Isomaltopentaose

The Screening and Identification of a Dextranase-Secreting Marine Actinmycete Saccharomonospora sp. K1 and Study of Its Enzymatic Characteristics.[Pubmed:38393040]

Mar Drugs. 2024 Jan 28;22(2):69.

In this study, an actinomycete was isolated from sea mud. The strain K1 was identified as Saccharomonospora sp. by 16S rDNA. The optimal enzyme production temperature, initial pH, time, and concentration of the inducer of this actinomycete strain K1 were 37 degrees C, pH 8.5, 72 h, and 2% dextran T20 of medium, respectively. Dextranase from strain K1 exhibited maximum activity at 8.5 pH and 50 degrees C. The molecular weight of the enzyme was <10 kDa. The metal ions Sr(2+) and (K+) enhanced its activity, whereas Fe(3+) and Co(2+) had an opposite effect. In addition, high-performance liquid chromatography showed that dextran was mainly hydrolyzed to isomaltoheptose and Isomaltopentaose. Also, it could effectively remove biofilms of Streptococcus mutans. Furthermore, it could be used to prepare porous sweet potato starch. This is the first time a dextranase-producing actinomycete strain was screened from marine samples.

A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis.[Pubmed:32240925]

J Pharm Biomed Anal. 2020 Jul 15;186:113267.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using Isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human alpha1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.

Carboxy-Terminal Region of a Thermostable CITase from Thermoanaerobacter thermocopriae Has the Ability to Produce Long Isomaltooligosaccharides.[Pubmed:31838796]

J Microbiol Biotechnol. 2019 Dec 28;29(12):1938-1946.

Isomaltooligosaccharides (IMOs) have good prebiotic effects, and long IMOs (LIMOs) with a degree of polymerization (DP) of 7 or above show improved effects. However, they are not yet commercially available, and require costly enzymes and processes for production. The Nterminal region of the thermostable Thermoanaerobacter thermocopriae cycloisomaltooligosaccharide glucanotransferase (TtCITase) shows cyclic isomaltooligosaccharide (CI)-producing activity owing to a catalytic domain of glycoside hydrolase (GH) family 66 and carbohydrate-binding module (CBM) 35. In the present study, we elucidated the activity of the C-terminal region of TtCITase (TtCITase-C; Met740-Phe1,559), including a CBM35-like region and the GH family 15 domain. The domain was successfully cloned, expressed, and purified as a single protein with a molecular mass of 115 kDa. TtCITase-C exhibited optimal activity at 40 degrees C and pH 5.5, and retained 100% activity at pH 5.5 after 18-h incubation. TtCITase-C synthesized alpha-1,6 glucosyl products with over seven degrees of polymerization (DP) by an alpha-1,6 glucosyl transfer reaction from maltopentaose, Isomaltopentaose, or commercialized maltodextrins as substrates. These results indicate that TtCITase-C could be used for the production of alpha-1,6 glucosyl oligosaccharides with over DP7 (LIMOs) in a more cost-effective manner, without requiring cyclodextran.

Characterization of a marine-derived dextranase and its application to the prevention of dental caries.[Pubmed:24197466]

J Ind Microbiol Biotechnol. 2014 Jan;41(1):17-26.

The dextranase added in current commercial dextranase-containing mouthwashes is largely from fungi. However, fungal dextranase has shown much higher optimum temperature than bacterial dextranase and relatively low activity when used in human oral cavities. Bacterial dextranase has been considered to be more effective and suitable for dental caries prevention. In this study, a dextranase (Dex410) from marine Arthrobacter sp. was purified and characterized. Dex410 is a 64-kDa endoglycosidase. The specific activity of Dex410 was 11.9 U/mg at optimum pH 5.5 and 45 degrees C. The main end-product of Dex410 was isomaltotriose, isomaltoteraose, and Isomaltopentaose by hydrolyzing dextran T2000. In vitro studies showed that Dex410 effectively inhibited the Streptococcus mutans biofilm growth in coverage, biomass, and water-soluble glucan (WSG) by more than 80, 90, and 95 %, respectively. The animal experiment revealed that for short-term use (1.5 months), both Dex410 and the commercial mouthwash Biotene (Laclede Professional Products, Gardena, CA, USA) had a significant inhibitory effect on caries (p = 0.0008 and 0.0001, respectively), while for long-term use (3 months), only Dex410 showed significant inhibitory effect on dental caries (p = 0.005). The dextranase Dex410 from a marine-derived Arthrobacter sp. strain possessed the enzyme properties suitable to human oral environment and applicable to oral hygiene products.

Dextran acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I as revealed by using uniformly 13C-labeled sucrose.[Pubmed:11423107]

Carbohydr Res. 2001 Jun 22;333(1):19-26.

A sucrose glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus transferred the uniformly 13C-labeled glucosyl residue ([U-(13)C]Glc) from [U-(13)C]sucrose to exogenous dextran T500 at the non-reducing-end, mostly by alpha-(1-->6) linkages and partially by alpha-(1-->3) linkages, as revealed by the 13C-(13)C NMR coupling pattern. With increasing amounts of [U-(13)C]sucrose, transfer of [U-(13)C]Glc to the alpha-(1-->3)-linked chain became predominant without increase in the number of chains. The transfer of [U-(13)C]Glc to an Isomaltopentaose acceptor occurred similarly to its transfer to T500. alpha-(1-->3)-branches in the [U-(13)C]dextran, specifically synthesized from [U-(13)C]sucrose by a Streptococcus bovis dextransucrase, were not formed by GTF-I, as judged by the observation that a newly-formed alpha-1,3,6-branched [U-(13)C]Glc was not detected, which could have been formed by transferring the unlabeled Glc from sucrose to the internal alpha-(1-->6)-linked [U-(13)C]Glc at C-3. The 13C-(13)C one-bond coupling constants (1J) were also recorded for the C-1--C-6 bond of the internal alpha-(1-->6)-linked [U-(13)C]Glc and of the non-reducing-end [U-(13)C]Glc.

Specificity of the glucan-binding lectin of Streptococcus cricetus.[Pubmed:3397177]

Infect Immun. 1988 Aug;56(8):1864-72.

The specificity of the glucan-binding lectin (GBL) of Streptococcus cricetus AHT was determined. Examination of the kinetics of aggregation of cell suspensions with glucans containing various percentages of alpha-1,6, alpha-1,4, alpha-1,3, and alpha-1,2 anomeric linkages revealed that only glucans with at least 80% alpha-1,6 linkages promoted strong aggregation. Moreover, only linear glucans with molecular weights greater than 5 X 10(5) were capable of causing rapid aggregation of the bacteria. The lectin was observed to be present on S. cricetus strains, on Streptococcus sobrinus, and on several Streptococcus mutants strains. Preincubation of suspensions of S. cricetus AHT with glucan T10 (molecular weight of 10,000) before the addition of high-molecular-weight glucan resulted in competitive inhibition in a concentration-dependent manner. Inhibition was achieved also with Isomaltopentaose, isomaltohexaose, and isomaltooctaose, but at higher concentrations than glucan T10. In contrast, no inhibition was observed with maltoheptaose, providing additional evidence for the specificity of GBL. Treatment of suspensions of S. cricetus AHT with trypsin before and after aggregation with high-molecular-weight glucan revealed a substantial level of protection of GBL when in a bound state. Collectively, these results indicated that GBL has an absolute affinity for glucans rich in alpha-1,6 linkages and possesses an active site which recognizes internal sequences and accommodates isomaltosaccharides of at least nine residues. This unusual specificity may contribute to the colonization of S. cricetus, S. sobrinus, and S. mutans in glucan-containing plaque in the oral cavity.

Cross-reaction of synthetic alpha-(1----3)-branched glucans with rabbit anti-N4 dextran.[Pubmed:2420785]

J Biochem. 1986 Jan;99(1):263-7.

Cross-reactions of four synthetic branched glucans (3-O-alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1----6)-alpha-D-glucopyranosyl linkages, and the maximum size of the alpha-(1----6)-specific antibody combining site corresponded to Isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1----6)-alpha-D-glucopyranan containing about 27% of randomly linked 3-O-alpha-D-mannopyranosyl side chains) also reacted with the same antibody.

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: production and characterization of mouse hybridoma antibodies specific for stearyl-isomaltosyl oligosaccharides.[Pubmed:2415810]

Mol Immunol. 1985 Sep;22(9):1021-37.

Twelve C57BL/6J hybridoma clones, 9, 2 and 1 from mice immunized with stearyl-isomaltotetraose, stearyl-Isomaltopentaose and stearyl-isomaltohexaose respectively were characterized. Seven produced IgA and 5 IgM. The specificities and sizes of their combining sites were determined by quantitative precipitin and precipitin inhibition assays. All 12 hybridoma antibodies precipitated with alpha 1----6 dextran B512 and linear dextran LD7, indicating that they recognize an internal -Glc alpha 1----6Glc alpha 1----6Glc- determinant. This in contrast with the results with rabbit antisera obtained in response to the same immunogen which recognize the non-reducing terminal determinant Glc alpha 1----6Glc alpha 1----6Glc-. Of the 12 hybridoma antibodies, 1 has an antibody combining site complementary to 4 alpha 1----6-linked glucoses while others have combining sites complementary to isomaltohexaose or isomaltoheptaose. The large combining-site sizes found in C57BL/6 hybridoma clones may be related to the pre-existing clonal repertoire in this strain. Binding constants of monomers of these antibodies for dextran B512 and isomaltoheptaose determined by affinity electrophoresis range from 1.4 X 10(3) to 4.6 X 10(5) ml/g and from 1.2 X 10(3) to 3.5 X 10(4) M-1 respectively, which is consistent with previous studies in the anti-dextran B512 system. The use of synthetic glycolipids as antigens enables us to study the gene control of antibody responses to glycolipids and to investigate the combining-site specificities of antibodies to a single antigenic determinant. Results so far show that all 12 hybridoma proteins are different despite the simplicity of the antigens. The findings provide further insight into the specificity of antibody combining sites.

Immunochemical studies on dextrans. Cross-reaction of synthetic linear dextrans with rabbit anti-N4 dextran.[Pubmed:6169705]

J Biochem. 1981 Mar;89(3):823-9.

Alpha (1 Leads to 6) specific anti-dextran antibody was raised in rabbits by injecting N4 dextran-concanavalin A conjugate, and the interactions of five synthetic linear dextrans (alpha(1 Leads to 6)-D-glucopyranans) with rabbit anti-N4 dextran were studied. The ability of glucans to precipitate antibody depended on their average molecular weight, samples with higher molecular weight precipitating more antibody nitrogen under the same conditions. This phenomenon was shown to be due to solubility of the antigen-antibody complex. Oligosaccharide inhibition assay indicated that the maximum size of the alpha(1 Leads to 6)-specific antibody combining site corresponded to Isomaltopentaose. The precipitated antibody class was shown to be IgG immunoglobulin, and it was mostly directed to linear non-terminal glucosidic linkages. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratio of antibody molecule to numbers of glucose residues was 1:16 in the extreme antibody excess region.

Hydrolysis of low molecular weight isomaltosaccharides by a p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase from a thermophile, Bacillus thermoglucosidius KP 1006.[Pubmed:365246]

Biochim Biophys Acta. 1979 Jan 12;566(1):62-6.

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase of a thermophile, Bacillus thermoglucosidius KP 1006, was purified to an electrophoretically-homogeneous state. Its molecular weight was estimated as 60 000 by gel electrophoresis. The molecular activity (ko) and the Km value at 60 degrees C and pH 6.8 for p-nitrophenyl-alpha-D-glucopyranoside were 233 s-1 and 0.24 mM, respectively. The enzyme cleft the non-reducing terminal alpha-1,6-glucosidic bonds of isomaltose, panose, isomaltotriose, isomaltotetraose, and Isomaltopentaose. The ko values were 72.4, 194, 208, 233 and 167 s-1, and the Km values were 3.3, 9.5, 11, 13 and 21 mM, respectively. Each isomaltosaccharide was hydrolyzed to glucose by the cleavage of single glucose units from its nonreducing end. The present study suggests that the enzyme is an oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) and an exo-glucosidase.

Binding properties of immunoglobulin combining sites specific for terminal or nonterminal antigenic determinants in dextran.[Pubmed:49389]

J Exp Med. 1975 Aug 1;142(2):435-59.

Binding constants of the dextran-reactive BALB/c mouse IgA myeloma proteins W3129 and QUPC 52 have been determined for each member of the isomaltose series of oligosaccharides and for methyl alphaDglucoside. Protein W3129 has maximum complementarity for Isomaltopentaose (IM5) deltaf degrees = 7,180 cal/mol) with 55-60% of the total binding energy directed against methylalphaDglucoside. Protein QUPC 52 gives maximum binding with isomaltohexaose (IM6) (deltaF degrees = -5,340 cal/mol) and has about 70% of its total binding energy for isomaltotriose (IM3), but at most only 5% for isomaltose (IM2) or methyl alphaDglucoside. Protein W3129 precipitates with branched dextrans high in alpha (1 yields 6) linkages and reacts with but does not precipitate a synthetic alpha (1 yields 6)-linked linear dextran. Protein QUPC 52 precipitates both branched and linear dextrans. Thus, the immunodominant group for protein W3129 is mimicked by methyl alphaDglucoside and this protein reacts exclusively at the terminal nonreducing ends of alpha (1 yields 6)-linked dextran chains. Protein QUPC 52 has an immunodominant group which is expressed by IM3 but not smaller oligosaccharides and this protein can react at nonterminal locations along alpha (1 yields 6)-linked dextran chains. Precipitation of linear dextran seems to be a valid although not quantitative assay for antidextrans with nonterminal specificity. Quantitative precipitin reactions with branched and linear dextrans suggest that alpha (1 yields 6)-specific human antidextrans are mixtures of molecules having terminal and nonterminal specificities and that the fraction of each type can vary among individuals. Rabbit antisera against IM3 or IM6 coupled to bovine serum albumin also appear to contain antibodies with nonterminal specificity for dextran chains although a large fraction has terminal specificity. Low molecular weight clinical dextran N-150N (congruent to 60,000) reacted more like linear dextran than like its parent native-branched dextran B512. This is thought to result from an abundance of nonterminal determinants in clinical dextran N-150N but a very small number of functional terminal determinants per molecule. An appreciation of terminal and nonterminal specificities and of the different immunodominant structures in isomaltosyl chains has proven to be of a great value in understanding the immunochemical reactions of dextrans. Moreover, certain previous findings with fructosan-reactive mouse myeloma proteins and human antilevans (55, 84) also suggest terminal and nonterminal specificities for levan chains.

The action pattern of Penicillium lilacinum dextranase.[Pubmed:1139551]

Carbohydr Res. 1975 Feb;39(2):303-15.

The product distributions resulting from the action of Penicillium lilacinum dextranase on end-labelled oligosaccharides of the isomaltose series have been determined. The initial rates of formation of labelled products were measured for isomaltotriose up to isomalto-octaose, and the molar proportions and radioactivity of the final products from isomaltotriose up to isomaltohexaose were determined. D-Glucose was released only from isomaltotriose and isomaltotetraose, by hydrolysis of the first linkage from the reducing end (linkage 1); the terminal bonds of higher members of the series were not attacked. All oligosaccharides except isomaltotriose were hydrolyzed at more than one linkage. The main points of attack on isomaltotetraose up to isomalto-octaose were at linkage 2, and at the third linkage from the non-reducing end; these two positions coincide for Isomaltopentaose. The degradation of isomaltotriose up to isomalto-octaose was entirely hydrolytic. The enzyme also catalyzed an extremely slow, concentration-dependent degradation of isomaltose, and this may have occurred via a condensation to isomaltotetraose, followed by hydrolysis of linkage 1 to give D-glucose and isomaltotriose.

[Test of cellulose and cellulose derivatives for carcinogenic activity in rats, mice and rabbits].[Pubmed:4460953]

Arch Geschwulstforsch. 1974;44(3):222-34.

Activated cellulose, p-aminobenzylcellulose, p-hydroxybenzylcellulose, cellulose-m-aminobenzoxymethylether and cellulose-m-hydroxybenzoxymethylether which are used as highmolecular inert components for the preparation of immunoadsorbents, have been tested for carcinogenic activity in Wistar-rats and CBA-mice by the subcutaneous and intramuscular route of administration. In the same manner the coupling products have been tested in rats, mice and rabbits of diazotized p-aminobenzylcellulose and cellulose-m-aminobenzoxymethylether with a) human gammaglobulin [(cellulose-p-benzylether)-azogammaglobulin and (cellulose-m-benzoxymethylether)-azogammaglobulin respectively] and b) 1-(m-hydroxyphenyl)-flavozole derivatives of saccharides of the isomaltodextrin- and maltodextrin series [saccharide-1-(3 ft.-hydroxy-4 ft.-(resp.-6 ft.-or-2 ft.-) [cellulose-p-benzylether]-azophenyl)-flavazoles and saccharide-1-(3 ft.-hydroxy-4 ft.-(resp. -6 ft;-or-2 ft.-) [cellulose-m-benzoxymethylether]-azophenyl)-flavazoles with isomaltotriose, isomaltotetraose, Isomaltopentaose, isomaltohexaose, isomaltooctaose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltooctaose as saccharide moieties]. No tumours were found in rabbits. No statistical significant difference could be stated between the tumour incidence of experimental animals and controls in the rat and mouse groups.

Immunochemical analysis of the idiotypes of mouse myeloma proteins with specificity for levan or dextran.[Pubmed:4128444]

J Exp Med. 1974 Jan 1;139(1):137-47.

This paper deals solely with idiotypic determinants, the configurations of which are modified when the antibody bearing them interacts with its ligand. This phenomenon is measured as an inhibition of the reaction between anti-idiotype and idiotype. Two points are made: (a) The assay for ligand-modifiable determinants can be used to determine the "size" of the combining site. This is illustrated here with the anti-alpha(1 --> 6) dextran mouse myeloma immunoglobulin W3129. Whether the interaction between a homologous series of alpha(1 --> 6) oligosaccharide ligands and the combining site of W3129 is measured by inhibition of precipitation with alpha(1 --> 6) dextran (4) or of binding of W3129 to anti-W3129 idiotype, the finding is the same. The order of inhibition is isomaltohexaose = Isomaltopentaose >> isomaltotetraose > isomaltotriose >>> isomaltose. The combining site is optimally complementary to Isomaltopentaose. (b) Cross-idiotypic specificity is closely correlated with cross-combining specificity; the converse is not true. This is illustrated here with three groups of mouse myeloma immunoglobulin, each specific for alpha(1 --> 3) dextran, alpha(1 -->6) dextran, beta(2 --> 1) or beta(2 --> 6) levan. If a given anti-idiotypic serum cross-reacted with several myeloma proteins, they always had similar combining specificity. Thus the three proteins, J558, MOPC 104E, and UPC 102, which cross-react with anti-J558 have combining specificity for alpha(1 --> 3) dextran; cross-reacting W3082, UPC 61, and Y5476 have specificity for levan; and cross-reacting W3129 and W3434 have specificity for alpha(1 --> 6) dextran. This extends previous studies with proteins specific for phosphorylcholine (7) or gamma-globulin (8). As expected, the converse is not true, for proteins may have combining specificity for alpha(1 --> 6) dextran e.g. QUPC 52, or levan e.g. J606, UPC 10 and yet not carry the above-mentioned reference idiotypes. The correlation between cross-idiotypic and combining specificity breaks down when idiotypic determinants which are not modifiable by ligand are studied. The implications of this are pointed out since most investigations deal with ligand-nonmodifiable determinants.