Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

l-Myoinositol-1-phosphate synthase from Neurospora crassa: Purification to homogeneity and partial characterization

The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH₄)₂SO₄ and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH₄)₂SO₄ and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg²⁺ and Mn²⁺ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD⁺ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD⁺-independent activity.     

Covalent attachment of epoxide hydrolase to dextran

Epoxide hydrolase (EC 3.3.2.3) purified from rat liver microsomes has been immobilized by covalent linking to dextran activated by imidazolyl carbamate groups, under mild conditions. K^app_m values of free and dextran bound epoxide hydrolase toward benzo(a)pyrene-4,5-oxide were 0.5 and 0.35 μM respectively, while V^app_max was lowered from 300 to 120 nmol min^?1mg^?1protein. The activity lost upon coupling could not be restored by digestion of the support by dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) treatment. This fact, along with the similarity of the activation energy values for both native and bound epoxide hydrolase, indicated that steric hindrance effects due to the polymer support played only a minor role in this loss of activity. Evidences of changes in the conformation of epoxide hydrolase were obtained by a comparative study of u.v. circular dichroism and tryptophan fluorescence emission spectra of the native and dextran bound enzymes. On the other hand, the enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation.     

Purification and some properties of the isomaltodextranase of Actinomadura strain r10 and comparison with that of Arthrobacter globiformis t6

A newly isolated soil-actinomycete, Actinomadura strain R10 (NRRL B-11411), produces an extracellular isomaltodextranase (optinal pH, 5.0) that was purified to homogeneity. It exolytically releases isomaltose and a minor trisaccharide product,α-d-Glcp-(1→3)-α-d-Glcp, from dextran B-512 and, in addition, forms transient transisomaltosylation products. This pattern of products is qualitatively similar to that previously reported for the isomaltodextranase (EC 3.2.1.94, optimal pH, 4-0) of Arthrobacter globiformis T6 (NRRL B-4425). The Arthrobacter isomaltodextranase is most active on the (1→6)-α-d-glucopyranosidic linkage, but the relative activity increases with the degrees of polymerization of isomalto-oligosaccharide substrates. In contrast, the relative activity of Actinomadura isomaltodextranase is almost constant throughout the same series of substrates, and is much higher on 3 O- and 4-O-α-isomaltosyl-oligosaccharides than that exhibited by the Arthrobacter enzyme; the activity of Actinomadura isomaltodextranase on the α-(1→4) linkage is 3-4 times greater than on the α-(1→6). These results indicate that, generically, the bacterial isomaltodextranase is a glycanase, whereas the actinomycetal enzyme is a glycosidase. This difference is reflected in the hydrolysis of dextrans, especially of dextran B-1355 (fraction S), which has a high content of unbranched α-(1→3) linked residues. In the digest of this dextran with Arthrobacter isomaltodextranse, short-chain fragments accumulated that were absent when the Actinomadura enzyme was employed.     

Pullulan 4-glucanohydrolase from Aspergillus niger

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6³-α-glucosylmaltotriose, 6²-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

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 [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]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 [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]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 [¹⁴C]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 C₃-OH on an acceptor dextran acts as a nucleophile on C₁ 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.     

Studies on fructosyl transferase from Agave americana

The possible role of fructosyl transferase in the biosynthesis of fructosans in Agave americana was investigated. This enzyme was extracted from A. americana stem and purified 17.5-fold by salt fractionation and DEAE-cellulose chromatography. The optimum conditions for the enzyme were pH 6. 1, temperature 37°, substrate concentration 20% and K_m 3.6 × 10^?1 M; Ag⁺, Pb ²⁺, Hg²⁺, Al³⁺, Sn²⁺, CN^? acted as inhibitors and Ca²⁺, Mg²⁺, Co²⁺ and Li⁺ actemd as activators. Only sugars of the type F ～ R (R-aidose), e.g. sucrose and raffinose acted as substrates for the enzyme. The donor acceptor specificity of the enzyme was studied extensively. Sugars sucrose. None of the intermediates of fructosan biosynthesis from sucrpse acted as fructose donors. The possible acceptors from sucrose and raffinose. The enzyme was capable of building up oligosaccharides up to FIOG from sucrose. None of the intermediates of fructosan biosynthesis from sucrose acted as fructose donors. The possible mechanism of fructosan biosynthesis from sucrose is discussed.     

Inverting character of α-glucuronidase A from Aspergillus tubingensis

α-Glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-α-1,2-D-xylopyranosyl-β-1,4-xylopyranosyl-β-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D₂O reaction mixture by ¹H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its β-anomer from which the α-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial α-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).     

Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-alpha-D-glucan from Streptococcus mutans serotype a

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.     

Purification and characterisation of 1F-fructosyltransferase from the roots of asparagus (asparagus officinalis L.)

A fructosyltransferase that transfers a terminal d-fructosyl group from a (2→1)-β-linked fructosaccharide to HO-1 of another d-fructosyl group has been purified from an extract of asparagus roots by successive chromatography with DEAE-cellulose, octyl-Sepharose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The disc-electrophoretically homogeneous enzyme was free from β-d-fructofuranosidase, sucrose:sucrose 1-fructosyltransferase, and 6^G-frutosyltransferase activity, and catalysed the d-fructosyl transfer from 1-kestose more rapidly to saccharides of the neokestose series [1^F(1-β-d-fructofuranosyl)_m-6^G(1-β-d-fructofuranosyl)_nsucrose] than to those of the 1-kestose series [1^F(1-β-d-fructofuranosyl)_nsucrose]. The enzyme was tentatively termed 1^F-fructosyltransferase. The general properties of the enzyme were as follows: mol. wt., ~64,000; optimum pH, ~5.0; stable at pH 5.0–5.5 at 45° for 20 min; stable at 30–45° for 10 min; inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

Purification and substrate specificity of an endo-dextranase of Streptococcus mutans K1-R

An extracellular endo-dextranase has been isolated from Streptococcus mutans K1-R. Incubation of cell-free culture fluid with sucrose permitted the removal of a large proportion of the extracellular d-glucosyltransferases by irreversible adsorption onto the insoluble glucans that these enzymes synthesize from sucrose. The remaining d-glucosyltransferases were separated from dextranase by precipitation with ammonium sulphate, chromatography on hydroxylapatite and DEAE-cellulose, followed by filtration on Ultrogel. The major products of action of the purified dextranase on (1→6)-α-d-glucans were isomaltotriose (IM₃), isomaltotetraose (IM₄), and isomaltopentaose (IM₅). Further hydrolysis of IM₄ and IM₅ occurred after prolonged incubation with excess of enzyme, to give d-glucose, IM₂, and IM₃. The relative rate of hydrolysis of isomaltose saccharides fell sharply with decreasing chainlength from IM₁₂ to IM₅. The hydrolysis of dextrans containing 96% or more of (1→6)-α-d-glucosidic linkages, expressed as apparent conversion into IM₃, was virtually complete, and substrates such as Streptococcus sanguis glucan, containing sequences of (1→6)-α-d-glucosidic linkages, were also effectively hydrolyzed. Dextranase activity towards the soluble glucan of Streptococcus mutans was limited, and there was no action on the insoluble glucan synthesized by S. mutans sucrose 3-d-glucosyltransferase.     

Glucofructosan metabolism in Cichorium intybus roots

A 10-fold purification of sucrose sucrose fructosyl transferase from Cichorium intybus roots was achieved by ammonium sulphate fractionation and DEAE-cellulose column chromatography. The energy of activation for this enzyme was ca 48 kJ/mol sucrose. Sucrose sucrose fructosyl transferase and invertase were prominent during early months of growth. Evidence obtained from: (1) the changes in carbohydrate composition at monthly intervals; (2) comparative studies on fructosyl transferase and invertase at different stages of root growth; and (3) incubation studies with [¹⁴C]glucose, [¹⁴C]fructose and [¹⁴C]sucrose revealed that, during the later stages of root growth, fructosan hydrolase is responsible for fructosan hydrolysis. No evidence for the direct transfer of fructose from sucrose to high M_r glucofructosans was obtained.     

Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically ¹³C-labeled d-glucose, 2-O-(α-d-glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α-d-gluco-pyranoside (glucosylglycerol), and α-d-glueopyranosyl β-d-fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L^-1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α-d-glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d-glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [¹³C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.     

Occurrence of an oxalate oxidase in Sorghum leaves

An oxalate oxidase found in the 15 000 g supernatant of 10-day-old sorghum leaves exhibited a pH optimum of 5 and a temperature optimum of 45° and was unaffected by Na⁺. The enzyme activity remained linear up to 10 min and the apparent K_m for oxalate was 2.4 × 10^?5 M. The enzyme activity was strongly inhibited by sodium dithionite and α,α′-dipyridyl. Inhibition by the latter was specifically reversed by Fe²⁺. The activity of the dialysed enzyme was restored by the addition of Fe²⁺ and FAD. Inhibition of the enzyme by iodoacetate, p-chloromercuribenzoate and N-methylmaleimide revealed that SH groups at the active site are essential.     

Acid invertase in germinating Lactuca sativa seeds: Evidence for de novo synthesis

Acid invertase activity in germinating lettuce seeds is first observed after 15 hr germination, from when it rises steadily at least till 30 hr of germination. The enzyme was purified about 500-fold using ammonium sulphate fractionation followed by isoelectric focussing. Labelling the enzyme with ³⁵SO₄ or leucine-¹⁴C during development of its activity, followed by purification suggests that acid invertase is synthesized de novo during germination. The possible significance of acid invertase in the metabolism of the seed is discussed.     

Dextransucrase and the mechanism for dextran biosynthesis

Remaud-Simeon and co-workers [Moulis, C.; Joucla, G.; Harrison, D.; Fabre, E.; Potocki-Veronese, G.; Monsan, P.; Remaud-Simeon, M. J. Biol. Chem., 2006, 281, 31254-31267] have recently proposed that a truncated Escherichia coli recombinant B-512F dextransucrase uses sucrose and the hydrolysis product of sucrose, d-glucose, as initiator primers for the nonreducing-end synthesis of dextran. Using ¹⁴C-labeled d-glucose in a dextransucrase-sucrose digest, it was found that <0.02% of the d-glucose appears in a dextran of M_n 84,420, showing that d-glucose is not an initiator primer, and when the dextran was treated with 0.01 M HCl at 80 °C for 90 min and a separate sample with invertase at 50 °C for 24 h, no d-fructose was formed, indicating that sucrose is not present at the reducing-end of dextran, showing that sucrose also was not an initiator primer. It is further shown that both d-glucose and dextran are covalently attached to B-512FMC dextransucrase at the active site during polymerization. A pulse reaction with [¹⁴C]-sucrose and a chase reaction with nonlabeled sucrose, followed by dextran isolation, reduction, and acid hydrolysis, gave ¹⁴C-glucitol in the pulsed dextran, which was significantly decreased in the chased dextran, showing that the d-glucose moieties of sucrose are added to the reducing-ends of the covalently linked growing dextran chains. The molecular size of dextran is shown to be inversely proportional to the concentration of the enzyme, indicating a highly processive mechanism in which d-glucose is rapidly added to the reducing-ends of the growing chains, which are extruded from the active site of dextransucrase. It is also shown how the three conserved amino acids (Asp551, Glu589, and Asp 622) at the active sites of glucansucrases participate in the polymerization of dextran and related glucans from a single active site by the addition of the d-glucose moiety of sucrose to the reducing-ends of the covalently linked glucan chains in a two catalytic-site, insertion mechanism.     

Synthesis of dextrans with controlled amounts of α-1,2 linkages using the transglucosidase GBD–CD2

GBD–CD2 is an α-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the α-1,2 transglucosylation of glucosyl moieties onto α-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD–CD2 follows a Ping Pong Bi Bi mechanism with a high k _cat value of 970 s⁻¹. The amount of the synthesised α-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled α-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of α-1,2 linkages ever reported.     

Synthesis and properties of some O-(2,2-dialkoxyethyl)glycolaldehydes

β-d-Mannosidase (β-d-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β-d-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α-d-mannosidase, β-d-galactosidase, 2-acet-amido-2-deoxy-d-glucosidase, (1→4)-β-d-mannanase, and (1→4)-β-d-glucanase activities, almost devoid of α-d-galactosidase activity, and contaminated with <0.02% of β-d-glucosidase activity. The purified enzyme had the same K_m for borohydride-reduced β-d-manno-oligosaccharides of d.p. 3–5 (12.5mm). The initial rate of hydrolysis of (1→4)-linked β-d-manno-oligosaccharides of d.p. 2–5 and of reduced β-d-manno-oligosaccharides of d.p. 3–5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β-d-mannopyranosides were readily hydrolysed. β-d-Mannobiose was hydrolysed at a rate ～25 times that of 6¹-α-d-galactosyl-β-d-mannobiose and 6³-α-d-galactosyl-β-d-mannotetraose, and at ～90 times the rate for β-d-mannobi-itol.     

Studies on dextranase from Penicillium aculeatum

A strain of Penicillium aculeatum has been found to synthesize large quantities of dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) in culture filtrate. Some of the conditions governing the enzyme production have been standardized. The enzyme in crude state was found to be highly stable, its activity being maximum at 50 to 60°C and at pH 5 to 6. About 90% of the substrate dextran was converted to isomaltose in a 4 h period at 40°C. The enzyme when purified by salt and solvent fractionation gave 1500 units per mg protein and retained its activity over a long period when stored at 4°C.