Highly hydrolytic reuteransucrase from probiotic Lactobacillus reuteri strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the alpha-(1-->4) glucosidic type ( approximately 70%). This reuteran also contains alpha-(1-->6)- linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing alpha-(1-->4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of alpha-(1-->4) linkages reported to date.     

Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the α-(1→4) glucosidic type. The glucan also contains α-(1→6)-linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M_r of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.     

4,6-α-glucanotransferase, a novel enzyme that structurally and functionally provides an evolutionary link between glycoside hydrolase enzyme families 13 and 70

Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-d-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional ¹H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (β/α)₈ barrel that use sucrose to synthesize α-d-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.     

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu⁹⁴⁰ in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu⁹⁴⁰ of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu⁹⁴⁰ mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu⁹⁴⁰ in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.     

Substrate Activation of beta-(1 --> 3) Glucan Synthetase and Its Effect on the Structure of beta-Glucan Obtained from UDP-d-glucose and Particulate Enzyme of Oat Coleoptiles

UDP-d-glucose, at a micromolar level in the presence of MgCl₂ and oat (Avena sativa) coleoptile particulate enzyme which contains both β-(1 → 3) and β-(1 → 4) glucan synthetases, produces glucan with mainly β-(1 → 4) glucosyl linkages. An activation of β-(1 → 3) glucan synthetase by UDP-d-glucose and a decrease in the formation of β-(1 → 3) glucan in the presence of MgCl₂ have been observed. However, at high substrate concentration (≥ 10⁻⁴m), the activation of β-(1 → 3) glucan synthetase is so pronounced that the formation of β-(1 → 3) glucosyl linkage predominates in synthesized glucan regardless of the presence of MgCl₂. These observations may explain the striking shift in the composition of glucan of particulate enzyme from a β-(1 → 4) to β-(1 → 3) glucosyl linkage when UDP-d-glucose concentration is raised from a low concentration (≤ 10⁻⁵m) to a higher concentration (≥ 10⁻⁴m).     

Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10⁷) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Cell Walls of Germinating Uredospores: II. Carbohydrate Polymers

Polymeric carbohydrates in ¹⁴C-labeled germ tube and uredospore walls of Uromyces phaseoli var. typica were studied by permethylation and by enzymatic hydrolysis. The native structure of the uredospore wall limited the effectiveness of both techniques with this wall, but evidence for two distinct polysaccharides was obtained. A linear (1→3) glucan, containing minor quantities of (1→6) linkages, may account for most of the glucose in the uredospore wall. A second uredospore polymer was a glucomannan similar to one reported for other rust fungi in that it consisted of approximately equal numbers of β(1→3) and β(1→4) mannosidic linkages with glucose as a minor component at the nonreducing end. Branching, most likely by (1→6) mannose links, was low. In contrast to uredospore wall, considerably more germ tube polysaccharide was accessible to enzymes and to methylation. Methylation studies indicate that (1→3) glucose and mannose bonds occur predominantly. Evidence from hydrolysis with exo- (β)-(1→3) glucanase suggests distinct wall regions of β(1→3) glycan, highly branched by (1→6) bonds, as well as wall regions of a glucomannan with alternating (1→3) glucose and (1→3) mannose residues. Polymer heterogeneity was indicated by differences in the proportions of mannose, glucose, and galactose as reducing end groups in different solubility fractions. In germ tube walls, but not in uredospore walls, glucosamine apparently existed as part of chitin polymer as evidenced by the isolation of N,N-diacetylchitobiose from chitinase digestion.     

The Formation of beta, 1 --> 4 Glucan from UDP-alpha-d-Glucose Catalyzed by a Phaseolus aureus Enzyme

Particulate enzyme preparations from Phaseolus aureus hypocotyls catalyze the formation of an alkali insoluble β, 1 → 4 linked [¹⁴C]-glucan using UDP-α-d [¹⁴C]-glucose as substrate. Particulate enzymes prepared from root tissue also catalyzed the production of β, 1 → 4 glucan. UDP-β-d-[¹⁴C]-glucose would not serve as a substrate for these enzymes. The presence or absence of β, 1 → 4 glucan synthetase activity was independent of tissue source, substrate concentration, or homogenization method.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

DEMONSTRATION OF A FIBRILLAR COMPONENT IN THE CELL WALL OF THE YEAST SACCHAROMYCES CEREVISIAE AND ITS CHEMICAL NATURE

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1 → 3)-glucanase and endo-β-(1 → 6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1 → 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1 → 3)- or endo-β-(1 → 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1 → 3)-glucanase it can be concluded that this fibrillar component is composed of β-(1 → 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.     

Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by Leuconostoc mesenteroides NRRL B-1299

When grown in glucose or fructose medium in the absence of sucrose, Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.     

A STUDY OF SIEVE ELEMENT STARCH USING SEQUENTIAL ENZYMATIC DIGESTION AND ELECTRON MICROSCOPY

The fine structure of plastids and their starch deposits in differentiating sieve elements was studied in bean (Phaseolus vulgaris L.). Ultrastructural cytochemistry employing two carbohydrases specific for different linkages was then used to compare the chemical nature of "sieve tube starch" (the starch deposited in sieve elements) with that of the ordinary starch of other cell types. Hypocotyl tissue from seedlings was fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon-Araldite. Treatment of thin sections on uncoated copper grids with α-amylase or diastase at pH 6.8 to cleave α-(1 → 4) bonds resulted in digestion of ordinary starch grains but not sieve element grains, as determined by electron microscopy. Since α-(1 → 6) branch points in amylopectin-type starches make the adjacent α-(1 → 4) linkages somewhat resistant to hydrolysis by α-amylase, other sections mounted on bare copper or gold grids were treated with pullulanase (a bacterial α-[1 → 6] glucosidase) prior to digestion with diastase. Pullulanase did not digest sieve element starch, but rendered the starch digestible subsequently by α-amylase. Diastase followed by pullulanase did not result in digestion. The results provide evidence that sieve element starch is composed of highly branched molecules with numerous α-(1 → 6) linkages.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of alpha-(1-->6)-glucosidase, its separation from transglucosylase, and the action of the two enzymes on branched oligosaccharides

1. An α-(1→6)-glucosidase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by adsorption of the latter on retrograded amylose. 2. The enzyme was detected in five of the six strains of S. mitis that were studied; α-(1→6)-glucosidase was not found in strain RB1633, a strain that did not store polysaccharide. 3. The glucosidase could act on compounds in which α-glucose is joined through an α-(1→6)-bond to either a maltosaccharide or an isomaltosaccharide. 6²-α-Glucosylmaltose (panose) and 6³-α-glucosylmaltotriose were hydrolysed more rapidly and isomaltodextrins more slowly than isomaltose. 4. Transferring activity towards isomaltose and panose was appreciable when the concentration of substrate was 2% or higher. 5. The enzyme had no action on α-(1→4)-glucosidic linkages. 6-α-Maltodextrinylglucoses were hydrolysed only after transglucosylase action had attenuated them to isomaltose.     

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GSand FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)₈ barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.     

Hydrolytic Activity and Substrate Specificity of an Endoglucanase from Zea mays Seedling Cell Walls

An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1→3),(1→4)-β-d-glucosyl sequences. Substrates with (1→4)-β-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1→3)-β-glucans such as laminarin were not hydrolyzed. When (1→3),(1→4)-β-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 × 10⁴). Activity against a water soluble (1→3),(1→4)-β-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1→3),(1→4)-β-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1→3),(1→4)-β-d-glucan that contained blocks of (1→4) linkages and/or more than a single contiguous (1→3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1→3),(1→4)-β-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase.     

Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron

Probiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) α-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for β-fructans and α-glucans. Reduced metabolism of reuteran and IMMP α-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.     

Tissue Slice and Particulate beta-Glucan Synthetase Activities from Pisum Epicotyls

β-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg²⁺ optimum, apparent K_m) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various β-glucanases all show that both β-1,3 and β-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. β-1,4 Linkages predominate at low substrate (5 μm) concentration. Kinetic data indicate that the capacity to synthesize β-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.     

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.     

Glucosyltransferase Mutants of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides NRRL B-1355 produces dextrans and alternan from sucrose. Alternan is an unusual dextran-like polymer containing alternating α(1→6)/α(1→3) glucosidic bonds. Cultures were mutagenized with UV and ethyl methanesulfonate, and colony morphology mutants were selected on 10% sucrose plates. Colony morphology variants exhibited changes from parent cultures in the production of one or more glucosyltransferases (GTFs) and glucans. Mutants were characterized by measuring resistance of glucan products to dextranase digestion, by electrophoresis, and by high-pressure liquid chromatography of maltose acceptor products generated from sucrose-maltose mixtures. Some mutants produced almost pure fraction L dextran, and cultures exhibited a single principal GTF band on sodium dodecyl sulfate-acrylamide gels. Other mutants produced glucans enriched for alternan. Colony morphology characteristics (size, smoothness, and opacity) and liquid culture properties (clumpiness, color, and viscosity in 10% sucrose medium) were explained on the basis of GTF production. Three principal GTF bands were detected.     

A New Way of Producing Isomalto-Oligosaccharide Syrup by Using the Transglycosylation Reaction of Neopullulanase

A new way of producing isomalto-oligosaccharide syrup from starch was developed. Isomalto-oligosaccharides contain one or more α-(1→6)-glucosidic linkages with or without α-(1→4)-glucosidic linkages. The isomalto-oligosaccharide syrups are receiving increased attention as food additives because it is thought that they help prevent dental caries and improve human intestinal microflora, acting as a growth factor for bifidobacteria. The new system for production of isomalto-oligosaccharide syrup is based on the strong α-(1→6)-transglycosylation reaction of neopullulanase. Bacillus subtilis saccharifying α-amylase was simultaneously used with neopullulanase to improve the yield of isomalto-oligosaccharides. The yield of isomalto-oligosaccharides was increased to more than 60%, compared with a yield of 45.0% obtained by the conventional system. To reduce the costs, the use of immobilized neopullulanase was investigated. Almost the same yield of isomalto-oligosaccharides was obtained by using immobilized neopullulanase.