Isomaltose Oligosaccharides Research Articles

Pengaruh Bubur Pisang Isomaltosa-oligosakarida dan Fibercreme terhadap Kadar Glukosa dan Lipida Darah serta Profil Digesta Tikus Diabetes

Dietary fiber and resistant starch are known to be beneficial in lowering both glucose and lipid concentration in human. Fibercreme, a fiber rich product has been developed. It is a commercial non-dairy creamer that uses various oligosaccharides such as Isomaltose-oligosaccharides (IMO) or inulin as fiber source to replace the glucose component of conventional creamer. In this study, IMO and fibercreme were used to replace sucrose of the banana porridge. Therefore, the purpose of this study was to determine porridge effects on glucose, triglyceride, serum cholesterol and digesta profile in diabetic STZ-NA induced rats. Twenty-five male Wistar rats were divided into five groups and fed four different diets including the standard (KS and KN), banana porridge with sweetener IMO (BP+IMO), fibercreme (BP+FC) and (BP + Sukrosa). In these diets, porridge subtituted 30% energy of the standard diet, and the intervention was conducted for 28 days. It was found that banana-IMO porridge showed the lowest decrease in serum glucose level with 56.09%, followed by banana-fibercreme (47.61%) and bananasucrose (36.61%). Furthermore, similar trend was found in their cholesterol-lowering effects with 36.82%, 25.68% and 7.86% respectively. However, triglyceride level effects were highest in banana-IMO porridge (9.06%), followed by banana-sucrose (6.47%) and banana-fibercreme (3.72%). In addition, sucrose replacement with IMO increases weight and water content, as well as decrease caecal digesta pH. However, fibercreme increases only water content and does not decrease caecal digesta pH.

Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose–Oligosaccharide Synthesis

Isomaltose–oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0–10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Specificities and reactivities of the antibodies formed in rabbits to stearyl-isomaltosyl oligosaccharides

The specificities and the sizes and shapes of the antibody combining sites of the 15 antisera raised against various stearyl-isomaltosyl oligosaccharides were studied by quantitative precipitin and precipitin inhibition. The antibodies precipitated well with dextrans B512 and B1424 but less well with B1299S and B1355S. Only 3 of the 15 antisera reacted with linear dextrans; however, with about 50% of the added antibodies being precipitated, showing that most of the antibodies cannot bind to internal determinants along the dextran molecules and are similar to myeloma protein W3129 in having cavity-type sites which bind only to terminal nonreducing ends of α1 → 6 dextran. Antibodies differing in the sizes of their antibody combining sites were elicited in different rabbits by the same antigen. Of the 15 antisera studied, four have antibody combining sites as large as IM3, five as large as IM4, three as large as IM5 and three as large as IM6. The association constants for various isomaltose oligosaccharides of an antiserum (R-862) showing fewest bands in isoelectric focusing gel were determined by affinity electrophoresis and were comparable to W3129.

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid. I. Antigenicity of the glycolipids and the production of specific antibodies in rabbits.

Isomaltose oligosaccharides varying in size from two sugars, isomaltose (IM2), to seven sugars, isomaltohepatose (IM7), were coupled to stearylamine by reductive amination with sodium cyanoborohydride. Each compound was purified by column chromatography to yield a series of glycolipids containing oligosaccharides differing in length. Stearyl-isomaltotriose to stearyl-IM7 could be incorporated into liposomes and could render them agglutinable by specific antibodies to alpha 1 leads to 6 dextran and could be lysed if complement was also added, whereas those containing stearyl-IM2 were not agglutinated or lysed, indicating that stearyl-IM2 may not be protruding from the liposome surface sufficiently to react with the antibody. Stearyl-isomaltosyl oligosaccharides by themselves or incorporated into liposomes were equally antigenic when emulsified in complete Freund's adjuvant. They elicited pauciclonal responses, and the antibodies were alpha 1 leads to 6 specific, cross-reacted with dextran, and gave semirestricted isoelectric focusing patterns.

Binding Constants of Dextrans and Isomaltose Oligosaccharides to Dextran-Specific Myeloma Proteins Determined by Affinity Electrophoresis

Abstract In “Binding Constants of Dextrans and Isomaltose Oligosaccharides to Dextran-Specific Myeloma Proteins Determined by Affinity Electrophoresis” by Kazusuke Takeo and Elvin A. Kabat in the December 1978 issue of the Journal of Immunology, Volume 121, Number 6, pages 2305 to 2310, the authors neglected to cite the work of V. Hořejší, M. Tichá, and J. Kocourek (1977) Biochim. Biophys. Acta 499: 290–300 and 301–308 on determination of binding constants of lectins by affinity electrophoresis.

Binding Constants of NZB Myeloma Antidextrans for Dextrans and Isomaltose Oligosaccharides Determined by Affinity Electrophoresis

Association constants of dextrans (Ka) and oligosaccharides (Kia) from NZB myeloma antidextrans (PC3858 and PC3936) were studied by affinity electrophoresis. With linear dextrans or with those with a low degree of branching, Ka ranged from 2.7 X 10(3) to 5.4 X 10(4) ml/g for PC3858 and from 1.3 X 10(4) to 2.6 X 10(5) ml/g for PC3936. Completely linear alpha-(1 leads to 6)-linked dextrans, LD7 and D3, showed relatively high affinities for the two NZB antidextrans. With oligosaccharides, the Kia value increased as the number oa alpha-(1 leads to 6)-linked glycosyl residues increased. Isomaltoheptaose (IM7) showed the highest Kia (1.9 X 10(4) M-1 for PC3858 and 1.63 X 10(4) M-1 for PC3936), whereas isomaltose (IM2) had the lowest Kia (2.36 X 10(2)M-1 for PC3858 and 1.32 X 10(2)M-1 for PC3936). Pullulan and glycogen showed very weak affinity for PC3936, but they did not react at all with PC3858. These findings indicate that NZB myeloma antidextrans, PC3858 and PC3936, are specific for internal chains of alpha-(1 leads to 6)-linked dextrans. Data on the precision with which Ka and Kia can be determined are presented.

Binding Constants of Dextrans and Isomaltose Oligosaccharides to Dextran-Specific Myeloma Proteins Determined by Affinity Electrophoresis

Abstract In polyacrylamide electrophoresis, mobilities of dextran-specific myeloma proteins W3129 and QUPC52 were retarded when dextrans were added to the separating gel. Retardation by dextran was reversed by adding isomaltose oligosaccharides to the gel. From measurements of the extent of retardation by dextran or of reversal by hapten as a function of concentration, association constants of dextrans (Ka) and of oligosaccharides (Kia) were determined. With W3129, NRRL dextrans B 742 FR.S, B 742 FR.L, N279, B 1355 FR.S, B 1399, B 1299 FR.S, and NRC fractions 6, 7, and 8 have Kα values ranging from 2 to 6 × 104 ml/g. With QUPC52, dextrans in general show lower Ka than with W3129; N279, NRC fractions 6, 7, and 8, and B 1399 have Ka values ranging from 1 to 7 × 104 ml/g, whereas B-742 FR.S, B-1299 FR.S, and B-1355 FR.S have Ka values ranging from 3 to 6 × 102 ml/g. Dextran D3, a completely linear α(1 → 6)-linked glucosyl chain having a m.w. of 36,500 has a relative high affinity for QUPC52 (Ka = 2.3 × 103 ml/g or 8.3 × 104 M-1), although it does not show any affinity for W3129. This result confirms that myeloma protein W3129 is specific for a terminal chain of α(1 → 6)-linked glucosyl residues, whereas QUPC52 reacts with the nonterminal portion of α(1 → 6)-linked glucosyl chains. Kia values obtained coincided with those by other methods reported earlier by Cisar et al. With W3129, affinity increases with chain length up to isomaltopentaose (IM5) with the highest affinity Kia = 6.7 × 104 M-1; Kia of isomaltohexaose (IM6) and isomaltoheptaose (IM7) being 6.3 × 104 and 2.3 × 104 ml/g, respectively. With QUPC52, affinity increases as the number of residues in the glucosyl chain increases, reaching an upper limit with IM6 and IM7, 1.2 and 1.4 × 104 M-1. This method of affinity electrophoresis is useful for obtaining binding constants ranging from 102 to 106 M-1. It may be performed on antibodies, lectins, and other binding proteins by using not only the purified proteins but also crude protein mixtures such as the ascitic fluid used in this study.

Immunochemical Studies on Dextrans

Abstract Rabbits were immunized with a hapten-protein conjugate corresponding to a terminal nonreducing chain of five α(1 → 6) linked glucose units plus an additional α-linkage to a six carbon open sugar chain. The antigen was prepared by oxidizing isomaltohexaose to isomaltohexaonic acid and coupling it to bovine serum albumin (BSA). Antisera cross-reacted with dextrans, the amount of dextran required for maximum precipitation being related to its content of α(1 → 6) linkages. This cross-reaction was independent of anti-BSA. Inhibition studies with the isomaltose oligosaccharides from the tri-(IM3) to hepta-(IM7) or octa-(IM8) saccharide with the six antisera showed that four had combining sites complementary to IM6 and two to IM5. For the four sera IM6, IM7, and IM8 were equally effective as inhibitors on a molar basis, better than IM5, IM4 and very much better than IM3. For the remaining two sera IM5, IM6, and IM7 were equivalent and gave better inhibition than IM4 and IM3. Inhibition studies using the aldonic acids series from IM4-COOH to IM7-COOH showed five sera to be maximally inhibited by the pentaonic acid (IM5-COOH) which corresponded to the inhibition obtained with IM6 to IM8 in three sera. The two sera that were maximally inhibited by IM5 showed better inhibition with IM5-COOH and higher members of this series. The last serum showed unique behavior, better inhibition being obtained with the hexasaccharide than with any of the acids. Inhibition by IM4-COOH was equivalent either to IM4, to IM3, or was intermediate for all sera. In one case the IM5-COOH was equivalent to IM4-COOH and IM4. Thus heterogeneous populations of antibodies were formed in each serum as indicated by differences in the relative ease with which inhibition with large and small oligosaccharides and their corresponding acids was obtained.

Studies on dextranases : Part IV. Mode of action of dextranase D 1, on oligosaccharides

The products of action of a purified, extracellular endo-dextranase D 1, isolated from a new species of Pseudomonas, on pure isomaltose oligosaccharides have been investigated. Reduced and tritiated oligosaccharides have also been studied, and a model is postulated for the enzyme active-site, based on substrate specificity.

Immunochemical studies on mouse myeloma proteins reactive with dextrans or with fructosans and on human antilevans.

Four BALB/c IgA mouse myeloma proteins (W3129, W3434, QUPC 52, and UPC 102) reactive with dextran, four myeloma proteins reactive with fructosans, three IgA (W3082, UPC 61, and Y5476), and one IgG2a (UPC 10), and two human antilevans were studied immunochemically. Quantitative precipitin and inhibition assays showed that W3129, W3434, and QUPC 52 had specificities for isomaltose oligosaccharides similar to those previously found with alpha(1 --> 6)-specific human antidextrans. W3129 and W3434 were most complementary to IM5 but W3129 reacted equally with IM4 and IM3 while W3434 had a greater affinity for IM4 than IM3. QUPC 52 had a larger combining region and was most complementary to IM6. Protein UPC 102 (IgA), like MOPC 104E (IgM) (27), was most complementary to the alpha(1 --> 3)-linked trisaccharide, nigerotriose, and thus differed from J558 (29), which was inhibited best by nigeropentaose. UPC 102 was similar to J558 but they differed from MOPC 104E in their reactions with non-alpha(1 --> 3)-linked disaccharides. The fructosan-specific myeloma proteins fell into two groups with different specificities. The first group, W3082 (IgA), UPC 61 (IgA), and the previously studied J606 (IgG3) (28, 29), reacted with inulin and W3082 and UPC 61 appeared to have identical specificities for beta(2 --> 1)-linked fructofuranosyl residues with maximum complementarity for the tetrasaccharide betaDfructofuranosyl (2 --> 1)betaDfructofuranosyl(2 --> 1)betaDfructofuranosyl(2 --> 6)Dglucose while protein J606 was inhibited best by the trisaccharide betaDfructofuranosyl(2 --> 1)betaDfructofuranosyl(2 --> 6)Dglucose. W3082 and UPC 61 also differed from J606 in their behavior toward sucrose and betaDfructofuranosyl(2 --> 6)Dglucose as compared with alphaDglucosyl(1 --> 3)Dfructose (turanose). The second group containing myeloma proteins UPC 10 (IgG2a) and Y5476 (IgA) behaved similarly to human antilevans in that neither reacted with inulin nor were they inhibited by the beta(2 --> 1)-linked fructose oligosaccharides. Unlike the beta(2 --> 1)-specific proteins, they reacted with perennial rye grass levan that contained over 90% beta(2 --> 6)links. The differences in specificity and site size among homogeneous mouse myeloma proteins reactive with the same antigenic determinant are completely consistent with the concept that they represent products of homogeneous clones selected from the known heterogeneous population of antibody-forming cells.

Linkage analysis of (1→6)-linked oligosaccharides by alkaline degradation

2- C-Methylglyceric acid was formed by sequential degradation of the chain in isomaltose oligosaccharides in aqueous barium hydroxide. The products of the stopping reaction, namely 6- O-substituted 3-deoxy- d-hexonic acids, were also obtained in considerable yield. The separation and determination of these products is of importance for the analysis of the linkage sequence in oligosaccharides.

Preparation of isomaltose oligosaccharides labelled with 14C in the non-reducing terminal unit, and their use in studies of dextranase activity

A series of end-labelled isomaltose oligosaccharides was prepared by the reaction of dextran-sucrase with sucrose- 14 C in the presence of excess of unlabelled isomaltose saccharides as alternative acceptor. The main product of each reaction contained one more D-glucose residue than the acceptor substrate, and the label was located at the non-reducing end. The end-labelled saccharides were used to determine the specificity of a bacterial dextranase that required five or more consecutive α-(1→6)- D-glucosidic linkages in the substrate. The third linkage from the reducing end of isomaltohexaose ( IM 6) and of other substrates with longer chains ( IM 7 and IM 8) was the most susceptible to attack, and the products from higher oligosaccharides were IM 3, IM 4, and IM 5. Isomaltopentaose ( IM 5) was further hydrolysed to IM 3 and IM 2 when a 35-fold excess of enzyme was added, but there was no action on IM 4, IM 3, or IM 2 under these conditions. It was concluded that the dextranase hydrolysed linkages penultimate to either end of the chain only with difficulty, and that end linkages were completely resistant to attack.

Studies on Human Antibodies

Abstract The 1-(m-nitrophenyl) flavazoles (NPF1) of the isomaltose oligosaccharides obtained by condensation of o-phenylenediamine, m-nitrophenyl hydrazine sulfate with the isomaltose oligosaccharides were used to study their interaction with human antidextran by the quantitative hapten inhibition and fluorescence-quenching techniques. The NPF1 haptens inhibited in the dextran-antidextran system in whole serum in the same order as did the isomaltose oligosaccharides; that is, NPF1 of isomaltoheptaose (NPF1IM7) was the best inhibitor, inhibitory potency decreased progressively to the NPF1 of isomaltotetraose (NPF1IM4). Studies with antidextrans purified on a specific immunoadsorbent and eluted sequentially by isomaltotriose (IM3) and isomaltohexaose (IM6) showed that NPF1IM4 and NPF1IM5 inhibited the IM3 antidextran eluate better than the IM6 eluate, confirming the previous finding that antidextrans consist of subpopulations with different size combining sites. All NPF1 of isomaltose oligosaccharides caused specific fluorescence quenching of the purified antidextrans which could be reversed specifically by addition of unlabeled IM6. Since relatively large amounts of nonspecific attenuation also occurred with γ globulin devoid of antidextran activity or with L-tryptophan solution, absolute values of maximum quenching (Qmax) could not be obtained, but the relative value of Qmax for each NPF1 was estimated from Scatchard plots by using various values of Qmax and taking the one which gave an extrapolated value of two combining sites since purified antidextrans are IgG and have two combining sites/molecule. NPF1IM3 with the NPF1 group closest to the immunodominant non-reducing end of IM3, which would be closest to the antibody-combining site, gave the largest Qmax and NPF1IM7 with the NPF1 group farthest from the immunodominant end gave the smallest Qmax. Differences in Qmax values were seen for the various antidextran fractions eluted by methyl α-D-glucoside, IM3 and IM6. Average intrinsic association constants, K0, of the purified antidextrans obtained from the Scatchard plots ranged from 5 to 8 × 104 for NPF1IM7 and from 1 to 1.3 × 104 for NPF1IM3. NPF1IM3 and NPF1IM4 gave smaller K0 values with the IM6 antidextran eluate than with the IM3 and methyl α-D-glucoside eluates, providing further evidence that small haptens are bound less effectively in combining sites of larger size.

A Simple Method for Quantitation of Haptens Using “Indirect” Single Radial Immunodiffusion

Abstract Single radial immunodiffusion has been adapted to the quantitation of low molecular haptens, making use of the principle of hapten inhibition of precipitation. As model haptens, isomaltose oligosaccharides were quantitatively estimated in a precipitating antidextran protein-dextran conjugate system. The antidextrans had been raised in sheep by immunization with a bovine serum albumin-dextran conjugate. After preparation of the agar plates containing the antidextran, the wells were filled with the haptenic oligosaccharide solutions. The latter were allowed to diffuse into the agar and to bind to the antidextran for 30, 120, or 300 min, and then the hapten inhibition halos were visualized or “developed” by immersing the plates in a solution of an ovalbumin-dextran conjugate for 24 hr. Thereafter, the diameters of the transparent hapten inhibition halos, contrasting against the remaining opaque parts of the plate, were measured. A linear relationship between area of hapten inhibition and concentration of individual isomaltose oligosaccharides was obtained, when sufficient time for complete hapten diffusion was allowed before visualizing hapten inhibition zones. In the examples studied, the smallest amount of oligosaccharide hapten measurable was about 10 µg/ml. The potential application of this procedure for quantitation of other low molecular monovalent haptens is discussed.

Hapten inhibition of passive antidextran dextran anaphylaxis in guinea pigs. Role of molecular size in anaphylactogenicity and precipitability of dextran fractions.

High titred antidextran antisera have been raised in rabbits by prolonged immunization with soluble conjugates between isomaltose oligosaccharides or dextran m<sub>w</sub> 4,400 and bovine serum albumin in complete Freund’s adjuvant. Guinea pigs sensitized with such antisera exhibited anaphylactic shock on i.v. challenge with fractions of hydrolyzed B512 dextran. The severity of anaphylaxis depended on the amount of sensitizing antibody and the dose and molecular size of the challenging dextran fraction. In maximally sensitized animals, fractions of m<sub>w</sub> 3,600 or larger were anaphylactogenic and smaller molecular size fractions were nonanaphylactogenic. In submaximally sensitized animals, dextran fractions of m<sub>w</sub> 10,500 or less were foundnonanaphylactogenic. In addition, nonanaphylactogenic dextran fractions produced desensitization, i.e. protection from anaphylaxis due to subsequent or simultaneous injection of more high molecular and anaphylactogenic dextran fractions. When the molar ratio in a mixture of nonanaphylactogenic/anaphylactogenic fractions was about 7.6, protection was complete with reduction of mortality from 100 to 0%. A molar ratio of 1.9, corresponding to a 13-percent by weight admixture of nonanaphylactogenic dextran, still significantly reduced mortality from 100 to 50%. From the ability to form precipitates with antidextrans and to elicit anaphylaxis in sensitized guinea pigs, and from the absence of these properties, the approximate molecular size range of bivalent and monovalent dextran B512 fractions was found to correspond to m<sub>w</sub> 3,600 and m<sub>w</sub> 1,430, respectively. The practical applicability of hapten inhibition is discussed.