NRRL B-512F Research Articles

Mathematical modeling characterization of mannitol production by three heterofermentative lactic acid bacteria

This study developed a non-structured mathematical model for describing the effect of carbon sources and culture pH on the mannitol production by three heterofermentative lactic acid bacteria (LAB), including Leuconostoc mesenteroides NRRL B-512F and NRRL B-523 as well as Lactobacillus fermentum NRRL B-1840. Mannitol production was studied in batch culture using four culture media containing the same carbon sources (glucose and fructose, but different glucose concentration) and different nitrogen sources (yeast extract, peptone, and meat extract) with diverse concentrations. The results obtained in the early stages of the estimation algorithm suggested that cell growth on fructose could be neglected. Therefore, the initially proposed mathematical model was redefined and subsequently confirmed. The strains NRRL B-512 F, NRRL B-523, and NRRL B-1840 reached the highest volumetric productivities (2.26 ± 0.05, 2.30 ± 0.05, and 1.95 ± 0.05 g L−1h−1; respectively) and mannitol yields (0.95 ± 0.01, 0.98 ± 0.04, and 1.00 ± 0.01 mol mannitol mol initial fructose−1; respectively) in medium A. The mannitol and biomass yields suggested a higher carbon concentration favored mannitol production and growth, but a lower nitrogen concentration directly impacted both yields. Mannitol production was associated with cell growth according to the estimated parameters.

Low molecular weight dextran production by Leuconostoc mesenteroides strains: Optimization of a new culture medium and the rheological assessments

Strains belonging to genus Leuoconostoc mesenteroides are able to produce dextran exo-polysaccharide. In this study, MRS broth and milk permeate were used for growth of three strains (NRRL B-512F, NCIB 8023 and NRRL B-512) of L. mesenteroides and the growth rate, dextran production, and its rheological properties were studied. Evaluation of molecular weight by high-performance size exclusion chromatography system showed that dextran produced in milk permeate had a lower molecular weight (<10000 Dalton) compared to the dextran produced in MRS broth (<40000 Dalton). The viscosity of dextran solution (2%) produced in MRS was 0.005–0.01 Pa.s while in the synthetic medium it was <0.005 Pa.s. Also in our study maximum ratio of growth factor and dextran yield belonged to NRRL B-512F, which was subjected to optimization of fermentation condition for dextran production. For optimization, response surface methodology by using sucrose, milk permeate and yeast extract in 5 levels were used. According to experiments, optimum concentrations for sucrose, milk permeate, and yeast extract were found to be 20, 15, and 15 (g/l), respectively.

Optimization of the expression of levansucrase L17 in recombinant E. coli

ABSTRACTLevansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose.

Efficient Production of Prebiotic Gluco-oligosaccharides in Orange Juice Using Immobilized and Co-immobilized Dextransucrase.

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was subjected to immobilization and co-immobilization with dextranase from Chaetomium erraticum. Immobilization has enhanced the operational and storage stability of dextransucrase. Two hundred milligrammes (2.4IU/mg) of alginate beads (immobilized and co-immobilized) were found to be optimum for the production of gluco-oligosaccharides (GOS) in orange juice with a high degree of polymerization. The pulp of the orange juice did not interfere in the reaction. In the batch process, co-immobilized dextransucrase (41g/L) produced a significantly higher amount of GOS than immobilized dextransucrase (37g/L). Alginate entrapment enhanced the thermal stability of dextransucrase for up to 3days in orange juice at 30°C. The production of GOS in semi-continuous process was 39g/L in co-immobilized dextransucrase and 33g/L in immobilized dextransucrase. Thus, immobilization technology offers a great scope in terms of reusability and efficient production of a value added functional health drink.

Combinatorial Engineering of Dextransucrase Specificity

We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.104 clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide 460DYVHT464 in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity.

Cashew juice containing prebiotic oligosaccharides.

The enzyme dextransucrase in a medium containing sucrose and an acceptor as substrate synthesizes prebiotics oligosaccharides. The cashew apple juice works as a source of acceptors because it is rich in glucose and fructose (enzyme acceptors). The use of cashew apple juice becomes interesting because it aims at harnessing the peduncle of the cashew that is wasted during the nut processing, which is the product of greater economic expression. The production of dextransucrase enzyme was done by fermentative process by inoculating the bacterium Leuconostoc mesenteroides NRRL B512F into a culture medium containing sucrose as the only carbon source. Thus, the aim of this work was the production of prebiotic oligosaccharides by enzymatic process with addition of the dextransucrase enzyme to the clarified cashew apple juice. Dextran yield was favored by the combination of low concentrations of sucrose and reducing sugars. The formation of oligosaccharides was favored by increasing the concentration of reducing sugars and by the combination of high concentrations of sucrose and reducing sugars, the highest concentration of oligosaccharides obtained was 104.73g/L and the qualitative analysis showed that at concentrations of 25g/L and 75g/L of sucrose and reducing sugar, respectively, it is possible to obtain oligosaccharides of degree of polymerization up to 12. The juice containing prebiotic oligosaccharide is a potential new functional beverage.

Structure and Property Engineering of α-d-Glucans Synthesized by Dextransucrase Mutants

Seven dextran types, displaying from 3 to 20% α(1→3) glycosidic linkages, were synthesized in vitro from sucrose by mutants of dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, obtained by combinatorial engineering. The structural and physicochemical properties of these original biopolymers were characterized. When asymmetrical flow field flow fractionation coupled with multiangle laser light scattering was used, it was determined that weight average molar masses and radii of gyration ranged from 0.76 to 6.02 × 10(8) g·mol(-1) and from 55 to 206 nm, respectively. The ν(G) values reveal that dextrans Gcn6 and Gcn7, which contain 15 and 20% α(1→3) linkages, are highly branched and contain long ramifications, while Gcn1 is rather linear with only 3% α(1→3) linkages. Others display intermediate molecular structures. Rheological investigation shows that all of these polymers present a classical non-Newtonian pseudoplastic behavior. However, Gcn_DvΔ4N, Gcn2, Gcn3, and Gcn7 form weak gels, while others display a viscoelastic behavior that is typical of entangled polymer solutions. Finally, glass transition temperature T(g) was measured by differential scanning calorimetry. Interestingly, the T(g) of Gcn1 and Gcn5 are equal to 19.0 and 29.8 °C, respectively. Because of this low T(g), these two original dextrans are able to form rubber and flexible films at ambient temperature without any plasticizer addition. The mechanical parameters determined for Gcn1 films from tensile tests are very promising in comparison to the films obtained with other polysaccharides extracted from plants, algae or microbial fermentation. These results lead the way to using these dextrans as innovative biosourced materials.

OPTIMIZATION OF ENZYMATIC SYNTHESIS OF ISOMALTO-OLIGOSACCHARIDES PRODUCTION

Glucosyltransferases can be applied in the synthesis of prebiotic oligosaccharides. Enzymatic synthesis using acceptors can be used to obtain these carbohydrates. When maltose is the acceptor, oligosaccharides containing one maltose moiety and up to eight glucose units linked by α-1,6-glycosidic bonds are obtained as the product of dextransucrase acceptor reaction. In this work, the enzymatic synthesis of isomalto-oligosaccharides using dextransucrase from Leuconostoc mesenteroides NRRL B-512F was optimized by response surface methodology. The effect of maltose and sucrose concentrations on the acceptor reaction was evaluated in a batch reactor system. Partially purified enzyme was used to reduce the enzyme purification cost. The results showed that high sucrose concentrations in conjunction with high maltose levels enhanced the isomalto-oligosaccharide synthesis. A productivity of 42.95 mmol/L.h of isomalto-oligosaccharides was obtained at the optimal operating condition (100 mmol/L of sucrose and 200 mmol/L of maltose). PRATICAL APPLICATIONS Oligosaccharides as prebiotic have a large application in food formulations, and their beneficial role in human health have been extensively studied. Although the acceptor mechanism of dextransucrase has already been extensively studied, an industrial process has not been developed yet for enzyme synthesis of isomalto-oligosaccharide. The process studied in this work allows the large-scale preparation of isomalto-oligosaccharide using partially purified enzyme.

Kinetic behaviour and specificity of β-fructosidases in the hydrolysis of plant and microbial fructans

Fructosidases, in particular exo-β-fructosidases, may act on fructans such as inulins and levans of plant and bacterial origin to produce fructose. In this paper, the kinetic properties of a commercial preparation (Fructozyme L) and a recombinant exoinulinase (BfrA) from Thermotoga maritima, were studied using fructan polymer substrates from various sources. Both enzymatic preparations preferentially hydrolyzed β2-1 linkages and low molecular weight fructans. We show that chicory inulin is degraded most efficiently by both preparations, followed by bacterial inulin, in spite of its high molecular weight and branching in β2-6 positions. All bacterial levans were more slowly hydrolyzed. Michaelis–Menten kinetics describe the hydrolysis of sucrose and low molecular weight fructans (≤8.3 kDa) by both enzyme preparations, while first order kinetics were observed with respect to bacterial fructans due to the high molecular weight and, therefore, low molar concentrations. Comparison of second order rate constants indicates that bacterial inulin ( Leuconostoc citreum CW28) is hydrolyzed more slowly with both enzyme preparations than chicory inulin by approximately one order of magnitude. For Leuconostoc mesenteroides NRRL B-512F levan, the second order rate constant for Fructozyme L is 200-fold lower than for chicory inulin. However, the second order rate constant for BfrA is only 22-fold lower than for chicory inulin. Taken together, our studies characterize the kinetics of fructan hydrolysis and also suggest that the kinetic parameters may be used to differentiate between fructan structures.

OPTIMIZATION OF CONDITIONS OFLEUCONOSTOC MESENTEROIDESNRRL B-640 FOR PRODUCTION OF A DEXTRANSUCRASE AND ITS ASSAY

ABSTRACT The optimum culture conditions for dextransucrase production from Leuconostoc mesenteroides NRRL B-640 were investigated. The dextransucrase production was studied in the temperature range, 22 to 30C under shaking and static flask conditions. The optimum temperature for dextransucrase production was 25C. The shaking flask culture gave higher enzyme activity by 20% than the static flask culture. The dextransucrase activity of L. mesenteroides NRRL B-640 was compared with L. mesenteroides NRRL B-512F. The results showed that the maximum dextransucrase activity achieved by L. mesenteroides NRRL B-640 at 25C under shaking flask culture, was 15% higher than that the enzyme activity given by L. mesenteroides NRRL B-512F grown at 23C under the static flask culture. The dextransucrase elaborated by L. mesenteroides NRRL B-640 displayed a maximum enzyme activity when assayed in the temperature range of 30 to 35C and at pH of 5.4. The enzyme was stable with in the 10–500 mM concentrations of sodium acetate buffer. PRACTICAL APPLICATIONS Dextransucrase synthesizes dextran that has several commercial applications in both its native and modified forms. Flocculation agent in waste-water treatment, blood volume expander, stabilizer in ice cream and a cryo-protective agent for cells are a few of dextran applications in its native form. It can serve as stabilizer in photographic, cosmetics and agriculture-based industries with generation of charged derivatives of dextran. Dextransucrases are reported to generate varieties of gluco-oligosaccharides that serve as prebiotics. Dextransucrases are reported in the glycoconjugation of several peptides for the development of vaccines, contraceptives and antibiotics. Dextran conjugates with iron and fluorophores are used in the clinical applications. Dextran hydrogels are used in pharmaceutical and biomedical applications such as contact lenses and in drug delivery. Looking at the wide applications of dextransucrase and growing demand there is a need for exploration of new strains for production of efficient and pure dextransucrase, producing better quality and yield of oligosaccharides and polysaccharides. The results of optimized conditions attain importance for large-scale production of dextransucrase.

Characterization of Glycansucrase Activities from Leuconostoc Mesenteroides LM17 and URE 13 Strains

ABSTRACT14 original strains from traditional Bulgarian fermented food have been studied in order to characterize new orginal glycosyltransferase activities In order to characterize the whole spectrum of functional glycosyltransferases in the studied strains, the EPS produced in liquid media where first purified and hydrolysed and the global enzymatic activities measured by DNS assay. Activities present in the culural supernatant and cell associated were determined using SD_SPAGE and in situ Periodic Acid Schiffs staining after incubation with sucrose and raffinose. Among the studied strains Lm17 and URE 13 strains shown interesting activities. Three polymers bands were detected during in situ EPS production in presence of sucrose associated respectively to ∼300 kD (only in URE 13) and 180 kDa and 120 kDa (in both Lm 17 and URE13) proteins The 180KDa band could correspond to a glucansucrase activity of these strains. Analysis of zymogram incubated in raffinose as substrate and hydrolysis of the EPS produced in liquid culture are in good correlation with the hypothesis of a fructosyltransferase activity that could be attributed to a 120 kDa extracellular and cell-associated protein‥ GTF specific motifs (YG repeats from the Glucan Binding Domain) from Lm 17 and URE 13 strains were amplified by PCR and sequenced. They share a high percentage of identity to each other, and to the YG-repeats from ATCC 8293 and NRRL B-512F. A conserved motif from the catalytic core of FTF genes from Lm 17 and URE 13 strains was also shown to be very conserved between these two strains and with sequences of levansucrase from Ln. mesenteroides NRRL B-512F.

One-step synthesis of isomalto-oligosaccharide syrups and dextrans of controlled size using engineered dextransucrase

Isomalto-oligosaccharides and dextrans of controlled molecular weight of about 10 and 40 kDa were produced using a simple one-step process using engineered L. mesenteroides NRRL B-512F dextransucrase variants. Isomalto-oligosaccharides were produced in a 58% yield by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units. Reaction conditions for optimal synthesis of dextrans of controlled molecular weight were defined, in respect of initial sucrose concentration and reaction temperature. Thus, we achieved synthesis with impressive yields of 69 and 75% for the 40 and 10 kDa dextran species, respectively. These two dextran sizes are particularly suitable for clinical applications, and are of great industrial demand. Compared with the traditional processes based on chemical hydrolysis and fractionation, which achieve only low yields, the new enzymatic methods offer improvement in quantity, quality and efficiency.

Alternansucrase acceptor products

The regioselectivity of alternansucrase (EC 2.4.1.140) differs from dextransucrase (EC 2.4.1.5) in ways that can be useful for the synthesis of novel oligosaccharide structures. For example, it has been recently shown that the major oligosaccharides produced when maltose is the acceptor include one trisaccharide structure, two tetrasaccharides, one pentasaccharide, two hexasaccharides, one heptasaccharide, and at least two octasaccharides, containing no adjacent α-(1→3) linkages and no more than two consecutive α-(1→6) linkages. This may shed some light on how the enzyme works to produce the alternating structure. Another characteristic of alternansucrase that distinguishes it from dextransucrase is its greater ability to use leucrose as an acceptor. Leucrose, produced by glucosyl transfer to fructose released from the initial sucrose substrate, represents a very poor substrate for Leuconostoc mesenteroides NRRL B-512F dextransucrase. Alternansucrase, however, continues to transfer glucosyl units to leucrose, resulting in some unusual glucosyl-fructose oligosaccharides.

Search for a dextransucrase minimal motif involved in dextran binding

Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C- or N- plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14 kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K d of 2.8 × 10 −9 M, this motif shows a very high affinity for isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl ® S300HR for rapid purification was evaluated and is discussed.

Identification of a single and non-essential cysteine residue in dextransucrase of Leuconostoc mesenteroides NRRL B-512F

Amino acid analysis of purified dextransucrase (sucrose: 1,6-α-D-glucan 6-α-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was carried out. The enzyme is virtually devoid of cysteine residue there being only one cysteine residue in the whole enzyme molecule comprising over 1500 amino acid residues. The enzyme is rich in acidic amino acid residues. The number of amino acid residues was calculated based on the molecular weight of 188,000 (Goyal and Katiyar 1994). Amino sugars were not found, implying that the enzyme is not a glycoprotein. It has been shown earlier that the cysteine residue in dextransucrase is not essential for enzyme activity (Goyal and Katiyar 1998). The presence of only one cysteine residue per enzyme molecule illustrates that its tertiary structure is solely dependent on other types of non-covalent interactions such as hydrogen bonding, ionic and nonpolar hydrophobic interactions.

Structural and functional features of fructansucrases present in Leuconostoc mesenteroides ATCC 8293

Glycosyltransferases produced by Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (equivalent to NRRL B-1118) were identified. Two glucansucrases and one fructansucrases were observed in batch culture while levC and levL genes, corresponding to two fructansucrases, were isolated from information obtained from the released draft sequence of this Leuconostoc strain genome and cloned in Escherichia coli. The recombinant enzymes were shown to be fructansucrases producing a polymer identified by NMR as levan, confirming our recent report stating that these are also mosaic levansucrases bearing structural features of glucansucrases in the amino and carboxy terminal regions, as is also the case of inulosucrase (IslA) from Leuconostoc citreum CW28 and levansucrase (LevS) from L. mesenteroides NRRL B-512F. The recombinant levansucrase LevC was purified and characterized in terms of pH, temperature, and kinetic properties. The enzyme exhibits Michaelis-Menten kinetic properties with a K(m) = 27.3 mM and a k(cat) = 282.9 s(-1). This levansucrase behaves mainly as a transferase as only 30% of the substrate is hydrolyzed in a wide range of sucrose concentrations, with higher hydrolytic activities at low substrate concentrations. With this report we experimentally confirm the unusual structural pattern displayed by fructansucrases present in Leuconostoc species that group as a novel sub family of fructansucrases.

Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various alpha-1,3 and/or alpha-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases.

Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents

The enzymatic glucosylation of luteolin was attempted using two glucansucrases: the dextransucrase from Leuconostoc mesenteroides NRRL B-512F and the alternansucrase from L. mesenteroides NRRL B-23192. Reactions were carried out in aqueous-organic solvents to improve luteolin solubility. A molar conversion of 44% was achieved after 24 h of reaction catalysed by dextransucrase from L. mesenteroides NRRL B-512F in a mixture of acetate buffer (70%)/bis(2-methoxyethyl) ether (30%). Two products were characterised by nuclear magnetic resonance (NMR) spectroscopy: luteolin-3′-O-α- d-glucopyranoside and luteolin-4′-O-α- d-glucopyranoside. In the presence of alternansucrase from L. mesenteroides NRRL B-23192, three additional products were obtained with a luteolin conversion of 8%. Both enzymes were also able to glucosylate quercetin and myricetin with conversion of 4% and 49%, respectively.

HIGH YIELD PURIFICATION OF DEXTRANSUCRASE FROM LEUCONOSTOC MESENTEROIDES NRRL B-512F BY PHASE PARTITIONING

The extracellular dextransucrase (sucrose: 1,6-α-D-glucan 6-α-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was purified by phase partitioning using poly(ethylene) glycol (PEG) and dextran generated by dextransucrase. Three steps of repeated phase partitioning by PEG 6000 and PEG 400 showed that the purification of dextransucrase by PEG 6000 was much greater than that obtained by PEG 400. Both the specific activity of 42.1 U/mg and the overall yield of 84% of dextransucrase activity obtained after the third step of phase partitioning by PEG 6000 were significantly higher than that of 23.8 U/mg and 46% overall yield, respectively, by PEG 400 or of that previously reported. The successive three steps, two-phase partitioning with a final concentration of 5% PEG 6000, reproducibly yielded a homogeneous preparation of dextransucrase.

Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase

Dextransucrase S (DSRS) is the only active glucansucrase that has been found in Leuconostoc mesenteroides NRRL B-512F strain. Native DSRS produces mainly 6-linked glucopyranosyl residue (Glc p), while Escherichia coli recombinant DSRS was observed to produce a glucan consisting of 70% 6-linked Glc p and 15% 3,6-Glc p. Lys residues were introduced at the N-terminal end of the core domain by site-directed mutagenesis. In glucans produced by the one-point mutants T350K and S455K, the amount of 6-linked Glc p was increased to about 85% of the total glucan produced, more similar in structure to native B-512F dextran. The double mutant T350K/S455K produced adhesive, water-insoluble glucan with 77% 6-linked Glc p, 8% 3,6-linked Glc p and 4% 2,6-linked Glc p. The T350K/S455K mutant exhibited a 10-fold increase in glucosyltransferase activity over those of the parental DSRS-His 6 and its T350K and S455K mutants. This is the first report demonstrating a change in the properties of a dextransucrase or a related glucosyltransferase through simple site-directed mutagenesis to create 2,6-linked Glc p.