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Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C-60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C-60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.

Mutanase, α-(1→3)-glucanase, belonging to the family glucanohydrolase, catalyses mutan [α-(1→3)-glucan] synthesized by cariogenic streptococci and hence has potential in caries prophylaxis. A novel bacterial strain with potential to produce higher mutanase (glucanohydrolase) activity was isolated from soils contaminated with cellulosic waste. One of the isolated strains, RSP-02, was subjected to biochemical and 16S rRNA molecular analysis, and we noticed that it belongs to the genus Paracoccus. The mutanase production (800- 1200 U l-1) in this strain was growth associated and substrate induced, and the activity was comparable with the strains reported earlier. The enzyme displayed a molecular mass of 138 kDa by native PAGE studies, showed endolytic activity and produced nigerose as end product. In vitro studies revealed production of 140±2.82 µg of glucose equivalents in 30 min from the biofilm formed on glass surface indicating its potentiality in dentistry. To the best of our knowledge, this is the first report on the production of mutanase by Paracoccus sp.; hence, this isolated bacterial strain is designated as Paracoccus mutanolyticus RSP-02.

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (

Functionalized rare sugars were synthesized with 2-, 3-, and 6-tosylated glucose derivatives as acceptor substrates by transglucosylation with sucrose and the glucansucrase GTFR from Streptococcus oralis. The 2- and 3-tosylated glucose derivatives yielded the corresponding 1,6-linked disaccharides (isomaltose analogues), whereas the 6-tosylated glucose derivatives resulted in 1,3-linked disaccharides (nigerose analogue) with high regioselectivity in up to 95 % yield. Docking studies provided insight into the binding mode of the acceptors and suggested two different orientations that were responsible for the change in regioselectivity.

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.

The most common type of carbohydrate-transport system in Streptococcus mutans is the phosphoenolpyruvate (PEP)-sugar phosphotransferase system (PTS). We previously showed that fourteen PTSs exist in S. mutans UA159 (Ajdic et al., 2002). Several studies have shown that microorganisms growing in biofilms express different genes as compared to their planktonic counterparts. In this study, we showed that one PTS of S. mutans was expressed in sucrose-grown biofilms. Furthermore, the same PTS was also responsible for the transport and metabolism of disaccharide nigerose (3-O-α-D-glucopyranosyl-D-glucose). Additionally, the results indicate that the studied PTS might be involved in the transport and metabolism of carbohydrates synthesized by glucosyltransferase B (GtfB) and glucosyltransferase C (GtfC) of S. mutans. To our knowledge, this is the first report that shows PTS transport of a disaccharide (and possibly extracellular oligosaccharides) with α-1,3 linkage.

We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze α-linked glucobioses, such as trehalose (α1-α1), kojibiose (α1-2), nigerose (α1-3), and maltose (α1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only l-rhamnose as the acceptor among various sugars examined with β-d-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-α-d-glucopyranosyl-l-rhamnose, indicating strict α1-3 regioselectivity. We propose 3-O-α-d-glucopyranosyl-l-rhamnose: phosphate β-d-glucosyltransferase as the systematic name and 3-O-α-d-glucopyranosyl-l-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase.

The gene (Ta0298) encoding a putative α-glucosidase from hyperthermophilic archaeon Thermoplasma acidophilum (AglA) was cloned and expressed in Escherichia coli. Gel filtration chromatography of the purified enzyme indicated that the native form was a pentamer with strong maltose (α-1,4 linkage)-hydrolyzing activity. AglA was optimally active at pH 5–6 and 80 °C and had a half-life of 16.8 h and 1.4 h at 80 °C and 85 °C, respectively. The enzyme also hydrolyzes kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6) to a lesser extent. Analysis of the reaction with maltooligosaccharides and panose as substrates show that AglA specifically liberates glucose from the non-reducing end indicating that it is typical of a glycoside hydrolase family 31 (GH31) α-glucosidase. Kinetic analyses revealed that the hydrolytic activity of AglA was greatly affected by the chain length of the substrate and the regiospecificity of the glucosidic linkages. The enzyme showed highest specificity for maltose and decreasing values of catalytic efficiency (kcat/Km) toward higher maltooligosaccahrides, although these still serve as substrates. The inhibition profile of AglA toward aesculin was revealed to be a mixed type of noncompetitive inhibition with a Ki value of 4.30 μM and K ′ i of 12.5 μM, whereas that toward acarbose showed a competitive inhibition pattern with a Ki of 2.99 μM. Structural analyses of two arbutin transglucosylation products using NMR indicated that the glucose unit of maltose was transferred to the C-3 and C-6 position in the glucose moiety of arbutin.

A soluble α-glucosidase was partially purified from Candida albicans cells by a three-step procedure consisting of size-exclusion, ion-exchange and adsorption chromatographies. After the last step, enzyme was enriched about 8.7-fold with a yield of 13% over the starting material and analysis of the purified preparation revealed two major polypeptides of 36 and 47 kDa. The latter was responsible for enzyme activity as visualized with a fluorescent substrate. Nigerose, an α-1,3-linked glucose disaccharide, was preferentially hydrolyzed by the purified enzyme over other glucosedisaccharides bearing distinct α-linkages. The purified α-glucosidase also converted the GlcMan9GlcNAc2 oligosaccharide into the Man9GlcNAc2 product in a time-dependent manner. These and other determined properties are consistent with a type GII α-glucosidase probably involved in N-glycan processing.

Two neutral disaccharides, gentiobiose [beta- D-Glc p-(1--6)- D-Glc] and nigerose [alpha- D-Glc p-(1--3)- D-Glc], were detected in tomato ( Lycopersicon esculentum Mill.) pericarp and locule. Gentiobiose was present in the locule of green fruit and ripe fruit at 0.88 and 5.8 micro mol (kg fresh weight)(-1), respectively. When vacuum-infiltrated into green tomato fruit, exogenous gentiobiose (50 or 200 micro g per fruit) hastened the initiation of ripening (as judged by colour change) by 1-3 days relative to fruit that were infiltrated with glucose or isomaltose. Nigerose plus gentiobiose was particularly effective, but nigerose alone had no significant effect. The endogenous disaccharides were found to be present in the apoplastic fluid of the fruit, compatible with a proposed intercellular signalling role. The origin and metabolic fate of the disaccharides were investigated. Phenolic esters of these disaccharides were not detectable in tomato fruit and it is therefore unlikely that the free disaccharides were formed from a pool of such esters. An alternative possible biosynthetic origin, via transglycosylation, is discussed. When [(14)C]gentiobiose was vacuum-infiltrated into unripe or ripe fruit, the disaccharide remained intact for at least 1 h but was largely degraded within 24 h. The results suggest that gentiobiose is a new, naturally occurring oligosaccharin with a rapid turnover rate.

Free proline was significantly (p

An extracellular alpha-glucosidase from the thermophilic archaebacterium Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration and Mono Q chromatography) with a yield of 4%. It is a monomer with a molecular mass of about 60 kDa and a pI around 5. At 98 degrees C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of 1% (w/v) dithiothreitol and 1% (w/v) BSA. Dithiothreitol (1%, w/v) and BSA (0.4%, w/v) also substantially increase the enzyme activity. The Km at 75 degrees C is 0.41 mM with pNP-alpha-D-glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-alpha-D-glucosidenigerosepanosepalatinoseisomaltosemaltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and beta-gentiobiose. A variety of techniques including immobolization (e.g., on epoxy and glass beads), chemical modification (cross- and cocross-linking) and the use of additives (including polyhydroxylic molecules, BSA, salts, etc.) were applied to enhance stability at temperatures above 100 degrees C. The half-life could be increased from about 4 min at 100 degrees C to 30-60 min at 130 degrees C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and 1% (w/v) BSA, and by cross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130 degrees C.

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of D-glucose and β-D-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k(cat) = 67 s(-1) and K(m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using D-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on D-xylose, 1,5-anhydro-D-glucitol, D-galactose, and methyl-α-D-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with D-xylose and methyl-α-D-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-D-glucosyl-D-glucose:phosphate β-D-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.

Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES-GroEL chaperones. Recombinant LJAG31 was an alpha-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k(cat)/K(m)) toward nigerose, maltulose, and kojibiose than other natural substrates having an alpha-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial alpha-1,3-glucosidase to be characterized with a high k(cat)/K(m) value for nigerose [alpha-d-Glcp-(1 --> 3)-d-Glcp]. Transglucosylation of 4-nitrophenyl alpha-d-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl alpha-nigeroside and 4-nitrophenyl alpha-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) alpha-1,3-glucosidase of glycoside hydrolase family 31.

Asp. niger α-glucosidase was crystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6B and TOYOPEARL HW-55 columns. The crystalline α-glucosidase, which was a glycoprotein containing 25.5% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.5 × 104 by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 × 104 and 9.8 × 104, respectively) separable only by reverse-phase HPLC causing irreversible inactivation. The optimum pH was 4.3. The enzyme also hydrolyzed α-glucans such as soluble starch and amylose. The ratios of V values (Km values, in parentheses, mm of nonreducing terminal) for maltose, kojibiose, nigerose, isomaltose, phenyl α-glucoside, phenyl α-maltoside, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, maltodextrin (DP = 17), and soluble starch were estimated to be 100 (0.75): 23 (4.6): 62 (12): 35 (8.0): 1....

Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative α-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was α,α-1,1-trehalose, a new feature among α-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (α-1,2 linkage), nigerose and turanose (α-1,3), leucrose (α-1,5), isomaltose and palatinose (α-1,6), and maltose (α-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous α-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the α-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the α-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the α-glucosidase), indicating that the α-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.

In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three alpha-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. Alpha-xylosidase encoded in rice genome was found for the first time.

Nigerose and nigerooligosaccharides served as acceptors for a glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus to give a series of homologous acceptor products. The soluble oligosaccharides (dp 5-9) strongly activated the acceptor reaction, resulting in the accumulation of water-insoluble (1-->3)-alpha-D-glucan. The enzyme transferred the labeled glucosyl residue from D-[U-13C]sucrose to the 3-hydroxyl group at the non-reducing end of the (1-->3)-alpha-D-oligosaccharides, as unequivocally shown by NMR 13C-13C coupling patterns. The values of the 13C-13C one-bond coupling constant (1J) are also presented for the C-1-C-6 of the 13C-labeled alpha-(1-->3)-linked glucosyl residue and of the non-reducing-end residue.

When Mucor javanicus was cultured with castanospermine, two alpha-glucosidases (CS-I, II) were produced. The two enzymes were purified by two CM-cellulofine column chromatographies, and compared with the alpha-glucosidase (C) from a culture without castanospermine. The molecular weights of the three enzymes were calculated to be 97,000 (CS-I, II) and 94,000 (C) from SDS-PAGE. CS-II was similar in hydrolysis of alpha-1,4-linkage to C, but hydrolyzed alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages at a lower rate than C. The Michaelis constant of CS-I for malto-oligosaccharides was several times as high as the constants of CS-II and C, but CS-I hydrolyzed nigerose at a faster rate than maltose.

A new α- D -glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri- O -methyl- D -glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di- O -methyl- D -glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α- D -glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2- O -α- D -glucosyl- D -erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O -α- D -glucopyranosyl-(1→3)- O -α- D -glucopyranosyl (1→4)- D -glucopyranose, O -α- D -glucopyranosyl-(1→4)- O -α- D -glucopyranosyl-(1→3)- D -glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α- D -Gl cp -(1→4)-α- D -Gl cp -(1→.The unique structural features of elsinan are discussed in comparison with other glucans.

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.

An attempt was made to search for the enzymes that degrade insoluble glucans (IG) produced by Streptococcus mutans strain JC2. By screening many soil microorganisms and a veriety of fungi strains, it was found that Streptomyces nigrifaciens ISP 5071 produces these enzymes.Culture conditions for the enzyme production were tested with the Streptomyces. The enzyme was produced only in the culture containing IG as an inducer. A maximum production of the enzymes was obtained in the culture supernatant when the spore was inoculated in the basic medium containing 0.3% IG, 0.4% glycerol and cultured at 27°C for 4 days under rotary shake of 160 per min.The crude enzyme solution was prepared from the culture supernatant by precipitation on 30-50% saturation of ammonium sulfate. The degradation activity of the crude enzyme solution toward some glucans was studied.The results were as follows.1) The crude enzyme solution readily degraded IG and a modified-IG which consisted only of α-(1→3) glucosidic bond.2) The crude enzyme solution split IG synergistically in combination with dextranase.3) Paper chromatographic analysis of the enzymatic digests from IG and modified-IG showed that nigerose was the main reaction product, and that practically no isomaltose and its oligosaccharides were present.4) The activity of dextranase was not detected in the crude enzyme solution. These results strongly suggested that the crude enzyme solution contains a certain α-(1→3) glucanohy drolase and not α(1→6) glucanohydrolase.

A transglucosidase, an α- d -glucosidase with a high transferase activity, has been purified to homogeneity from culture broths of A. niger. The enzyme, which gave a single protein band on SDS-gel electrophoresis (mol. wt. 116,000) and two protein bands on isoelectric focusing (pI values 5.1 and 5.0), is a glycoprotein, containing 27.6% of carbohydrate, most of which is mannose, has an optimal pH of 4.0–4.5, and is stable in the pH range 4.0–6.0 and at m ) for the following substrates were obtained: maltose (73.5, 1.7), isomaltose (31.9, 7.7), nigerose (63.6, 20.0), kojibiose (22.1, 3.6), phenyl α- d -glucopyranoside (2.5, 2.4), p-nitrophenyl α- d -glucopyranoside (2.1, 0.7), and methyl α- d -glucopyranoside (1.0, 26.3). The effect of the enzyme on the conversion, by amyloglucosidase, of wort saccharides into glucose has been quantified. A procedure, based on the use of phenyl α- d -glucopyranoside as substrate, is recommended for the assay of this enzyme in crude culture filtrates.

The acceptor reaction of dextransucrase consists of the transfer of d -glucosyl groups from sucrose to other carbohydrates, and occurs at the expense of dextran synthesis. In the present study, solutions of [ 14 C]sucrose and of each of seventeen acceptor sugars were digested with highly purified Leuconostoc mesenteroides B-512F dextransucrase. The products were separated by paper chromatography, and quantitated by liquid scintillation counting. Maltose was the most effective acceptor; its products, members of an isomaltodextrinyl-maltose series (d.p. 3 to 6), accounted for >75% of the d -glucosyl groups of sucrose. Other acceptors giving rise to a similar series of oligosaccharide products were (in order of decreasing effectiveness): isomaltose, nigerose, methyl α- d -glucoside, 1,5-anhydro- d -glucitol, d -glucose, turanose, methyl β- d -glucoside, cellobiose, and l -sorbose. Lactose, raffinose, melibiose, d -galactose, and d -xylose each gave a single, mono- d -glucosylated product; d -fructose and d -mannose each gave a pair of mono- d -glucosylated (disaccharide) products. Another series of digests contained sucrose and various proportions of maltose. As the level of maltose increased, the size of the largest oligosaccharide acceptor-product decreased, and less dextran was produced. The virtual absence of high-d.p. (8 to 13) oligosaccharide products in all acceptor digests is interpreted as evidence against a role for acceptors as primers of dextran synthesis.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.

Srome properties were examined of purified α-1, 3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8. The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1, 3-glucanase was stable up to 50°C on heating for 10min. This enzyme hydrolyzed the substrate α-1, 3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1, 3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1, 3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-1, 3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.

Fragmentation by controlled acetolysis of an alkali-soluble d -glucan, isolated from the fruit body of Laetiporus sulphureus , followed by fractionation of the products on a column of silica gel, provide a simple, preparative approach to the peracetates of 3- O -α- d -glucopyranosyl- d -glucose (nigerose) and of O -α- d -glucopyranosyl-(1→3)- O -α- d -glucopyranosyl-(1→3)- d -glucose (nigerotriose). The synthesis of methyl β-, benzyl β-, phenyl α-, and phenyl β-nigeroside, and of methyl β-nigerotrioside is described.

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [ 14 C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [ 14 C]sucrose. After extensive washings with buffer, the 14 C label remained attached to BGD. This labeled material was previously shown to be [ 14 C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [ 14 C]dextran whereas essentially no [ 14 C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [ 14 C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [ 14 C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C 3 -OH on an acceptor dextran acts as a nucleophile on C 1 of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.

The substrate specificity of flint corn ;-glucosidase was investigated. The enzyme was active especially on nigerose, phenyl-α-maltoside and malto-oligosaccharides. The action on isomalto-oligosaccharides was very weak. The α-glucans, such as amylopectin, β-limit dextrin, glycogen and amylose besides soluble starch, also were hydrolyzed, though no activity on dextran was observed.The ratio of hydrolysis rate for maltose, nigerose, phenyl-α-maltoside, maltotriose, maltotetraose and maltopentaose was estimated to be 100: 105: 110: 97:100: 90 in this order. The extrapolated Km values for maltose, nigerose, phenyl-α-maltoside, maltotriose, maltotetraose and maltopentaose were 1.25 mm, 1.43 mm, 1.00 mm, 0.71 mm, 0.60 mm and 1.25 mm, respectively. The enzyme is considered to be essentially an acid α-glucosidase with a preferential activity on malto-oligosaccharides to α-glucans.

Extracellular glucansucrases from various bacterial sources, including Leuconostoc mesenteroides, have been shown to catalyze the transfer of glucosyl groups from sucrose to low-molecular-weight acceptor sugars, forming a series of oligosaccharides. An extracellular glucansucrase recently isolated from Leuconostoc mesenteroides NRRL B-1355 synthesizes a polysaccharide consisting of alternating α-(1→6)- and α-(1→3)-linked d-glucose residues. We have found that this enzyme preparation is capable of forming both α-(1→6)- and α-(1→3)-linked acceptor-products in the presence of a number of low-molecular-weight acceptor sugars. d-Glucose gave isomaltose only, no nigerose being formed. Similarly, methyl α-d-glucoside, methyl β-d-glucoside, maltose, and nigerose gave methyl α-isomaltoside, methyl β-isomaltoside, panose, and 62-O-α-d-glucosylnigerose, respectively. However, isomaltose gave both isomaltotriose and 32-O-α-d-glucosylisomaltose. These initial acceptor-products may also act as acceptors, and the structures of the products of higher d.p. show that a (1→3)-α-d-glucosidic bond is formed only when the nonreducing glucose acceptor group is linked through an α-(1→6) bond to another glucose residue. Experiments using [14C]sucrose and unlabeled acceptors showed that acceptor reactions occur by transfer of a glucosyl group from sucrose to O-3 or O-6 of the glucosyl group at the nonreducing end of the acceptor saccharide. If a “good” acceptor is defined as one that gives rise to a greater amount of oligosaccharide and less polysaccharide, then the acceptors tested may be listed as follows: maltose ≥ nigerose > methyl α-d-glucoside > isomaltose >d-glucose > methyl β-d-glucoside.

Cladosporium resinae (1→3)-α-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1→3)-α-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1→3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1→3)-α-D-glucosidic linkages that occur in mycodextran, isolichenin, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucans synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1→6)-α-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1→3)-α-D-glucanase to determine the relative proportions of(1→6)-linked to (1→3)-linked sequences of D-glucose residues in the insoluble glucans produced by oral streptococci. The simultaneous action of the two D-glucanases was highly effective in solubilizing the glucans.

A glucoamylase was purified from the culture broth of Paecilomyces varioti AHU 9417 by precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was estimated to be 6.9×104 by SDS-disc electrophoresis, and the optimum pH was 4.5. The enzyme preferentially hydrolyzes α-1,4-glucosidic linkages in a series of maltooligosaccharides, and is capable to slowly hydrolyzing nigerose, isomaltose, and panose, but is not active on kojibriose. Soluble starch, amylopectin, glycogen, and β-limit dextrin are rapidly degraded. Activity toward raw starches is very low, but rice and waxy corn raw starches are relatively subject to attack. The subsite affinities (Ai) of each subsite (i) in the active site of the enzyme were evaluated from the Michaelis constants (Km) and the molecular activities (k0) for a series of maltooligosaccharides according to the subsite th...

The substrate specificity of sugar beet α-giucosidase was investigated. The enzyme showed a relatively wide specificity upon various substrates, having α-1,2-, α-1,3-, α-1,4- and α-l,6-glucosidic linkages.The relative hydrolysis velocity for maltose (G2), nigerose (N), kojibiose (K), isomaltose (I), panose (P), phenyl-a-maltoside (ɸM) and soluble starch (SS) was estimated to be 100:130: 10.7: 22.6: 54.6: 55.8: 120 in this order; that for malto-triose (G3), -tetraose (G4), -pentaose (G5), -hexaose (G6), -heptaose (G7), -octaose (G8), amyloses (G13) and (G17), 91: 91: 91: 91: 80: 57: 75: 73. The Km values for N, K, I, P, and SS were 16.7 mM, 1.25 mM, 10.8 mM, 8.00 mM, 4.12 mM and 1.90 mg/ml, respectively; that for G2, G3, G4, G5, G6, G7, G8, G13 and G17 were 20.0 mM, 3.67 mM, 2.34 mM, 0,64 mM, 0.42 mM, 0.32 mM, 0.23 mM, 0.36 mM and 0.26 mM, respectively.The enzyme, though showed higher affinity and activity toward soluble starch than toward maltose, was considered essentially to be an α-glucosidase.

Cell walls of the Basidiomycete fungus Polyporus tumulosus (Cooke) were fractionated, and the polysaccharide content of the fractions investigated. The major constituents of the cell wall include four polysaccharides, chitin, a β-1, 3-glucan and the alkali soluble α-glucan and xylomannan. The glucan is highly dextrotatory with an [α] D 2 1 of + 221° and gave on partial acid hydrolysis and acetolysis an homologous series of oligosaccharides. The disaccharide was shown to be nigerose 3-0-α-D-glucopyranosyl-D-glucose. Periodate oxidation and methylation studies provided supporting evidence that the polysaccharide is an essentially unbranched polymer of 1,3-linked glucose residues. The other alkali-soluble polysaccharide, a xylomannan, is a polymer of mannose and xylose in the approximate molar proportions of 1.2:1. It has an [ α ] D = + 56° and on partial acid hydrolysis and acetolysis gave an homologous series of 1,3-linked mannodextrins but no oligosaccharides containing xylose were obtained. An α-1,3-linked mannan was prepared from the xylomannan by degradation with mild acid or by degradation of the periodate-oxidased and reduced xylomannan. The structure therefore is visualised as having a backbone of 1,3-linked mannan, to which xylose residues are attached. Methylation studies showed that branching occurs at C-4 of the mannopyranose units; the presence of 2,3-di- o -methyl- d -xylose in the hydrolysate of the methylated polysaccharide indicated that some of the xylose residues are 1,4-linked. The possible structure of the fungal cell wall is discussed in the light of the results obtained.

Srome properties were examined of purified α-l,3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8.The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1,3-glucanase was stable up to 50°C on heating for 10 min. This enzyme hydrolyzed the substrate α-l,3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1,3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1,3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-l,3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.

An enzyme in glucosyltransferase preparations from Streptococcus mutans catalyzed the transfer of [14C]glucopyranoside from purified isomaltosaccharides, each containing [14C]glucopyranoside at its non-reducing terminus, to acceptor dextran, in the absence of sucrose. Half of the radioactivity present in the resulting [14C]dextrans was resistant to hydrolysis by amylo-1,6-glucosidase. Treatment of the [14C]dextrans with endodextranase resulted in extensive hydrolysis and produced [14C]-labeled limit oligosaccharides containing branch sites. Acetolysis of the [14C]-labeled limit oligosaccharides yielded [14C]nigerose, thus indicating the formation of branch sites on dextran in the absence of sucrose. The enzyme catalyzing this reaction has not been identified but appears to be independent of the major extracellular glucosyltransferases of S. mutans .

The topics from the results of the studies conducted by the author during the past three decades are reviewed. The studies are mainly related to oligo- and polysaccharides and include the following projects: 1. Glucobioses in Japanese rice wine (Sake) and Koji extract. 2. Chemical synthesis of kojibiose and nigerose. 3. Acetolysis of gluco-oligosaccharides and glucans. 4. Structural studies of Konjac glucomannan. 5. Biochemical studies of dextrans. 6. Biosynthesis of fungal glycogen. 7. Structural studies of cell wall polysaccharides. 8. Biochemical studies of plant cell wall polysaccharides. 9. Some polysaccharide degrading enzymes including an α-1, 3- glucanase of a Streptomyces species, a dextranase capable of splitting α-1, 2-linkage at the branch point, and a /3-glucosidase of Pyricularia rr yzae.

Concanavalin A, a protein isolated from the jack bean (Canavalia ensijormis), has previously been shown to form a precipitate with glycogen and yeast mannan, and more recently with dextrans and amylopectin. Double-diffusion precipitation studies in agar gel have been found to afford a very sensitive and satisfactory method for investigating the interaction of concanavalin A with certain types of branched, glucose- and mannose- containing polysaccharides. In this manner concanavalin A can be used as a reagent for the detection and preliminary characterization of various types of polysaccharides. Bacterial levans as well as certain plant fructans also form precipitation bands with concanavalin A. This observation represents the first report of such an interaction. The method may also provide an indication as to the heterogeneity of polysaccharide preparations that react with concanavalin A as well as affording preliminary information on the relative molecular size of a series of reactive polysaccharides. Inhibition of the precipitation bands by low molecular weight carbohydrate molecules may be demonstrated. These inhibition studies reveal the highly specific nature of the combining sites of the concanavalin A molecule.

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.

1. A highly dextrorotatory polysaccharide (alpha(D)+232 degrees in n-sodium hydroxide), previously isolated as a fragment of Aspergillus niger cell walls, was prepared from whole mycelium and subjected to partial acid hydrolysis. 2. Fractionation of the hydrolysate on a charcoal column with a linear gradient of ethanol yielded a series of oligosaccharides. The disaccharide member was shown to be nigerose (3-O-alpha-d-glucopyranosyl-alpha-d-glucopyranose), although a small proportion of the disaccharide peak (10%) was present as maltose despite the fact that all the nigeran had been removed from the starting material. 3. The oligosaccharides forming the main peaks from the column were shown to be members of a polymer-homologous series (nigerodextrins) by (a) the relationship between the logarithm of their chromatographic mobility and degree of polymerization, (b) obeying the Freudenberg relationship, and (c) partial acid hydrolysis.

The transglucosidation action of Schizosaccharomyces pombe α-glucosidas e has been investigated by paperchromatography.The enzyme sythesized some oligosaccharides, consisting mainly of isomaltose, together with panose, isomaltotriose, nigerose, kojibiose and maltotriose by transferring glucosyl moiety from maltose. A small amount of isomaltose was also formed even from free glucose.The transglucosidation mechanism of the enzyme and the process of the further converison of the transglucosidation products were examined. The data indicated that the transglucosidation action of Schizosacch. pombe α-glucosidase also was similar to those of mould α-glucosidase.

Four α-glucosidases have been fractionated from sugar-beet cells. They are divisible into two groups in terms of soluble starch-hydrolysing activity. One group hydrolyses soluble starch at a faster rate than maltose. The enzymes of this group are not detected during the phase of starch accumulation in cells, but undergo a marked increase with the decrease of starch content. A possible role for these enzymes is in the metabolism of starch in the cell. Another group hydrolyses soluble starch only very weakly. The former group are highly active in the release of α-1,3-linked glucose from nigerose.

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.

The structure of a fungal (1→3)-α- d -glucan, pseudonigeran (DP 330, [α] d 25 +252° in m sodium hydroxide), has been investigated by methylation, periodate oxidation, and partial acid hydrolysis. Pseudonigeran was isolated, by alkali extraction, in 4% yield from the mycellium of Aspergillus niger NNRL 326 together with small amounts of nigeran and other polysaccharides. Pseudonigeran was further purified through its acetate. Hydrolysis of the methylated pseudonigeran (1 mole) yielded 2,3,4,6-tetra- (1 mole) and 2,4,6-tri-O-methyl- d -glucose (330 moles). Periodate oxidation of reduced pseudonigeran, followed by reduction and partial acid hydrolysis yielded the corresponding degraded pseudonigeran, glycerol, and traces of 2-hydroxyethylidene acetal of glucosylerythritol, indicating the presence of about 1.4% of (1→4) linkages which are either present in pseudonigeran or arise from contaminating material. Partial acid hydrolysis yielded glucose, nigerose, nigerotriose, nigerotetraose, and nigeropentaose. The structure of pseudonigeran is discussed on the basis of these findings.

THE occurrence of 1,3-α linkage in some dextrans has already been suggested by many workers1–5. Barker et al.3,4 have fractionated the partial acid hydrolysate of the dextran produced by Betacoccus arabinosaseous (Birmingham strain) on a charcoal column and recognized the presence of a trace of nigerose by paper chromatography. Jeanes et al.5 have characterized dextrans from 96 strains of bacteria by periodate oxidation analysis and classified them into several groups on the basis of the proportions of 1,3 like linkages. According to their results, 29 of them appeared to contain more than 10 per cent 1,3 -linkages and a few of them showed an extremely high content of 1,3-linkages (more than 30 per cent).

IT is generally agreed that the iodine-staining polysaccharide isolated from many red algae is a glucan of the amylopectin type in which the chain-forming links are α-1 : 4 and the branch links, α-1 : 6-glucosidic. The floridean starch from Dilsea edulis appears to differ, in finer details of structure, from one sample to another. This difference turns mainly on the presence or the absence of a third type of link, namely, the 1 : 3-glucosidic linkage1. The majority of investigations favour the view that 1 : 3-glucosidic linkages are in fact not present in Dilsea starch2. We therefore think it desirable to record some observations made in the course of a comprehensive linkage analysis of floridean starch which establish that a small proportion of 1 : 3-links is an integral part of the starch structure and, moreover, that these links have the α-configuration.