Mikihiko Kobayashi

Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses. Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100. The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state. The ]urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule. EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture. The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran. The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.

The dextransucrase isoenzymes of Leuconostoc mesenteroides NRRL B-1299

Abstract Dextransucrase (EC 2.4.1.5) activity of Leuconostoc mesenteroides NRRL B-1299 strain was shown to be inducible by sucrose, and detected both in the culture supernatant and centrifuged residue fractions. The total enzyme activity reached a maximum in 18–24 h after inoculation, with the extracellular and intracellular enzymes in the ratio of 2:3. From the sediment, 41.3% of the total intracellular activity was solubilized by successive treatments with disruption, lysozyme digestion, and deoxycholate extraction. The extracellular enzyme gave a number of active bands on acrylamide-disc electrophoresis, whereas the intracellular enzyme gave only two bands. Furthermore, the multiple forms of the extracellular enzyme, with the monomer having a molecular weight of 42 000, acted as the oligomeric isoenzymes by gel electrophoresis according to Hedrick, J.L. and Smith, A.J. ((1968) Arch. Biochem. Biophys. 126, 155–164). On the other hand, the intracellular enzymes with a similar molecular weight of 74 000, were characterized as the charge isomer proteins. These isoenzymes also differed from each other in their enzymatic characteristics (i.e. optimum pH, temperature, and K m ).

Isolation of enzymes from polyacrylamide disk gels by a centrifugal homogenization method.

A sliced segment of polyacrylamide gel was quickly homogenized without any loss of gel pieces. The gel segment was placed on a disposable pipet tip, which was packed with a small amount of lumped copper wires and held in a microfuge tube. The gel was homogenized by centrifugation for 15 s at 15,000 g at 0 to 4 degrees C. Almost 70% of endodextranase activity could be recovered from homogenized gel within 30 min at 4 degrees C, whereas only 20% of activity was eluted from gel slices. If necessary, copper wire could be replaced by fine stainless-steel wire or by the nylon string used in fishing lines. Proteins could also be recovered from the homogenized gel by charging electric current for 1 h at 4 degrees C.

Dextran α-(1→2)-debranching enzyme from Flavobacterium Sp. M-73. Properties and mode of action

Abstract The general properties and specificity of a dextran α-(1→2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5–6.5, and 45°, respectively. The enzyme was stable up to 40° on heating for 10 min, and over a pH range of 6.5–9.0 on incubation at 4° for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1→2)-α- d -glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces d -glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the K m value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21 ; B-1299 soluble, 31.5%, 0.27 ; and B-1397, 11.8%, 0.91 . The debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran α-(1→2)-debranching enzyme, and its systematic name is also discussed.

Enzymic degradation of water-soluble dextran from Leuconostoc mesenteroides NRRL B-1299

Abstract Structural studies on the water-soluble dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 gave several important results that not only supported previous results but afforded an insight into the association of average repeating-units in the whole molecule. Sequential degradation of soluble dextran from its nonreducing terminals was achieved with two different enzymes, namely, α- d -(1→2)-debranching enzyme and d -glucodextranase. The debranching enzyme removed four separate residues of α-(1→2)-branched d -glucose from the average repeating unit consisting of 15 d -glucosyl residues. d -Glucodextranase continuously produced d -glucose from the nonreducing terminals by an exo type of action, but internal branches greatly restricted its action. Extensive digestion of soluble dextran B-1299 with the two enzymes released 74.3% of d -glucose and gave a limit dextrin of high molecular weight containing 8.4% of 2,6-di- O -substituted d -glucosyl residues. 13 C-N.m.r. studies indicated a characteristic pattern of the α- d -(1→2)-branched structure, which was significantly changed on treatment with the debranching enzyme. Moreover, an ∼8-fold increase in the degree of linearity was observed after action of the debranching enzyme. The possible structure of water-soluble dextran B-1299 is discussed, based on a comparison of the limit dextrin with the native dextran, in regard to chemical structure, molecular-weight distribution, and degree of hydrolysis with two exo-enzymes. The native dextran might be constructed with at least ∼8,200 “twigs” of repeating unit, and there are 14 steps of connected twigs between the reducing and nonreducing terminals. Upon consecutive hydrolysis with the two exo-enzymes, most of the twigs located at the 14th step ( i.e., nonreducing terminals) were hydrolyzed to d -glucose.

Pronounced hydrolysis of highly branched dextrans with a new type of dextranase

Abstract Two distinct dextranase activities, I and II, were isolated from the culture supernatant of a soil bacterium (strain M-73). Among the various α-glucans tested, only dextrans containing (1→2)-α-linkage at the branch points were susceptible to dextranase I. The hydrolyzate of the B-1299 dextran with this enzyme was constituted of glucose as a sole low molecular-weight product and limit dextrin. Therefore, it was suggested that dextranase I has a definite specificity to (1→2)-α-linkage at the branch points.

Use of water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide for the fluorescent determination of uronic acids and carboxylic acids

Reaction between glucuronic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was monitored by the o-phthalaldehyde (OPA) method, which was developed for the fluorescent assay of compounds containing an amino group. About 1 nmol of glucuronic acid was detected by this method. This EDC-OPA method was effective in detecting not only acidic sugar but also carboxylic acid. Although the sensitivity of the EDC-OPA method was somewhat lower than that of amino acid determination by OPA, a very simple and convenient assay was attained for compounds containing a carboxyl group.

Acetolysis of Leuconostoc mesenteroides NRRL B-1299 dextran. Isolation and characterization of oligosaccharides containing secondary linkages from the borate-soluble fraction

Abstract Fractionation of the deacetylated acetolyzate of the borate-soluble fraction of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 gave, after chromatography on charcoal—Celite, preparative paper-chromatography, and paper electrophoresis, four trisaccharide fractions and four tetrasaccharide fractions. The isolated oligosaccharides were characterized by their paper-chromatographic mobility, examination of partial acid-hydrolyzates of the oligosaccharides and their corresponding alditols, and methylation analysis. These oligosaccharides were shown to be ( a ) kojitriose ( 1 ), ( b ) isomaltotriose ( 2 ), ( c ) a mixture of 2- O -α-isomaltosyl- D -glucose ( 3 ), 2 1 - O -α- D -glucosylisomaltose ( 4 ), and 2- O -α-nigerosyl- D -glucose ( 5 ), ( d ) 6- O -α-kojibiosyI- D -glucose ( 6 ), ( e ) isomaltotetraose ( 7 ), ( f ) a mixture of 2- O -α-isomaltotriosyl- D -glucose ( 8 ) and 2 1 - O -α- D -glucosylisomaltotriose ( 9 ), ( g ) 6- O -α-kojitriosul- D -glucose ( 10 ), and ( h ) 6 3 - O -α- D -glucosylkojitriose ( 11 ), respectively. Some of these oligosaccharides are newly isolated and characterized.

Application of Periodate Oxidized Glucans to Biochemical Reactions

ABSTRACT Periodate oxidation of glucans afforded a dialdehyde structure, which was highly reactive with various compounds containing amino groups. A covalent Schiff base linkage was readily formed at the alkaline pH of 8-9 and cyclodextrin dialdehyde gave both positively and negatively charged derivatives upon incubation with hexamethylenediamine and ɛ-aminocaproic acid, respectively. Binding of the amino group containing a fluorescent probe of ethylenediaminonaphthalene yielded fluorescent glycogen, which was hydrolyzed with Taka-amylase A. By gel filtration with a Bio-Gel P-2 column, hydrolyzed oligosaccharides containing a fluorescent probe were strongly retained to the column. Dextran dialdehyde was useful in producing a covalent linkage with trypsin under very mild conditions, and the enzyme-dextran complex formed was recovered in a high-molecular weight and active form. Thus, various glucan dialdehydes may serve as useful cross-linking reagents for enzymes.

Inhibition of dextran synthesis by glucoamylase and endodextranase

Abstract In a model experiment, glucoamylase was shown to inhibit α- D -glucan synthesis as catalyzed by potato phosphorylase. Both glucoamylase and endodextranase inhibited dextran synthesis with dextransucrases of Leuconostoc mesenteroides . The inhibition could be ascribed to competition between glucoamylase and dextransucrase for the glucosyl groups at the non-reducing end of dextran. The inhibition caused by endodextranase may result from rapid and random hydrolysis of acceptor dextrans. Moreover, significantly low units of glucoamylase, as compared with endodextranase, effectively inhibited dextran synthesis. These results thus present evidence that bio-synthesis of dextran occurs by the addition of glucosyl groups at the non-reducing end of the growing dextran. The measurement of initial velocity suggested that the ping-pong Bi-Bi mechanism proposed for the levansucrase of Bacillus subtilis is also applicable to dextransucrase.