Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35 degrees C and the enzyme exhibited stability up to 50 degrees C. The enzyme hydrolyzed cycloalternan [CA; cyclo(-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(-->3)-alpha-d-Glcp-(1-->)] as the best substrate, to produce only isomaltose via an intermediate, alpha-isomaltosyl-(1-->3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates, such as panose, alpha-isomaltosyl-(1-->4)-maltooligosaccharides, alpha-isomaltosyl-(1-->3)-glucose, and alpha-isomaltosyl-(1-->3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1-->4)- or (1-->3)-linkages. The K(m) value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4s(-1)) was not significantly different from that of cycloalternan (10.8 s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.     

Cyclic tetrasaccharide-synthesizing enzymes from Arthrobacter globiformis A19

A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil. The enzymes, 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity. The molecular and enzymatic properties of IMT from A. globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75. Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B. globisporus. The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A. globiformis A19. The two genes linked together in tandem and formed a gene cluster, ctsYZ. Both of the gene products showed similarities to alpha-glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes. The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria.     

A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195

Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein. The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end. CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-(1-->4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan. Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch.     

Enzymatic synthesis of two novel non-reducing oligosaccharides using transfructosylation activity with β-fructofuranosidase from Arthrobacter globiformis

Two novel non-reducing oligosaccharides together with tri- and tetra-saccharides were synthesized by transfructosylation activity from sucrose as a donor and cellobiose or cellotriose as an acceptor with a purified beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and these oligosaccharides were identified as O-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside and O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside by spectrometric analyses. Both oligosaccharides were stable under condition at 100 degrees C for 30 min, and showed no degradation at pH 2.     

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.     

Purification, characterization, and gene cloning of a novel maltosyltransferase from an Arthrobacter globiformis strain that produces an alternating alpha-1,4- and alpha-1,6-cyclic tetrasaccharide from starch

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50 degrees C and was stable from pH 5.0 to 9.0 and up to 30 degrees C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45 degrees C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of > or =3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose --> 6(4)-O-alpha-maltosyl-maltotetraose + maltose and (ii) 6(4)-O-alpha-maltosyl-maltotetraose --> CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular alpha-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to alpha-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.     

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) seedlings

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.     

Alternansucrase acceptor reactions with methyl hexopyranosides

Alternansucrase (EC 2.4.1.140, sucrose: (1-->6), (1-->3)-alpha-D-glucan 6(3)-alpha-D-glucosyltransferase) is a D-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked D-glucan from sucrose. It also synthesizes oligosaccharides via D-glucopyranosyl transfer to various acceptor sugars. We have studied the acceptor products arising from methyl glycosides as model compounds in order to better understand the specificity of alternansucrase acceptor reactions. The initial product arising from methyl beta-D-glucopyranoside was methyl beta-isomaltoside, which was subsequently glucosylated to yield methyl beta-isomaltotrioside and methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside. These products are analogous to those previously described from methyl alpha-D-glucopyranoside. The major initial acceptor product from methyl alpha-D-mannopyranoside was methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranoside, but several minor products were also isolated and characterized, including a 3,6-di-O-substituted mannopyranoside. Methyl alpha-D-galactopyranoside yielded two initial products, methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-galactopyranoside and methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside, in a 2.5:1 molar ratio. Methyl D-allopyranosides were glucosylated primarily at position 6, yielding methyl alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. The latter subsequently gave rise to methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. In general, the methyl alpha-D-hexopyranosides were better acceptors than the corresponding beta-glycosides.     

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Enzymatic characterization and substrate specificity of thermostable beta-glycosidase from hyperthermophilic archaea, Sulfolobus shibatae, expressed in E. coli

Enzymatic properties and substrate specificity of recombinant beta-glycosidases from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at 95 degrees C and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at 75 degrees C was 15 h whereas it drastically decreased to 3.9 min at 95 degrees C. The addition of 10 mM of MnCl2 enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG. rSSG apparently preferred laminaribiose (beta1-->3Glc), followed by sophorose (beta1-->2Glc), gentiobiose (beta1-->6Glc), and cellobiose (beta1--4Glc). Various intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.     

Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Le x and relevant compounds

From the beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (1) prepared by the transglycosylation of beta-galactosidase from Bacillus circulans, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (9) and alpha-D-Neu5Ac-(2-->6)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (10) were effectively synthesized with an equimolar ratio of CMP-Neu5Ac by recombinant rat alpha-(2-->3)-N-sialyltransferase and rat liver alpha-(2-->6)-N-sialyltransferase, respectively. The former enzyme also transferred effectively the Neu5Ac residue from CMP-Neu5Ac to the location of OH-3 in the non-reducing terminal of beta-D-Gal-(1-->4)-beta-D-Gal-OC6H4NO2-p or beta-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p, while the latter enzyme did not. In the case of equimolar ratio of GDP-Fuc/acceptor, 1 and 9 were further fucosylated quantitatively to form beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (14) and alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (13) by recombinant human alpha-(1-->3)-fucosyltransferase VII, respectively.     

The hydrolytic and transferase action of alternanase on oligosaccharides.

Alternanase is an enzyme which endo-hydrolytically cleaves the alpha-(1-->3), alpha-(1-->6)-linked D-glucan, alternan. The main products are isomaltose, alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc and the cyclic tetrasaccharide cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. It is also capable of acting on oligosaccharide substrates. The cyclic tetrasaccharide is slowly hydrolyzed to isomaltose. Panose and the trisaccharide alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Glc both undergo transglycosylation reactions to give rise to the cyclic tetrasaccharide plus D-glucose, with panose being converted at a much faster rate. The tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc is hydrolyzed to D-glucose plus the trisaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc. Alternanase does not act on isomaltotriose, theanderose (6(Glc)-O-alpha-D-Glcp sucrose), or alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glc. The enzyme releases 4-nitrophenol from 4-nitrophenyl alpha-isomaltoside, but not from 4-nitrophenyl alpha-D-glucopyranoside, 4-nitrophenyl alpha-isomaltotrioside, or 4-nitrophenyl alpha-isomaltotetraoside.     

Isolation and structural analysis of ajugose from Vigna mungo L

The hexasaccharide ajugose, alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside, generally uncommon in legumes, was detected in the seeds of Vigna mungo L. by TLC and paper chromatography. Ajugose was then isolated by silica gel chromatography and its structure was established by acid and enzymatic hydrolysis, fast atom bombardment mass spectrometry and both one- and two-dimensional 1H and 13C NMR techniques.     

Purification and characterization of branching specificity of a novel extracellular amylolytic enzyme from marine hyperthermophilic Rhodothermus marinus

An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.     

alpha-Mannosidase-catalyzed synthesis of a (1-->2)-alpha-D-rhamnodisaccharide derivative

Enzymatic transglycosylation using p-nitrophenyl alpha-D-rhamnopyranoside as the glycosyl donor and 6equiv of ethyl 1-thio-alpha-D-rhamnopyranoside as the glycosyl acceptor yielded a D-rhamnooligosaccharide derivative. The reaction was catalyzed by jack bean alpha-mannosidase in a 1:1 (v/v) mixture of 0.1 M sodium citrate buffer (pH4.5)-MeCN at 25 degrees C. The enzyme exhibited high catalytic activity for the reaction, to afford in 32.1% isolated yield (based on donor substrate) ethyl alpha-D-rhamnopyranosyl-(1-->2)-1-thio-alpha-D-rhamnopyranoside, which is a derivative of the common oligosaccharide unit of the antigenic lipopolysaccharides from Pseudomonas.     

Catalytic properties and mode of action of endo-(1-->3)-beta-D-glucanase and beta-D-glucosidase from the marine mollusk Littorina kurila

A complex of the enzymes from the liver of the marine mollusk Littorina kurila that hydrolyzes laminaran was investigated. Two (1-->3)-beta-d-glucanases (G-I and G-II) were isolated. The molecular mass of G-I as estimated by gel-permeation chromatography and SDS-PAGE analysis was 32 and 40kDa, respectively. The G-II molecular mass according to SDS-PAGE analysis was about 200kDa. The pH optimum for both G-I and G-II was pH 5.4. The G-I had narrow substrate specificity and hydrolyzed only the (1-->3)-beta-d-glucosidic bonds in the mixed (1-->3),(1-->6)- and (1-->3),(1-->4)-beta-d-glucans down to glucose and glucooligosaccharides. This enzyme acted with retention of the anomeric configuration and catalyzed a transglycosylation reaction. G-I was classified as the glucan endo-(1-->3)-beta-d-glucosidase (EC 3.2.1.39). G-II exhibited both exo-glucanase and beta-d-glucoside activities. This enzyme released from the laminaran glucose as a single product, but retained the anomeric center configuration and possessed transglycosylation activity. The hydrolysis rate of glucooligosaccharides by G-I decreased with an increase of the substrate's degree of polymerization. In addition to (1-->3)-beta-d-glucanase activity, the enzyme had the ability to hydrolyze p-nitrophenyl beta-d-glucoside and beta-d-glucobioses: laminaribiose, gentiobiose, and cellobiose, with the rate ratio of 50:12:1. G-II may correspond to beta-d-glucoside glucohydrolase (EC 3.2.1.21).     

Kinetic investigation of a new inhibitor for human salivary alpha-amylase

This study is the first report on the effectiveness and specificity of alpha-acarviosinyl-(1-->4)-alpha-D-glucopyranosyl-(1-->6)-D-glucopyranosylidene-spiro-thiohydantoin (PTS-G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosyl-maltoside (GalG2CNP) and amylose hydrolysis catalysed by human salivary alpha-amylase (HSA). Synthesis of PTS-G-TH was carried out by transglycosylation using acarbose as donor and glucopyranosylidene-spiro-thiohydantoin (G-TH) as acceptor. This new compound was found to be a much more efficient HSA inhibitor than G-TH. The inhibition is a mixed-noncompetitive type on both substrates and only one molecule of inhibitor binds to the enzyme. Kinetic constants calculated from secondary plots are in micromolar range. Values of K(EI) and K(ESI) are very similar in the presence of GalG2CNP substrate; 0.19 and 0.24 microM, respectively. Significant difference can be found for K(EI) and K(ESI) using amylose as substrate; 8.45 and 0.5 microM, respectively. These values indicate that inhibition is rather uncompetitive than competitive related to amylose hydrolysis.     

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

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.     

Purification and characterization of a beta-glucuronidase from Aspergillus niger.

A beta-glucuronidase from Pectinex Ultra SP-L, a commercial pectolytic enzyme preparation from Aspergillus niger, was purified 170-fold by ion-exchange chromatography and gel filtration. Apparent M(r) of the purified enzyme, estimated by denaturing gel electrophoresis and size-exclusion chromatography, were 68,000 and 71,000, respectively, indicating that the enzyme is a monomeric protein. It released uronic acids not only from p-nitrophenyl beta-glucosiduronic acid (PNP-GlcA) but also from acidic galactooligosaccharides carrying either beta-D-glucosyluronic or 4-O-methyl-beta-D-glucosyluronic residues at the nonreducing termini through beta-(1-->6)-glycosidic linkages. The enzyme exhibited a maximal activity toward these substrates at pH 3.0. A regioisomer, 3-O-beta-glucosyluronic acid-galactose, was unsusceptible to the enzyme. The enzyme did act on a polymer substrate, releasing uronic acid from the carbohydrate portion of a radish arabinogalactan-protein modified by treatment with fungal alpha-L-arabinofuranosidase. The enzyme produced acidic oligosaccharides by transglycosylation, catalyzing the transfer of uronic acid residues of PNP-GlcA and 6-O-beta-glucosyluronic acid-galactose to certain exogenous acceptor sugars such as Gal, N-acetylgalactosamine, Glc, and xylose.