucose from 2:1 to 1:2 (w/w), the formation of cyclodextrins was completely inhibited, and a homologous series of malto-oligosaccharides (Gn) was produced. The conversion of starch into acceptor products was in the range of 63-79% in 48 h. The degree of polymerisation of malto-oligosaccharides formed could be modulated by the ratio of starch: D-glucose provided; at a ratio of 1:2 (w/w), the reaction was quite selective for the formation of G2-G3.

Production of 2-O-α-d-glucopyranosyl l-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp.

Transglycosylation to produce a 2-O-α-d-glucopyranosyl l-ascorbic acid (AA-2G) was studied using cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. A series of maltooligosaccharides substituted 2-O-derivatives of l-ascorbic acid (AA) were analyzed by HPLC. The maltooligosaccharides were hydrolyzed by glucoamylase to give AA-2G. CGTase also produced AA-2G using dextrin as a glycosyl donor and AA as an acceptor. CGTase utilized α-, β-, and γ-CDs, amylose, soluble starch and corn starch as glycosyl donors but not glucose.

Substrate Specificity of Human and Bovine Pancreatic .ALPHA.-Amylases Using End-blocked Maltooligosaccharides.

β-D-Galactosyl and β-N-acetylglucosaminyl maltooligosaccharide derivatives, which are substituted at the nonreducing-end glucosyl residue of p-nitrophenyl α-maltooligosaccharides (maltotriose to maltopentaose), were regioselectively synthesized using the transglycosylation reaction of β-D-galactosidase from Bacillus circulans and hen-egg white lysozyme, respectively. On porcine and human pancreatic α-amylases, the hydrolytic pattern and kinetic parameter (ko/Km) of both end-blocked maltooligosaccharides, p-nitrophenyl 45 -O-β-D-galactosyl-α-maltopentaoside (Ga14 G5P), p-nitrophenyl 44 -O-β-D-galactosyl-α-maltotetraoside (Ga14 G4P) and p-nitrophenyl 43 -O-β-D-galactosyl- α-maltotrioside (Ga14 G3P), were very close to those of p-nitrophenyl α-maltohexaoside (G6P), p-nitrophenyl α-maltopentaoside (G5P) and p-nitrophenyl α-maltotetraoside (G4P), respectively, corresponding to their degrees of polymerization, while p-nitrophenyl 35 -O-β-N-acetylglu-cosaminyl-α-maltopentaoside (NG5P) and p-nitrophenyl 34 -O-β-N-acetylglucosaminyl-α-mal-totetraoside (NG4P) behaved as G5P and G4P, respectively, as if the G1cNAc group is ignored. On the contrary, p -nitrophenyl 65 -O-β-D-galactosyl-α-maltopentaoside (Ga16 G5P) caused a remarkable decrease in the ko/Km value compared with G5P, and p -nitrophenyl 64 -O-β-D-galactosyl-α-mal-totetraoside (Ga16 G4P) acted only slightly for the α-amylases. A series of maltooligosaccharides with both ends blocked were shown to be advantageous as probes for substrate recognition at the active site in porcine and human pancreatic α-amylases.

Di- and Oligosaccharide Substrate Specificities and Subsite Binding Energies of Pig Intestinal Glucoamylase-Maltase

The substrate specificity of pig intestinal glucoamylase-maltase was investigated. The α-1,β-2-glycosidic bond of the disaccharide sucrose was not hydrolyzed. Various substrates with α-1,4-glycosidic bonds (maltose, maltooligosaccharides) were hydrolyzed with high maximal reaction velocities. Reduction lowered the rate of hydrolysis drastically:k0decreases from 75 s−1for maltose to 3 s−1for maltitol while theKmvalue increases from 4.2 to 50 mM. Leucrose with α-1,5-glycosidic bond was hydrolyzed with ak0value of 8 s−1and aKmvalue of 74 mM. Disaccharides with α-1,6-glycosidic bonds were hydrolyzed with extremely low rates: for isomaltose and isomaltulosek0values of 5 and 3 s−1, respectively, andKmvalues of 90 and 42 mM, respectively, were observed. Again reduction lowers thek0values: The corresponding disaccharide alcohols α-d-glucopyranosyl-1,6-sorbitol and α-d-glucopyranosyl-1,6-mannitol, like isomaltooligosaccharides, were not hydrolyzed. Regarding the conformation of sucrose, leucrose, and maltose previously determined by molecular dynamics simulations, a reasonable explanation for the different rates of hydrolysis could be postulated. Based on the enzyme kinetic parameters for the series of maltooligosaccharides, subsite affinities (Ai) according to the subsite theory were calculated as 7.5 (A1), 17 (A2), 3.4 (A3), and 1.3 kJ/mol (A4) for subsites 1, 2, 3, and 4, respectively. The intrinsic rate constantkintwas estimated at 76 s−1.

Multiple neutral alkali halide attachments onto oligosaccharides in electrospray ionization mass spectrometry

Oligosaccharides perform essential functions in a variety of biological and agricultural processes. Recent approaches to characterization of these molecules by mass spectrometry have utilized mainly soft-ionization methods such as electrospray ionization (ESI) and thermospray (TS), as well as fast atom bombardment (FAB). The behavior of a series of maltooligosaccharides with α-(1 → 4) linkages, maltose (G 2) through maltoheptaose (G 7), under ESI conditions, has been investigated here. The oligosaccharides were dissolved in N,N-dimethylacetamide containing lithium chloride (DMAc/LiCl) prior to analysis by ESI-MS. A highly unusual feature, evident in all mass spectra obtained using this solvent system, was the presence of multiple ‘neutral’ salt attachments onto lithium adducts of the sugars. Resultant ions took the form of [G x + Li + nLiCl +, where n may reach a value as high as eight. Compared to LiCl, the propensity for alkali halide attachment using other alkali chlorides or lithium halides was greatly reduced. An investigation of this phenomenon is presented in which the organic and inorganic portions of the employed solvent were systematically varied, and semi-empirical computer modeling was performed to better understand lithium coordination by the sugars.

Elucidation of the subsite structure of bacterial saccharifying alpha-amylase and its mode of degradation of maltose

The subsite structure of bacterial saccharifying alpha-amylase (BSAm) was elucidated by two methods using a series of maltooligosaccharides labeled with [ 14C] d-glucose at the reducing end. The rate parameter k o K m and the cleavage frequency were obtained using the labeled substrates at sufficiently low concentrations to eliminate transglycosylation and condensation. This evaluation showed that the active center is composed of five subsites, with the catalytic site located between the 3rd and the 4th subsites from the nonreducing end. The evaluated affinity values of a subsite varied with the set of data used, which suggests some stimulation factor resulting from the chain length effect. The appearance of a time lag during the digestion of the poor substrate, maltose, was studied using radioactively labeled maltose (81.6 mM). Radioactive oligosaccharides larger than maltose were found at a significant level of more than 2% of the initial substrate in the digests, including a product peculiar to condensation, G-G ∗-G, as 8–10% of the maltotriose in the digests. This indicates that transglycosylation is a main side reaction (ca. 90%). A degradation pathway for maltose via maltosyl transfer was proposed, in which G 3 behaves as a kind of catalyst.

Synthesis of a polymeric-based stationary phase for carbohydrate separation by high-pH anion-exchange chromatography with pulsed amperometric detection

Spherical highly cross-linked styrene-divinylbenzene copolymer particles were chemically modified by direct nitration, followed by reduction with tin metal and quaternization with iodomethane to produce a superficial layer of quaternized amino functions. Besides being simple and economical, the proposed method of synthesis leads to an anion exchanger that allows the use of shorter columns (75 × 4.6 mm I.D.) than those currently employed for carbohydrate separation by high-performance anion-exchange chromatography with pulsed amperometric detection under alkaline conditions. The new sorbent has proved to be highly selective for isomeric disaccharides and individual oligomers of an homologous series of maltooligosaccharides of up to 21 glucose residues. Extended exposure of columns packed with this sorbent to high pressure and strong alkaline solutions did not have any untoward effect on mechanical stability and Chromatographic performance.

Substrate specificity and subsite affinities of rabbit liver acid alpha-glucosidase.

The substrate specificity of rabbit liver acid alpha-glucosidase was investigated. The enzyme showed a wide specificity for various substrates, and hydrolyzed alpha-glucans such as glycogen and soluble starch. The k0 values (s-1) for maltose, kojibiose, nigerose, isomaltose, phenyl alpha-glucoside, panose, phenyl alpha-maltoside, soluble starch, beta-limit dextrin, amylopectin, shellfish glycogen, and rabbit liver glycogen were estimated to be 94.8, 18.8, 143, 3.6, 11.8, 27.8, 115, 99.2, 155, 83.5, 126, and 108, and the Km values (concentration of non-reducing terminal) for these substrates were 2.1, 1.8, 7.5, 36, 5.4, 1.9, 1.2, 0.90, 9.1, 1.0, 16, and 13 mM, respectively. Isomaltose and phenyl alpha-glucoside were unfavorable as substrates. The acid alpha-glucosidase is characterized by a relatively high activity toward glycogen. The k0 values (s-1) for maltotriose, -tetraose, -pentaose, -hexaose, -heptaose, and -octaose, and maltodextrin (n = 17) were 140, 140, 131, 132, 134, 132, and 74.3, and the Km values, 2.1, 1.8, 1.9, 3.4, 5.0, 4.9, 4.9 and 2.6 mM, respectively. Based on the rate parameters for the series of maltooligosaccharides, the subsite affinities (Ais) in the active site were evaluated as 0.54 (A1), 5.34 (A2), and 0.34 (A3) kcal/mol for subsites 1, 2, and 3, respectively. These three subsites were considered to be predominantly responsible for the binding of substrates to the active site.

The pH dependence of the action pattern in porcine pancreatic α-amylase-catalyzed reaction for maltooligosaccharide substrates

Porcine pancreatic α-amylase (EC 3.2.1.1; abbreviated PPA), which hydrolyzes α- d-(1,4) glucosidic bonds in starch and amylose, displays an optimum at pH 6.9 for the majority of substrates. The optimum pH, however, shifted to 5.2 for the hydrolysis of some low molecular substrates ( Ishikawa, K., et al., 1990 , Biochemistry 29, 7119–7123). Details of the substrate-dependent shift of the optimum pH in PPA were studied by use of a series of maltooligosaccharides with 14C-labeled reducing end glucose as substrates. The optimum pH for maltotriose was 5.2, whereas that for maltopentaose and maltohexaose was unchanged at pH 6.9. The pH profile for the intermediate size substrate maltotetraose showed abnormality; the apparent optimum pH was broadened between 5.5 and 6.5 and the bond cleavage pattern depended on pH, unlike that for the other substrates examined. These results were independent of either buffer systems or substrate concentration. Analyses of the hydrolysates of the maltooligosaccharides revealed that the shift of the optimum pH to the neutral region occurred only when the fifth subsite of PPA in the productive binding modes was occupied by a glucosyl residue of a substrate. The three-catalytic residue model of PPA deduced from the analysis of the hydrolysis of some modified maltooligosaccharides ( p-nitrophenyl-α- d-maltoside, γ-cyclodextrin, maltopentaitol, and maltohexaitol) (Ishikawa, K., et al., 1990, Biochemistry 29, 7119–7123) was successfully adapted to the linear maltooligosaccharides used in this work. These results indicate that the different productive binding modes of the linear oligosaccharide substrates affect directly the catalytic power and the optimum pH of PPA.