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Title:
BRANCHED ALPHA GLUCANS
Document Type and Number:
WIPO Patent Application WO/2017/046040
Kind Code:
A1
Abstract:
The present invention relates to the field of poly-and oligosaccharides and their dietary effects. In particular it relates to a method of producing a branched α-glucan. Further aspects of the invention are a branched α-glucancomprising alternating α(1→4) and α(1→6) glucosidic linkages and having α(1→4,6) branching points, a food composition, and the use of an α-glucanotransferase enzyme for reducing the digestible carbohydrates of a starch containing food material.

Inventors:
GANGOITI MUÑECAS, Joana (Faculty of Mathematics and Natural Sciences, Microbial Physiology Research GroupGroningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 7 9747 AG Groningen, 9747, NL)
VAN LEEUWEN, Sander Sebastiaan (Faculty of Mathematics and Natural Sciences, Microbial Physiology Research GroupGroningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 7 9747 AG Groningen, 9747, NL)
DIJKHUIZEN, Lubbert (Faculty of Mathematics and Natural Sciences, Microbial Physiology Research GroupGroningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 7 9747 AG Groningen, 9747, NL)
VAFEIADI, Christina (1B Chemin des Molliettes, 1000 Lausanne 26, 1000 Lausanne 26, 1000, CH)
LAMOTHE, Lisa (22 Rue Saint Martin, 1003 Lausanne, 1003 Lausanne, 1003, CH)
Application Number:
EP2016/071474
Publication Date:
March 23, 2017
Filing Date:
September 12, 2016
Export Citation:
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Assignee:
NESTEC S.A. (IP Department, Av. Nestlé 55, 1800 Vevey, 1800, CH)
RIJKSUNIVERSITEIT GRONINGEN (Broerstraat 5, Gronningen, 9712 CP, 9712 CP, NL)
International Classes:
C08B30/00; A61K31/715; C08B31/00
Domestic Patent References:
WO2004022076A12004-03-18
WO2001090372A12001-11-29
Foreign References:
EP1943908A12008-07-16
EP2427565A22012-03-14
EP1943908A12008-07-16
EP2248907A12010-11-10
EP2427565B12014-01-15
US20060100171A12006-05-11
US20090297663A12009-12-03
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Attorney, Agent or Firm:
COUZENS, Patrick (IP Department, Av. Nestlé 55, 1800 Vevey, 1800, CH)
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Claims:
Claims

1 . Method of producing an a-glucan with a ratio of branching of at least 8% comprising contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an α-glucanotransferase enzyme capable of cleaving a(l->4) glucosidic linkages and making new alternating a(l->4) and a(l->6) glucosidic linkages with a(l->4,6) branching points without forming consecutive a(l->6) glucosidic linkages, wherein said α-glucanotransferase is selected from the group consisting of GTFB, GTFC and GTFD types of enzyme, or a functional homolog thereof having the specified enzymatic activity.

2. A method according to claim 1 wherein the α-glucanotransferase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:l.

3. A method according to claim 1 or claim 2 wherein the α-glucanotransferase is an Azotobacter chroococccum GTFD enzyme.

4. A method according to claim 1 wherein the α-glucanotransferase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:92.

5. A method according to claim 1 or claim 4 wherein the α-glucanotransferase is a Paenibacillus beijingensis GTFD enzyme.

6. A method according to any one of claims 1 to 5 wherein the substrate has a degree of polymerization of at least three.

7. A method according to any one of claims 1 to 6 wherein the substrate is selected from the group consisting of starch, starch derivatives, malto-oligosaccharides, amylose, amylopectin, maltodextrins, a(l->4) glucans and combinations thereof.

8. A method according to any one of claims 1 to 7 wherein the production of a-glucan is stopped before the reaction between the substrate and the α-glucanotransferase enzyme has reached completion.

9. A method according to any one of claims 1 to 8 wherein the polysaccharide or oligosaccharide substrate is contacted with an a-glucanotransferase enzyme at a temperature of between 30 °C and 75 °C and a pH of between 4.8 and 8.0.

10. An α-glucan comprising alternating a(l->4) and a(l->6) glucosidic linkages and having a(l->4,6) branching points wherein the α-glucan has a ratio of branching of at least 12%, has more than 1 wt.% maltopentose units, has less than 0.5% consecutive a(l->6) glucosidic linkages, and has an average molecular mass between 1 x 10s Da and 40 x 10s Da.

1 1 . An α-glucan according to claim 10 wherein the ratio of branching is at least 15% and the a- glucan has an average molecular mass between 22 x 10s Da and 35 x 10s Da.

12. Food composition comprising the α-glucan of claim 10 or claim 11.

13. A food composition according to claim 12 wherein the food composition is a beverage; a breakfast cereal; a pet food product; a baked dough product; or a confectionery product.

14. Use of a GTFD a-glucanotransferase enzyme for reducing the digestible carbohydrates of a food material.

15. Use according to claim 14 wherein the α-glucanotransferase enzyme comprises an amino acid sequence having at least 80% identity to SEQ ID NO:l, or at least 80% identity to SEQ ID NO:92.

Description:
Branched alpha glucans

Field of the Invention

The present invention relates to the field of poly- and oligosaccharides and their dietary effects. In particular it relates to a method of producing a branched a-glucan. Further aspects of the invention are a branched a-glucan comprising alternating a(l->4) and a(l->6) glucosidic linkages and having a(l->4,6) branching points, a food composition, and the use of an a-glucanotransferase enzyme for reducing the digestible carbohydrates of a starch containing food material.

Background of the Invention

The prevalence of obesity and being overweight is rapidly increasing worldwide. The development of foods with high satiating capacities and low energy densities may help to prevent weight gain and to stimulate weight loss. Consumption of food and drinks containing non-digestible carbohydrates instead of sugars induces a lower blood glucose rise after meals compared to sugar-containing food and drinks.

The most common carbohydrate in human diets is starch. This polysaccharide is produced by most green plants as an energy store. It is contained in large amounts in such staple foods as potatoes, wheat, maize, rice, and cassava. Various methods have been proposed for the chemical modification of starch and malto-oligosaccharides into non-digestible carbohydrates.

EP2427565 describes the use of a glucanotransferase enzyme of Lactobacillus reuteri 121 GTFB to convert starch into linear gluco-oligosaccharides containing relatively long isomalto-oligosaccharide side chains. Such materials are partially resistant to digestion and hence give less glucose production on consumption, contributing to the prevention of obesity and type II diabetes.

It has been observed that highly branched a-glucans can combine a reduced digestibility with a thickening effect triggered by the low pH conditions of the stomach. This thickening leads to feelings of satiety. The production of highly branched a-glucans from sucrose has been described, for example the synthesis of reuteran by the Lactobacillus reuteri 121 GTFA glucansucrase (Kralj et al., 2002).

It would be desirable to provide further means for the enzymatic modification of starch, starch derivatives and malto-oligosaccharides in order to change their functional properties and enhance their nutritional value. In particular it would be beneficial to provide enzymes to perform such modifications which are suitable for use in food manufacture and exhibit good enzyme activity and thermostability.

Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field. As used in this specification, the words "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to".

Summary of the invention

An object of the present invention is to improve the state of the art and to provide an improved solution for the enzymatic modification of starch and other polysaccharide or oligosaccharide into materials having reduced digestibility, or at least to provide a useful alternative. The object of the present invention is achieved by the subject matter of the independent claims. The dependent claims further develop the idea of the present invention.

Accordingly, the present invention provides in a first aspect a method of producing an a-glucan with a ratio of branching of at least 8% comprising contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an a-glucanotransferase enzyme capable of cleaving a(l->4) glucosidic linkages and making new alternating a(l->4) and a(l->6) glucosidic linkages with a(l->4,6) branching points, wherein said a-glucanotransferase is selected from the group consisting of GTFB, GTFC and GTFD types of enzyme, or a functional homolog thereof having the specified enzymatic activity.

In a second aspect, the invention relates to an α-glucan comprising alternating a(l->4) and a(l->6) glucosidic linkages and having a(l->4,6) branching points wherein the α-glucan has a ratio of branching of at least 15% and an average molecular mass between 1 x 10 s Da and 40 x 10 s Da. A third aspect of the invention relates to a food composition comprising an α-glucan comprising alternating a(l->4) and a(l->6) glucosidic linkages and having a(l->4,6) branching points wherein the α-glucan has a ratio of branching of at least 15% and an average molecular mass between 1 x 10 s Da and 40 x 10 s Da.

A still further aspect of the invention is the use of an α-glucanotransferase enzyme that comprises an amino acid sequence having at least 80% identity to SEQ ID NO:l, or has an amino acid sequence of SEQ ID NO:l, for reducing the digestible carbohydrates of a starch containing food material. Recently, the inventors have identified two novel glycoside hydrolase subfamilies of enzymes inactive on sucrose, but displaying 4,6-a-glucanotransferase (4,6-a-GTase) activity on starch/maltodextrin substrates, and represented by the Lactobacillus reuteri 121 GTFB (S. Kralj et al., 2011) and the Exiguobacterium sibiricum 255-15 GTFC enzymes (Gangoiti et al., 2015). Both GTFB and GTFC enzymes catalyze the cleavage of the a(l->4) and the synthesis of linear a(l->6)-glucan chains. Like glucansucrases, GTFB type of enzymes are exclusively found in lactic acid bacteria and possess a catalytic (a/ )8-barrel domain that is circularly permuted with respect to that of the GH13 a-amylase enzymes. In contrast, the GTFC type of enzymes is present in non-lactic acid Gram-positive bacteria, and has a non-permuted domain organization resembling that of GH13 enzymes. GTFC enzymes thus appear to represent further evolutionary intermediates between GH13 a-amylases and GH70 glucansucrases/ GTFB-like 4,6-a-glucanotransferases.

The inventors have identified a novel GH70 family protein in the genome of the nitrogen-fixing bacterium Azotobacter chroococccum NCIMB 8003. This enzyme is designated GTFD and is most closely related to GTFC type of enzymes, sharing the same domain architecture and displaying hydrolase/transglycosylase activity with starch/maltodextrin substrates. However, the A chroococcum GTFD enzyme is unable to synthesize consecutive a(l->6) glucosidic bonds, and instead it forms a higher molecular mass and relatively highly branched a-glucan with alternating a(l->4) and a(l->6) glucosidic linkages from amylose and starch. Therefore this polymer is similar to reuteran synthesized by the L. reuteri 121 GTFA glucansucrase from sucrose, regarded as a health promoting food ingredient. The GTFD enzyme shows its maximum activity at pH 6.5 and 60°C, and exhibits a higher thermostability than the GTFA glucansucrase and the GTFB and GTFC 4,6-a-glucanotransferases. An enzyme that converts starch into a reuteran-like α-glucan polymer has not been described before.

ID Χ Η NMR analysis of the branched α-glucan formed by the A. chroococcum GTFD enzyme revealed the formation of a(l->4) and a(l->6) linkages. Methylation analysis of the α-glucan revealed the presence of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted glucopyranose residues. The presence of 6-substituted, and 4,6-disubstituted glucopyranose residues means that the GTFD enzyme forms a(l->6) linkages in linear and branched orientation. No evidence was observed for two consecutive a(l->6)-linked glucopyranose residues by 2D NMR spectroscopy analysis. Thus, all the branched residues are a(l->4,6)-a-D-Glcp-a(l->4)- residues. Also, all 6-substituted glucopyranose residues detected by methylation analysis must be a(l->4)-linked and are connecting a(l->4) glucan chains forming alternating α(1->6)/α(1->4) linkages in the linear part of the α-glucan structure. This is in contrast to the action of branching enzymes with E.C. 2.4.1.18 activity disclosed in EP1943908. Such branching enzymes only create a(l->4,6) branching points but do not create a(l->6) linkages in the linear part of the a-glucan structure, and so do not form alternating a(l->4) and a(l->6) glucosidic linkages.

The inventors have identified a second example of a GH70 family protein displaying this novel reaction and product specificity in the genome of the bacterium Paenibacillus beijingensis DSM 24997. This enzyme is designated GTFD and is closely related to the GTFC subfamily of enzymes present in Bacillus, Geobacillus and Exiguobacterium strains. It displays 4,6-a-glucanotransferase activity with starch/maltodextrin substrates but, like A. chroococcum GTFD, is unable to synthesize consecutive a(l->6) glucosidic bonds. The P. beijingensis GTFD activity on amylose results in the synthesis of a high- molecular (HMM) and low-molecular mass (LMM) polysaccharides, highly branched a-glucans with alternating a(l->4) and a(l->6) glucosidic linkages.

ID Χ Η NM analysis of the branched α-glucan formed by the P. beijingensis GTFD enzyme revealed the formation of a(l->4) and a(l->6) linkages. Methylation analysis of the α-glucan revealed the presence of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted glucopyranose residues. The presence of 6-substituted, and 4,6-disubstituted glucopyranose residues means that the GTFD enzyme forms a(l->6) linkages in linear and branched orientation. No evidence was observed for two consecutive a(l->6)-linked glucopyranose residues by 2D NMR spectroscopy analysis. Thus, all the branched residues are a(l->4,6)-a-D-Glcp-a(l->4)- residues. Also, all 6-substituted glucopyranose residues detected by methylation analysis must be a(l->4)-linked and are connecting a(l->4) glucan chains forming alternating α(1->6)/α(1->4) linkages in the linear part of the α-glucan structure. This is in contrast to the action of branching enzymes with E.C. 2.4.1.18 activity disclosed in EP1943908. Such branching enzymes only create a(l->4,6) branching points but do not create a(l->6) linkages in the linear part of the α-glucan structure.

Description of the Drawings

Figure 1 shows a linear schematic representation of the domain organization of GH70 family proteins. From left to right: Lactobacillus reuteri 121 GTFA-ΔΝ glucansucrase (Pijning et al., 2012), Lactobacillus reuteri 121 GTFB-ΔΝ 4,6-a-glucanotransferase (Bai et al., 2015b), Exiguobacterium sibiricum 255-15 GTFC 4,6-a-glucanotransferase (Gangoiti et al., 2015), Azotobacter chroococcum NCIMB 8003 GTFD enzyme and P. beijingensis DSM 24997 GTFD. The crystal structure of the GTFA-ΔΝ glucansucrase (Pijning et al., 2012) (left) and the B. licheniformis a-amylase (Machius et al., Mol Biol 246:545-559 (1995)) (right) are included showing the unusual "U-fold"domain structure of GH70 glucansucrases, in which 4 of the 5 domains are built up form two discontinuous segments of the polypeptide chain, i.e. are formed from the N- and C-terminal halves. Domains A, B, C and IV were identified in the A. chroococcum GTFD primary structure by sequence comparisons with the L. reuteri 121 GTFB and E. sibiricum GTFC sequences, using Clustal W2. GTFD enzyme lacks the Ig2-like domains identified in E. sibiricum GTFC and domain V exclusively found in glucansucrases and GTFB-like 4,6-a- glucanotransferases. Domains A, B, C and IV were assigned in P. beijingensis GTFD by sequence comparison with L. reuteri 121 GTFB. The order of the conserved regions l-IV in glucansucrases and GTFB-like 4,6-a-glucanotransferases is ll-lll-IV-l, reflecting their circularly permuted domain organization. The amino acid residue numbers indicate the start of each domain. Conserved regions I- IV are represented by dark grey rectangles.

Figure 2 is a phylogenetic tree of representative family GH70 and GH13 protein sequences identified via BLASTp searches using the Exiguobacterium sibiricum 255-15 GTFC 4,6-a-glucanotransferase protein as query (shown in bold). The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. The bar corresponds to a genetic distance of 0.2 substitutions per position (20% amino acid sequence difference). The bootstrap values adjacent to the main nodes represent the probabilities based on 1000 replicates. The protein sequences are annotated by their GenBank Gl number. The names of the bacterial species are provided in Table 1. The novel Azotobacter chroococcum NCIMB 8003 GTFD enzyme is highlighted with a grey background.

Figure 3 shows the sequence alignment of conserved motifs l-IV in the catalytic domain of Azotobacter chroococcum NCIMB 8003 GTFD enzyme (A), (putative) GTFC-like 4,6-a-glucanotransferase enzymes (B), (putative) GTFB-like 4,6-a-glucanotransferase enzymes (C), glucansucrase enzymes (D), and GH13 a-amylase enzymes (E). The seven strictly conserved amino acid residues in GH70 enzymes (indicated by the numbers 1 to 7 above the sequences) are also conserved in the novel Azotobacter chroococcum NCIMB 8003 GTFD protein. Amino acids that constitute the catalytic triad are indicated with triangles. Residues forming acceptor subsites -1, +1 and +2 in GTF180-AN (Vujicic-Zagar et al., 2010) are shown lightly shaded. Symbols: NU = nucleophile, A/B = general acid/base, TS = transition state stabilizer.

Figure 4 shows an SDS-PAGE analysis of the Azotobacter chroococcum NCIMB 8003 GTFD protein purified from E. coli BL21 Star (DE3). Lane M, molecular mass standards; lane 1, sample of cell free extract; lane 2, sample of the insoluble fraction after centrifugation of lysed cells; lane 3, pooled fractions after Ni-NTA agarose column chromatography. Bands corresponding to the GTFD protein are marked with an arrow. Figure 5 shows biochemical properties of the purified Azotobacter chroococcum NCIMB 8003 GTFD enzyme. (A) Effect of pH on GTFD activity. The assays were carried out at 37°C and relative enzyme activity was compared with enzyme activity at pH 6.5 (100% value). (B) Effect of temperature on GTFD activity. Experiments were performed at pH 6.5 and relative activity was compared with the enzyme activity at 60°C (100% value). (C) Effect of temperature on GTFD stability. GTFD enzyme (0.5 mg ml "1 ) was incubated for 10 min at the indicated temperature in 20 mM Tris-HCI pH 8.0 buffer containing 1 mM CaC . Residual activity was assayed at 50°C in the standard conditions described in experimental section.

Figure 6 shows a TLC analysis of the product mixtures synthesized by 40 μg ml 1 of the Azotobacter chroococcum NCIMB 8003 GTFD enzyme from incubations with 25 mM malto-oligosaccharides (DP2- DP7), 0.6 % (w/v) amylose V, 0.6 % potato soluble starch, and 0.6 % amylopectin. The reaction mixtures were incubated at 37 °C and pH 6.5 during 24 h. S, standard; Gl, glucose; G2-G7, maltose to maltoheptaose; AMV, amylose V; STR, starch; AMP, amylopectin; Pol, polymer.

Figure 7 shows a 500-MHz 1-dimensional X H NMR analysis of reaction mixtures obtained by the incubation of 0.6% (w/v) amylose V (A) and potato starch (B) with 40 μg ml 1 of Azotobacter chroococcum NCIMB 8003 GTFD enzyme (pH 6.5, 24 h), recorded in D2O. Chemical shifts are given in parts per million relative to the signal of internal acetone (δ 2.225).

Figure 8 shows HPSEC profiles of the reaction products obtained from 0.6% (w/v) amylose V, incubated with 40 μg ml "1 of Azotobacter chroococcum NCIMB 8003 GTFD enzyme (pH 6.5) and Lactobacillus reuteri 121 GTFB enzyme (pH 4.7) for 24 h at 37 °C. The dashed line corresponds to the elution profile of the amylose V. The solid black and grey lines correspond to the elution profiles of products synthesized by GTFD and GTFB enzymes, respectively.

Figure 9 shows a 500-MHz ID X HNMR spectrum, 2D H TOCSY spectra (mixing time 150 ms), and 2 D ^C^H HSQC spectrum of the void-volume Bio-Gel P-2 polysaccharide fraction, obtained after 24 h incubation of 0.6% (w/v) amylose V with the Azotobacter chroococcum NCIMB 8003 GTFD enzyme (40 μg ml "1 ) recorded at 300K in D2O. Peaks for a(l->4) and a(l->6) anomeric signals have been indicated. Structural reporter peaks a. H-4 for 6-substituted Glc, b. H-4 for terminal Glc, c. for H-4 for 4- substituted Glc, d. H-6a for 6-substituted Glc and e. H-6b for 6-substituted Glc.

Figure 10 shows an HPAEC-PAD profile of the oligosaccharide mixture formed upon incubation of the Azotobacter chroococcum NCIMB 8003 GTFD enzyme (20 μg ml "1 ) with (A) maltohexaose and (B) amylose V for t = 10 min, 30 min, 3 h, and 24 h (pH 6.5, 37 °C). The identity of peaks was assigned using commercial oligosaccharide standards. * Unidentified carbohydrate structure. Gl, glucose; G2-G6, maltose to maltohexaose; iso-G2, isomaltose; Pa, panose.

Figure 11 shows an HPAEC-PAD profile of the oligosaccharide mixture formed upon the incubation of Azotobacter chroococcum NCIMB 8003 GTFD enzyme (40 μg ml "1 ) (A) and Lactobacillus reuteri 121 GTFB enzyme (40 μg ml "1 ) (B) with 0.35% amylose V (AMV) (donor substrate) or amylose V with 25 mM maltose or 25 mM isomaltose (acceptor substrates) for 24 h at 37°C. The identity of peaks was assigned using commercial oligosaccharide standards. Gl, glucose; G2-G4, maltose to maltotetraose; iso-G2- iso-G5, isomaltose to isomaltopentaose; Pa, panose.

Figure 12 shows a TLC analysis of the Azotobacter chroococcum NCIMB 8003 GTFD polymer, Lactobacillus reuteri 121 GTFA reuteran polymer and Lactobacillus reuteri 121 GTFB isomalto/malto- polysaccharide (IMMP), after treatment with (A) Aspergillus oryzae a-amylase, (B) Chaetomium erraticum dextranase and (C) Klebsiella planticola pullulanase Ml. Lanes 1-3: product mixtures generated by the enzymatic hydrolysis of the GTFD polymer, reuteran and IMMP, respectively. Lane 4, positive controls for the a-amylase, dextranase and pullulanase treatments: starch (A), dextran (B) and pullulan (C). Lane S, standard: glucose (Gl) to maltoheptaose (G7); Pol, polymer.

Figure 13 shows an HPAEC-PAD profile of the oligosaccharides formed after treatment of the Azotobacter chroococcum NCIMB 8003 GTFD polymer with pullulanase Ml. Established oligosaccharide structures are included. The identity of peaks 1- 6 and 8 was assigned using commercial oligosaccharide standards and by comparison with the profile of the pullulanase hydrolysate of reuteran. Peak 7 corresponds to an oligosaccharide of DP5 with at least one a(l->6) linkage (van Leeuwen et al., 2008).

Figure 14 is a visual representation of composite structures for the Lactobacillus reuteri 121 GTFA product from sucrose and the Azotobacter chroococcum NCIMB 8003 GTFD product from amylose. The composite structures contain all structural features established for the respective products. Quantities of each structural element fit with the combined data of ID Χ Η NM integration and methylation analysis.

Figure 15 shows the sequence alignment of conserved motifs l-IV in the catalytic domains of Azotobacter chroococcum NCIMB 8003 and Paenibacilus beijingensis DSM 24997 GTFD enzymes. The seven strictly conserved amino acid residues in GH70 enzymes are indicated by the numbers 1 to 7 above the sequences. Residues forming acceptor subsites -1, +1 and +2 in GTF180-AN (Vujicic-Zagar et al., 2010) are shown lightly shaded. Symbols: NU = nucleophile, A/B = general acid/base, TS = transition state stabilizer.

Figure 16 shows SDS-PAGE analysis of P. beijingensis GTFD 4,6-a-glucanotransferase at different stages of purification. Lane M, molecular mass standards; lane 1, sample of E. coli cell free extract; lane 2, sample of the insoluble fraction after centrifugation of lysed cells; lane 3, purified GTFD enzyme after Ni-NTA agarose column chromatography. Bands corresponding to the P. beijingensis GTFD protein are indicated with an arrow.

Figure 17 shows biochemical properties of the purified GTFD enzyme from P. beijingensis. (A) Effect of pH on GTFD activity. Experiments were carried out at 40°C and the relative enzyme activity was compared with enzyme activity at pH 7.0 (100% value). (B) Effect of temperature on GTFD activity. The assays were performed at pH 7.0 and relative activity was compared with the enzyme activity at 50°C (100% value). The results are the mean of two replicates, and the bars indicate the standard error of two replicates.

Figure 18 shows TLC analysis of the products produced by the P. beijingensis GTFD (A) and A. chroococcum GTFD (B) enzymes at 40 μg ml "1 from 25 mM malto-oligosaccharides (DP2-DP7), 0.6 % (w v "1 ) amylose V, and 0.6 % (w v "1 ) amylopectin. The reaction mixtures were incubated at 37°C and pH 7.0 (P. beijingensis GTFD) or pH 6.5 (A. chroococcum GTFD) during 24 h. S, standard; Gl, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; G6, maltohexaose; G7, maltoheptaose; AMV, amylose V; AMP, amylopectin; Pol, polymer. Figure 19 shows one-dimensional 500-MHz Χ Η NM spectra of the product mixtures generated after the incubation of 0.6% (w v 1 ) amylose V with the P. beijingensis GTFD 4,6-a-glucanotransferase enzyme at 40 μg ml "1 for 24 h at 37°C and pH 7.0. The spectrum was recorded in D2O at 25°C. Chemical shifts are shown in parts per million relative to the signal of internal acetone (δ = 2.225). Ga/β and Ra/β indicate the anomeric signals corresponding to the D-Glcp units and the reducing -(l->4)-D-Glcp units, respectively.

Figure 20 shows HPSEC chromatograms of the product mixtures obtained by the incubation of 0.6% (w v "1 ) amylose V with 40 μg ml 1 of A. chroococcum GTFD enzyme (pH 6.5) and P. beijingensis GTFD enzyme (pH 7.0) for 24 h at 37°C. The dashed line corresponds to the elution profile of the starting amylose V. The solid black and grey lines correspond to the elution profiles of products synthesized by P. beijingensis and A. chroococcum GTFD enzymes, respectively. Figure 21 shows 500-MHz X H NM spectra of the high- (HMM) (A) and low molecular mass (LMM) (B) polysaccharides produced by the P. beijingensis GTFD enzyme, isolated by size-exclusion chromatography on a Sephadex S200 column. The reaction products were obtained from 0.6% (w v "1 ) amylose V, incubated with 40 μg ml "1 of the P. beijingensis GTFD enzyme for 24 h at 37°C and pH 7.0. The spectra were recorded in D2O at 25°C. Chemical shifts are shown in parts per million relative to the signal of internal acetone (δ = 2.225). Peaks for a(l->4) and a(l->6) anomeric signals have been indicated.

Figure 22 shows a 500-MHz ID X H NMR spectrum, 2D H TOCSY spectra (mixing time 150 ms), and 2D ^C^H HSQC spectrum of the HMM product of P. beijingensis GTFD enzyme incubated with amylose V, recorded at 298K in D2O. Peaks for a(l->4) and a(l->6) anomeric signals have been indicated. Structural reporter peaks a: H-4 for 6-substituted Glcp, b: H-4 for terminal Glcp, c: for H-4 for 4- substituted Glcp, d: H-6a for 6-substituted Glcp and e: H-6b for 6-substituted Glcp.

Figure 23 shows enzymatic treatment of the P. beijingensis DSM 24997 GTFD HMM and LMM polymers, A. chroococcum NCIMB 8003 GTFD reuteran-like polymer, and L. reuteri 121 GTFB isomalto/malto-polysaccharide (IMMP). Reaction mixtures containing 5 mg ml 1 of a-glucans were incubated separately with a high dose of (A) Aspergillus oryzae a-amylase, (B) Chaetomium erraticum dextranase and (C) Klebsiella planticola pullulanase Ml for 48 h at 37°C and subjected to TLC analysis. Lanes 1-4: reaction products generated by the enzymatic treatment of the P. beijingensis GTFD HMM polymer, P. beijingensis GTFD LMM polymer, reuteran-like polymer, and IMMP, respectively. Lane 5, positive controls for the a-amylase, dextranase and pullulanase digestions: amylose (A), dextran (B) and pullulan (C). Lane S, standard: glucose (Gl) to maltoheptaose (G7); Pol, polymer.

Figure 24 shows HPAEC-PAD profiles of the oligosaccharides formed after treatment of the P. beijingensis GTFD HMM polymer (A), P. beijingensis GTFD LMM polymer (B), and A. chroococcum GTFD polymer (C) with pullulanase Ml. Established oligosaccharide structures are included. The identity of peaks 1-16 was assigned using commercial oligosaccharide standards and by comparison with the profile of the pullulanase hydrolysate of reuteran.

Figure 25 is a visual representation of composite structures for the A. chroococcum NCIMB 8003 GTFD polymer, the HMM P. beijingensis GTFD polymer and the LMM P. beijingensis GTFD polymer formed from amylose V. The composite structures contain all structural features established for the respective products. Quantities of each structural element fit with the combined data of ID X H NMR integration and methylation analysis, as well as enzymatic degradation studies with a-amylase, dextranase and pullulanase.

Figure 26 shows HPAEC-PAD profiles of the oligosaccharide mixtures formed upon incubation of maltoheptaose with P. beijingensis GTFD (A) and A. chroococcum (B) GTFD enzymes (20 μg ml "1 ) for t = 10 min, 30 min, 3 h, and 24 h, at 37 °C and pH 7.0 and pH 6.5, respectively. The identity of peaks was assigned using commercial oligosaccharide standards. * Unidentified carbohydrate structures. Gl, glucose; G2-G6, maltose to maltohexaose; iso-G2, isomaltose; Pa, panose.

Figure 27 is a plot of in vitro digestibility of gelatinized wheat starch treated with A. chroococcum GTFD (B) compared to untreated gelatinized wheat starch (A). The graph plots % hydrolysed sample versus time in minutes.

Figure 28 is a plot of in vitro digestibility of A. chroococcum GTFD - high molecular mass (C), P. beijingensis GTFD - high molecular mass (D), and P. beijingensis GTFD - low molecular mass (E) products from Amylose V compared to gelatinized wheat starch (A). The graph plots % hydrolysed sample versus time in minutes. Detailed Description of the invention

Consequently the present invention relates in part to a method of producing an a-glucan with a ratio of branching of at least 8% (for example at least 12%) comprising contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an a-glucanotransferase enzyme capable of cleaving a(l->4) glucosidic linkages and making new alternating a(l->4) and a(l->6) glucosidic linkages with a(l->4,6) branching points, wherein said a-glucanotransferase is selected from the group consisting of GTFB, GTFC and GTFD types of enzyme (for example a GTFD type of enzyme), or a functional homolog thereof having the specified enzymatic activity. For example, the α-glucanotransferase enzyme in the method of the invention may be capable of cleaving a(l->4) glucosidic linkages and transferring malto-oligosaccharides up to DP5, resulting in the formation of single a(l->6) linkages, in both alternating and branching patterns. Polysaccharides are polymeric carbohydrate molecules composed of long chains of monosaccharide units bound together by glycosidic linkages. Oligosaccharides are saccharide polymers containing a small number (typically three to nine) of monosaccharides. An example of a substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units is amylose. The method of the invention may comprise contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an a-glucanotransferase enzyme capable of cleaving a(l->4) glucosidic linkages and making new alternating a(l->4) and a(l->6) glucosidic linkages with (for example together with) a(l->4,6) branching points without forming consecutive a(l->6) glucosidic linkages, wherein said α-glucanotransferase is selected from the group consisting of GTFB, GTFC and GTFD types of enzyme, or a functional homolog thereof having the specified enzymatic activity. In the present specification, the abbreviation GTF refers to glucanotransferase.

The alternating a(l->4) and a(l->6) glucosidic linkages may be interspersed with some consecutive a(l->4) glucosidic linkages, such a structure can also be described as chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages. Single a(l->6) glucosidic linkages between one or more a(l->4) glucosidic linkages as may be formed in the method of the invention are sometimes referred to as "bridging" a(l->6) linkages. The notation (al->4) may be used instead of a(l->4) to refer to a l->4 a linkage, but these are equivalent, as are (al->6) and a(l->6). The method of the invention may comprise contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an α-glucanotransferase enzyme capable of cleaving a(l->4) glucosidic linkages and making new a(l->6) glucosidic linkages to form structures having chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages and having a(l->4,6) branching points, without forming consecutive a(l->6) glucosidic linkages, wherein said α-glucanotransferase is selected from the group consisting of GTFB, GTFC and GTFD types of enzyme, or a functional homolog thereof having the specified enzymatic activity.

In the context of the present invention, the ratio of branching is defined as the total number of branching anhydroglucose units (AGU), i.e. AGU being bound to three other units, with respect to the total number of AGU of a molecule. The ratio of branching can be determined by methods known in the art, such as methylation with gas chromatography. The a-glucan produced by the method of the invention may have a ratio of branching of at least 8%, for example at least 10%, for example at least 15%.

The α-glucan produced by the method of the invention may comprise at least one a(l->4) glucosidic linkage adjacent to an a(l->6) glucosidic linkage and at least one a(l->4,6) branching point. The a- glucan produced by the method of the invention may comprise alternating a(l->4) and a(l->6) glucosidic linkages and have a(l->4,6) branching points, for example the a-glucan produced by the method of the invention may comprise between 40 and 50 percent consecutive a(l->4) glucosidic linkages, between 12 and 21 percent single a(l->6) glucosidic linkages in alternating pattern and between 8 and 25 percent a(l->4,6) branching points, for example between 12 and 21 percent a(l->4,6) branching points, for example between 15 and 20 percent a(l->4,6) branching points. The α-glucan produced by the method of the invention may have less than 1 % consecutive a(l->6) glucosidic linkages, for example it may have less than 0.5 % consecutive a(l->6) glucosidic linkages, for further example it may have no consecutive a(l->6) glucosidic linkages.

The α-glucanotransferase in the method of the invention may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:l (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:l). The α-glucanotransferase in the method of the invention may consist of an amino acid sequence having at least 80% identity to SEQ ID NO:l (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:l). mraspsqffa isllsiaisg llsgaavaap aptaleqvpd gkggvkwqev thdasaeeeq kgqdpkkflg iqaittepdg svkvemgkpe vrqpasgdvf vsnekldehv ifqafalyqp ndnatykalt enapqlaqwg itdvwspppy raasdskyge gyaiadrydl gaydkgptky gtadelkaai galhnndiri qvdvvpnqii glnerhvlpv tgvdmygkpm npfldhylys tyskgsapgq aehgvikewd yfhfhgtttq yqglfrvlsd anstlyrylg pnhpenylpa flaesdaaky gkintidgyl ladtwfaven aesenavyap Iflyyeeprn gvveqtfmdf arengytgsd ediratmlae Irmtpnpigp Imdeylaaqp gyskksedda kvtalrydgp endashigtn vldfeflvgn dldtiredvq qeqlnwqkyl Idfgfdgfri daashintdm Irdevtqrln hfagedvneh Isyiesyvtq qvdflqsnny gqmamdagpf sglmfsfgrd waplryafea slidrvnggp alpnwsfvnn hdqehnilvt vplteeeagg yepnsqpyel rqlekydadr nsvekqwaph nvpamyaill ttkdtvptvf ygdmfvsskp ymstptpyrd divnilklrk qfakgeqvir yensntgsng edlvsnirlg ndrktgvavv agnnpaldtt itvdmgaqhr nqwfvdamgy qperlktdkd grltvqvkgt qnvdvkgyla awvpdlqaqe (SEQ ID NO:l)

The α-glucanotransferase in the method of the invention may be an Azotobacter chroococccum GTFD enzyme for example the α-glucanotransferase in the method of the invention may be an Azotobacter chroococccum NCIMB 8003 GTFD enzyme. The method of the invention for producing an α-glucan with a ratio of branching of at least 8% may comprise contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an Azotobacter chroococccum GTFD enzyme. A further aspect of the invention provides a method for producing an α-glucan with a ratio of branching of at least 8%, the method comprising contacting a polysaccharide or oligosaccharide substrate, comprising at its non-reducing end at least two a(l->4) linked D-glucose units, with an a-glucanotransferase enzyme comprising (for example consisting of) an amino acid sequence having at least 80% identity to SEQ ID NO:92 (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:92). SEQ ID NO:92 is the sequence of Paenibacillus beijingensis DSM 24997 GFTD enzyme, Genbank accession WP_052702730.1.

The a-glucanotransferase in the method of the invention may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:92 (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:92). The α-glucanotransferase in the method of the invention may consist of an amino acid sequence having at least 80% identity to SEQ ID NO:92 (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:92).

The α-glucanotransferase in the method of the invention may be a Paenibacillus beijingensis GTFD enzyme for example the α-glucanotransferase in the method of the invention may be a Paenibacillus beijingensis DSM 24997 GFTD enzyme. Paenibacillus beijingensis DSM 24997 is the type strain of P. beijingensis, also known as ACCC 03082, and may be obtained from Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The method of the invention for producing an a-glucan with a ratio of branching of at least 8% may comprise contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with a Paenibacillus beijingensis GTFD enzyme.

An aspect of the invention provides a method for producing an α-glucan with a ratio of branching of at least 8%, the method comprising contacting a polysaccharide or oligosaccharide substrate, comprising at its non-reducing end at least two a(l->4) linked D-glucose units, with a-glucanotransferase comprising an amino acid sequence having at least 80% identity to SEQ ID NO:92.

The substrate in the method of the invention may have a degree of polymerization of at least three, for example it may comprise at least three D-glucose units. The degree of polymerization is the number of monomeric units in a polymer or oligomer molecule. For example, the substrate in the method of the invention may have a degree of polymerization of at least four, for example it may comprise at least four D-glucose units. The substrate in the method of the invention may be selected from the group consisting of starch (for example waxy starch or high amylose starch), starch derivatives, malto- oligosaccharides, amylose, amylopectin, maltodextrins, a(l->4) glucans and combinations thereof. Starch derivatives are prepared by physically, enzymatically, or chemically treating native starch to change its properties. The substrate in the method of the invention may be comprised within another material, for example the substrate may be starch provided in the form of flour. It is advantageous to be able to convert polysaccharides or oligosaccharides comprised within food ingredients into a-glucans with lower digestibility, for example branched a-glucans. Such a conversion may increase the fibre content of the ingredients and/or may aid in reducing the calorie content of the ingredients. The method of the invention may be performed as part of a food processing operation, for example the a-glucanotransferase enzyme may be applied to food ingredients during a process to produce a food product. The substrate may be comprised within a material which already has a positive nutritional profile, for example the substrate may be comprised within wholegrain flour. The extent to which the polysaccharide or oligosaccharide substrate may be converted by the α-glucanotransferase enzyme in the method of the invention can be adjusted by limiting the time of reaction. Partially converted substrates will provide different physical properties. The production of a- glucan in the method of the invention may be stopped before the reaction between the substrate and the α-glucanotransferase enzyme has reached completion, for example it may be stopped by denaturing (e.g. by heat) or removing the enzyme.

The α-glucanotransferase enzyme in the method of the invention may be immobilized, for example immobilized before contacting the polysaccharide or oligosaccharide substrate. Such immobilization techniques are well known in the art. Removal of the enzyme (discussed above) may be facilitated by immobilization of the enzyme. Immobilization techniques may be selected from the group consisting of covalent binding, entrapment, physical adsorption, cross-linking and combinations of these. In immobilization by covalent binding, enzymes are covalently linked to a support through the functional groups in the enzymes that are not essential for the catalytic activity. Oxides materials such as alumina, silica, and silicated alumina can be used for covalent binding of the enzyme. In immobilization by entrapment the enzyme is localized within the lattice of a polymer matrix or membrane. Entrapment methods are classified into five major types: lattice, microcapsule, liposome, membrane, and reverse micelle. The enzyme is entrapped in the matrix of various synthetic or natural polymers. Alginate, a naturally occurring polysaccharide that forms gels by ionotropic gelation is one such immobilization matrix. Immobilization by physical adsorption is the simplest and the oldest method of immobilizing enzymes onto carriers. Immobilization by adsorption is based on the physical interactions between the enzymes and the carrier, such as hydrogen bonding, hydrophobic interactions, van der Waals force, and their combinations. Adsorption is generally less disruptive to the enzymes than chemical means of attachment. Immobilization by cross-linking utilizes bi- or multifunctional compounds, which serve as the reagent for intermolecular cross-linking of the enzymes. Cross-linking may be used in combination with other immobilization methods such as adsorption or entrapment.

The polysaccharide or oligosaccharide substrate may be contacted with an a-glucanotransferase enzyme in the method of the invention at a temperature of between 30 °C and 75 °C (for example between 40 °C and 75 °C, for example between 50 °C and 70 °C, for example between 35 °C and 45 °C) and a pH of between 4.8 and 8.0 (for example between 5.5 and 7.5, for example between 6.0 and 7.0).

In a further embodiment the present invention pertains to an a-glucan comprising alternating a(l->4) and a(l->6) glucosidic linkages and having a(l->4,6) branching points wherein the α-glucan has a ratio of branching of at least 12% (for example at least 15%), has more than 1 wt.% maltopentose units and has an average molecular mass between 1 x 10 s Da and 40 x 10 s Da, for example, an average molecular mass between 5 x 10 s Da and 30 x 10 s Da, for example, an average molecular mass between 10 x 10 s Da and 20 x 10 s Da. The α-glucan according to the invention may comprise between 40 and 50 percent consecutive a(l->4) glucosidic linkages, between 12 and 21 percent single a(l->6) glucosidic linkages in alternating pattern and between 12 and 25 percent a(l->4,6) branching points, for example between 12 and 21 percent a(l->4,6) branching points, for example between 15 and 20 percent a(l->4,6) branching points. The α-glucan according to the invention may have less than 1 % consecutive a(l->6) glucosidic linkages, for example it may have less than 0.5 % consecutive a(l->6) glucosidic linkages, for further example it may have no consecutive a(l->6) glucosidic linkages. The a- glucan of the invention is similar to the reuteran synthesized by the L. reuteri 121 GTFA glucansucrase from sucrose, regarded as a health promoting food ingredient, but has some important differences. The ratio of branching is higher, for example in the samples analysed in table 3 it can be seen that Azotobacter chroococcum NCIMB 8003 GTFD enzyme produces 18 % branching compared to 11 % for L. reuteri 121 GTFA enzyme. The more branching is present in an a-glucan, the less access is provided to digestive enzymes and so the α-glucan could present a lower digestibility. The α-glucan of the invention also comprises maltopentose units in its structure (see Figure 14) which are not present in reuteran, for example reuteran produced by the L. reuteri 121 GTFA enzyme. Higher amounts of consecutive a(l->4) linkages, for example maltopentose units, may provide particular physical properties such as gelling and viscosity modification which provide useful applications, for example in foodstuffs. The α-glucan of the invention may have a(l->4,6) branching points, a ratio of branching of at least 12%, more than 1 wt.% maltopentose units, less than 1 % consecutive a(l->6) glucosidic linkages (for example less than 0.5 % consecutive a(l->6) glucosidic linkages, for further example no consecutive a(l->6) glucosidic linkages), an average molecular mass between 1 x 10 s Da and 40 x 10 s Da and may comprise structures (for example linear structures) having chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages. The a-glucan of the invention may comprise at least 1 % of structures having chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages, for example at least 10 % structures having chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages, for further example at least 20 % structures having chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages. The α-glucan of the invention may have at least 5 % a(l->6) glucosidic linkages. The Paenibacillus beijingensi GTFD enzyme synthesizes a high and a low molecular mass polymer from amylose V (see Fig. 20). Both polymer fractions have high degrees of branching of around 17-21%. The Paenibacillus beijingensi GTFD enzyme can synthesize polymers with long a(l->4) sequences, for example maltohexaose and maltoheptaose units (see Fig.24). Under the experimental conditions used, the high molecular mass polymer has an average molecular mass of 27 x 10 s Da and the low molecular mass polymer has an average molecular mass of 19 x 10 3 Da. This provides the possibility of creating reuteran-like polymers of different sizes. The α-glucan of the invention may have a ratio of branching of at least 15% (for example at least 17%, for further example at least 20%) and have an average molecular mass between 22 x 10 s Da and 35 x 10 s Da. The α-glucan of the invention may have a ratio of branching of at least 15% (for example at least 17%, for further example at least 20%) and have an average molecular mass between 15 x 10 3 Da and 25 x 10 3 Da. The α-glucan of the invention may have more than 1 wt.% maltohexaose units. The α-glucan of the invention may have more than 1 wt.% maltoheptaose units. The α-glucan of the invention may have a ratio of branching of at least 17%, an average molecular mass between 15 x 10 3 Da and 25 x 10 3 Da, more than 1 wt.% maltohexaose units and more than 1 wt.% maltoheptaose units. In a further aspect, the invention provides an α-glucan obtainable (for example obtained) by contacting a polysaccharide or oligosaccharide substrate comprising at its non-reducing end at least two a(l->4) linked D-glucose units with an a-glucanotransferase enzyme comprising an amino acid sequence having at least 80% identity to SEQ ID NO:l, or at least 80% identity to SEQ ID NO:92.

The α-glucan of the invention can be regarded as a dietary fiber. Due to its highly branched structure, the α-glucan will resist enzymatic degradation in the upper gastrointestinal tract and end up in the large intestine where it can be fully fermented by the colonic microflora. In addition, such dietary fibres enhance satiety in humans or animals. Blood sugar levels rise after a meal. As the a-glucans of the invention display reduced digestibility compared to materials such as starch, meals prepared containing them will cause a reduced blood glucose response compared to the equivalent meal with starch, and will provoke a lower insulin response. A composition comprising the α-glucan of the invention may be for use in the control of postprandial blood glucose and insulin levels in a subject. The subject may be a human or a pet. A composition comprising the α-glucan of the invention may be for use in the treatment or prevention of a disorder linked to an increase in postprandial blood glucose and insulin levels in a subject. The disorder may be selected from the group consisting of diabetes, for example gestational diabetes; impairment of glucose metabolism; hyperinsulinemia or insulin resistance. The subject may be a diabetic or pre-diabetic human or pet. Typically, postprandial hyper-insulinemia may promote the development of insulin resistance, metabolic syndrome, glucose intolerance and type-2 diabetes [Kopp W., Metabolism. 2003, Jul; 52(7):840-844]. Lowering the insulin demand after a meal however, can reduce on one hand the deterioration of the glycemic control in type-2 diabetes and on the other hand reduce the risk of developing type-2 diabetes in predisposed subjects. A "pre-diabetic patient" is a subject showing insulin resistance or impaired glucose metabolism and is predisposed, for example by family history, lifestyle or genetics, for developing diabetes later in life. Reducing insulin secretion reduces the risk of the pancreas becoming exhausted in the long term, and so is beneficial for management of the pancreas in pre-diabetes or patients with metabolic disorders. The use of a composition comprising the α-glucan of the invention would consequently reduce the risk and/or the development of diabetes, impaired glucose metabolism, hyperinsulinemia or insulin resistance in those subjects.

Prevalence of diabetes, insulin resistance or glucose intolerance is mostly observed in adult humans. However, more and more children are affected, or predisposed or at risk of developing such a disorder later in life. Hence, advantageously, prevention and/or treatment of those disorders is started already in young age. Alternatively, and similarly as observed with humans; diabetes, hyperinsulinemia or insulin resistance are more and more widespread among animals, particularly with animals kept as pet animals. Hence, the invention also pertains to cats and dogs.

A composition comprising the α-glucan of the invention may be for non-therapeutic use to decrease plasma postprandial glucose and insulin levels. It is advantageous that a composition comprising the α-glucan of the invention can also be administered to subjects, for example healthy subjects, which may be at risk of developing diabetes type-2, insulin resistance or glucose intolerance at some later time. A composition comprising the a-glucan of the invention, as disclosed herein, provides a reduced insulin level after consumption. Many healthy people desire to lose weight. Consuming meals which contain dietary fibre can increase satiety and therefore help people consume fewer digestible calories. A composition comprising the a-glucan of the invention may be for non-therapeutic use to lose weight. Another aspect of the invention relates to a food composition comprising the α-glucan of the invention. The food composition may for example comprise between 1 and 20 wt.% of the α-glucan of the invention. The food composition may be a beverage, for example a powdered beverage mix or a beverage creamer; a breakfast cereal; a pet food product; a baked dough product, for example a bread, a pizza or a filled savoury turnover; or a confectionery product. The confectionery product may be a frozen confectionery product such as an ice-cream; a baked confectionery product such as a biscuit, for example a filled biscuit or wafer; a chocolate confectionery product; or a sugar-style confectionery product such as a gum, a jelly, a hard-boiled sweet or a chewy sweet. The term "sugar-style confectionery product" or "sugar-style candy" refers to confectionery products which would traditionally have been based on sugar, but may be manufactured with alternative sweeteners and/or sugar substitutes.

In a further embodiment, the invention provides for the use of a GTFD a-glucanotransferase enzyme for reducing the digestible carbohydrates of a food material. The invention provides for the use of an a-glucanotransferase enzyme that comprises an amino acid sequence having at least 80% identity to SEQ ID NO:l (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:l), or has an amino acid sequence of SEQ ID NO:l, for reducing the digestible carbohydrates of a food material, for example a starch-containing food material. In the scope of the current invention, digestible carbohydrates correspond to the fraction of the total carbohydrates that is digestible and available to provide energy to body cells.

The invention further provides for the use of a GTFD α-glucanotransferase enzyme that comprises an amino acid sequence having at least 80% identity to SEQ ID NO:92 (for example at least 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:92), or has an amino acid sequence of SEQ ID NO:92, for reducing the digestible carbohydrates of a food material, for example a starch-containing food material.

The invention provides for the use of a GTFD α-glucanotransferase enzyme for reducing the glycemic index of a food material, for example α-glucanotransferase enzyme that comprises an amino acid sequence having at least 80% identity (for example at least 90, 95, 96, 97, 98, or 99% identity) to SEQ ID NO:l or SEQ ID NO:92. The glycemic index is a number associated with a particular type of food that indicates the food's effect on a person's blood glucose (also called blood sugar) level. A value of 100 represents the standard, an equivalent amount of pure glucose.

Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the method of the present invention may be combined with the product of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. Further advantages and features of the present invention are apparent from the figures and experimental section.

Experimental Section

Introduction

Glycoside Hydrolase (GH) family 70 originally was established for glucansucrase (GSs) enzymes (http://www.cazy.org/), which solely are found in lactic acid bacteria (Lombard et al., 2014). GSs use sucrose to synthesize a diversity of linear and branched a-glucans, differing in the type of glycosidic linkages, the degree and type of branching, the length of the glucan chains, molecular mass, and the conformation of the polymers. Depending on the main glucosidic linkage present in their glucans products, they can be divided into dextran [a(l->6) linkages], mutan [a(l->3) linkages], reuteran [a(l->4) and a(l->6) linkages], and alternan [alternating a(l->3) and a(l->6) linkages]. Enzymes from GH70 belong to clan GH-H (also known as a-amylase superfamily), together with enzymes from families GH13 (mainly starch modifying enzymes) and GH77 (e.g. 4-a-glucanotransferases) (Stam et al., 2006). All GH-H members employ a similar catalytic mechanism, involving a covalent glucosyl intermediate and retention of the a-configuration in their products, but they vary widely in their product and reaction specificity. To date, 60 GS enzymes have been biochemically characterized and three-dimensional structures are available for four proteins with different product specificity (Vujicic-Zagar et al., 2010; Ito et al., 2011; Brison et al., 2012; Pijning et al., 2012). These crystal structures showed that GSs are structurally similar to the closely related GH13 and GH77 starch modifying enzymes, confirming earlier secondary- structure predictions (MacGregor et al., 1996; 2001). However, GSs also possess unique structural features. Firstly, compared to GH13 and GH77 enzymes, their catalytic (a/ )8-barrel domain is circularly permuted. As a consequence, in GSs the order of the four signature sequence motifs l-IV of clan GH-H is ll-lll-IV-l (Fig. 1), and does not correspond to the order found in GH13 and GH77 enzymes (l-ll-lll-IV). Secondly, these crystal structures revealed that in GS proteins the peptide chain contains 5 domains (A, B, C, IV, and V) and that the peptide chain follows an unusual "U" fold domain structure, in which 4 of the 5 domains are built up from two discontinuous segments of the polypeptide chain (Fig. 1). The A, B, and C domains form the catalytic core and are also found in GH13 enzymes. In contrast, domains IV and V are unique to GS. The role of domain IV remains to be elucidated, whereas domain V appears to be important for polysaccharide synthesis. Indeed, truncation of domain V in the Lactobacillus reuteri 180 GTF180 glucansucrase enzyme yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. This truncated protein instead produced a large amount of oligosaccharides (Meng et al., 2015). Recently, a GH70 subfamily of enzymes inactive on sucrose, but displaying clear hydrolase/transglycosylase activity on malto-oligosaccharides (MOS) and starch has been identified, which also reflects the evolutionary relationships between families GH70 and GH13 (Kralj et al., 2011). Members of this subfamily have clear sequence similarity with GS enzymes, and are only found in lactic acid bacteria. The Lactobacillus reuteri 121 GTFB enzyme is the first characterized member of this subfamily cleaving a(l->4) linkages and synthesizing a(l->6) linkages, designated as a 4,6-a- glucanotransferase (4,6-a-GT) enzyme. As a result of GTFB enzyme activity, linear isomalto-/malto- polysaccharides (IMMPs) are produced which consist of linear a(l->6)-glucan chains attached to the non-reducing ends of MOS or starch fragments. IMMPs represent a new type of starch derived soluble dietary fiber according to in vitro studies, and as such they are potentially valuable food ingredients (Bai et al., 2015a; Dobruchowska et al., 2012; Dijkhuizen et al., 2010; Leemhuis et al., 2014). The exact in vivo role of GTFB-like enzymes remains unknown. It should be noted that the gtfB gene was identified upstream of the gtfA gene from L. reuteri 121 encoding a regular GH70 glucansucrase that converts sucrose into reuteran, a branched glucan with alternating a(l->4) and a(l->6) linkages) (Kralj et al., 2002; van Leeuwen et al., 2008a, Dobruchowska et al., 2013). More recently a second GH70 subfamily with 4,6-a-glucanotransferase activity in non-lactic acid bacteria has been identified and the Exiguobacterium sibiricum 255-15 GTFC enzyme (Gangoiti et al., 2015) characterized. Exiguobacterium is a genus of bacilli and the data shows that biochemically this GTFC enzyme is rather similar to the L. reuteri GTFB enzymes. A main difference observed is that this GTFC enzyme synthesizes lsoMalto-/Malto-Oligosaccharides (IMMOs), instead of a (modified) polymer (IMMP) from malto-oligosaccharides and amylose V. Regarding their amino acid sequences, the L. reuteri 121 GTFB and the E. sibiricum GTFC share only 30% identity, but they display high conservation in their signature motifs l-IV. However, the order of these conserved motifs in GTFC enzymes is l-ll-lll- IV reflecting their non-permuted domain organization, similar to GH13 proteins but differing from the permuted order ll-lll-IV-l characteristic for glucansucrases and GTFB-like 4,6-a-glucanotransferases (Fig. 1). GTFC enzymes share the A, B and C domains with GH13 enzymes. GTFC enzymes additionally possess a domain IV inserted into domain B, however, and in several cases Ig-like2 domains that may allow cell surface binding. In view of its activity and domain organization, the E. sibiricum GTFC 4,6-a- glucanotransferase enzyme represents an evolutionary intermediate between GH13 a-amylases and GH70 glucansucrases/ GTFB-like 4,6-a-glucanotransferases (Gangoiti et al., 2015).

This experimental section reports the biochemical characterization of a novel GH70 enzyme with unique product specificity and a different microbial origin, Azotobacter chroococcum NCI MB 8003. This protein was designated GTFD and resembles 4,6-a-glucanotransferases in using maltodextrins and starch as substrates. However, the A. chroococcum GTFD enzyme catalyzes the synthesis of a high molecular mass and relatively highly branched a-glucan with alternating a(l->4) and a(l->6) glucosidic bonds from amylose. This product does not contain consecutive a(l->6) linkages, clearly differing from the IMMPs and IMMOs synthesized by the L reuteri 121 GTFB and E. sibiricum GTFC, respectively. The structure of the polymer formed by the A. chroococcum GTFD enzyme is more similar to that of the reuteran synthesized by the L. reuteri 121 GTFA glucansucrase from sucrose (Kraj et al., 2004; van Leeuwen et al., 2008a; Dobruchowska et al., 2013). With the discovery of this novel Azotobacter GTFD 4,6-a-glucanotransferase it appears that the distribution of GH70 enzymes is not limited to Gram- positive bacteria. A. chroococcum NCIMB 8003 is a soil-dwelling, l\h fixing Gram-negative bacterium that forms thick-walled cysts. Based on the analysis of its recently elucidated genome, this bacterium was predicted to produce extracellular polysaccharides such as alginate, levan and cellulose ( obson et al., 2015). Our work suggests that A chroococcum also is able to synthesize a reuteran-like a-glucan from starch/maltodextrins. The in vivo role of the polymer produced by the A. chroococcum GTFD 4,6- a-glucanotransferase enzyme remains to be investigated.

A. chroococcum GTFD showed 48% identity in amino acid sequence to a hypothetical GH70 enzyme encoded by Paenibacillus beijingensis DSM 24997 (Genbank accession WP_052702730.1). With the aim of further expanding the repertoire of starch-converting GH70 family enzymes, the GTFD enzyme of the plant-growth promoting rhizobacterium Paenibacillus beijingensis DSM 24997 was characterized. Our data shows that the P. beijingensis GTFD is also a reuteran-like polymer-forming 4,6-a-GTase, providing the first example of this novel reaction and product specificity in a Gram- positive bacterium. Besides, differences between the products synthesized by the action of the A. chroococcum GTFD and P. beijingensis GTFD were found, enlarging the range of reuteran-like polymers that can be synthesized from amylose. Finally, A. chroococcum GTFD and P. beijingensis GTFD isolated reuteran-like polymers, and the reaction mixtures obtained from starch incubations were subjected to in vitro digestibility studies with digestive enzymes (porcine pancreatin and rat intestinal powder extracts) GCT Fliki i t e pr oensn- other to evaluate the potential use of these enzymes for the production of less easily digestible starchy food.

Materials and methods Phylogenetic analysis

The E. sibiricum 255-15 sequence was retrieved from the GH70 database (http://www.cazy.org) and used as query in BLASTp searches (http://www.ncbi.nlm.nih.gov/BLAST/). Phylogenetic analysis was performed using MEGA, version 6 (Tamura et al., 2013) with a total of 71 amino acid sequences corresponding to representative GH70 and GH13 sequences identified via BLASTp. The GenBank accession numbers of the sequences used in this section are listed in Table 1. Sequences were aligned by MUSCLE, using default parameters. A phylogenetic tree was constructed by the Maximum Likelihood method based on the JTT matrix based model using in MEGA6. Partial deletion of the positions containing alignment gaps and missing data was conducted. Statistical confidence of the inferred phylogenetic relationships was assessed by performing 1,000 bootstrap replicates. Table 1. GenBank accession numbers of the family GH70 and GH13 protein sequences used in the phylogenetic tree of figure 2. gi|5013398771 dextransucrase Exiguobacterium sibiricum 255- 15

gi|654644457| dextransucrase Exiguobacterium undae

gi|504784268| dextransucrase Exiguobacterium antarcticum B7

gi|657229007| dextransucrase Exiguobacterium acetylicum gi|737429885| dextransucrase Exiguobacterium sp. RIT341

gi|737339900| dextransucrase Bacillus kribbensis

gi|737335335| dextransucrase partial Bacillus coagulans gi|757632476| dextransucrase partial Bacillus coagulans gi|335366490| dextransucrase Bacillus coagulans 2-6

gi|753712235| alpha amylase catalytic domain protein Bacillus coagulans DSM 1 ATCC 7050

GTFD protein→ gi|747128849| dextransucrase Azotobacter chroococcum NCIMB 8003

gi|489780009| dextransucrase partial Lactobacillus fermentum ATCC 14931 θ £ ώ = <D ο- ο gi|490699763| dextransucrase partial Lactobacillus fermentum 28-3-CHN gi|738781828| glycosyl hydrolase family 70 partial Pediococcus pentosaceus gi|767167586| glycosyl hydrolase family 70 Lactobacillus delbrueckii gi|312280789| dextransucrase Lactobacillus delbrueckii subsp. bulgaricus ND02 gi|357208772| putative glucansucrase Lactobacillus reuteri

gi|737661728| hypothetical protein partial Lactobacillus acidipiscis

gi|754206103| dextransucrase partial Lactobacillus sanfranciscensis gi| 1894857841 inactive glucansucrase Lactobacillus reuteri

gi|752446660| hypothetical protein partial Lactobacillus plantarum

gi|335350722| inactive glucansucrase Lactobacillus salivarius GJ-24 gi| 148531598| dextransucrase Lactobacillus reuteri DSM 20016

gi|515741611 glucansucrase Lactobacillus parabuchneri

gi| 1 184325731 dextransucrase Oenococcus oeni ATCC BAA-1 163

gi|599001939| glucosyltransferase GTFG Lactobacillus sucicola DSM 21376 JCM 15457

gi|68160983I Ir1943 Lactobacillus reuteri

gi|7684297| glucosyltransferase Streptococcus oralis

gi| 146741366| glucosyltransferase Streptococcus criceti

gi|390484468| glucansucrase Weissella confusa LBAE C39-2

gi|3130095| glucosyltransferase-SI Streptococcus mutans

gi|251825496| glucansucrase Leuconostoc lactis

gi|217330914| glucansucrase Weissella cibaria

gi|444737522| dextransucrase Lactobacillus curvatus

gi| 1456664401 dextransucrase Lactobacillus reuteri

gi|6328140811 glucosyltransferase-l Lactobacillus kunkeei EFB6

gi|599000389| glucosyltransferase GTFG Lactobacillus sucicola DSM 21376 JCM 15457

gi|51574173| glucansucrase Lactobacillus reuteri

gi|296470401 | dextransucrase Leuconostoc citreum gi|221388451 glucosyltransferase Streptococcus sobrinus

gi|679092076| alternansucrase Leuconostoc citreum

gi|51574154| glucansucrase Lactobacillus reuteri

gi|3740743311 chain A Crystal Structure Of Leuconostoc Mesenteroides NrrI B-1299 N gi|755167313| alpha-amylase Streptococcus pneumoniae

gi|565861005| alpha-amylase Streptococcus mitis

gi|494939858| alpha-amylase Bacteroides intestinalis DSM

gi|492283664| alpha-amylase Bacteroides fragilis

gi|806522612| alpha-amylase Parabacteroides goldsteinii DSM 19448 WAL 12034 gi|446718150| alpha-amylase Escherichia fergusonii

gi|737197682| alpha-amylase Bacillus coagulans

gi|499280363| alpha-amylase Agrobacterium tumefaciens

gi|753800608| alpha-amylase Odoribacter splanchnicus

gi|657310130| cytoplasmic alpha-amylase Escherichia coli

gi|82501 15| maltohexaose-forming alpha-amylase Geobacillus stearothermophilus gi|228609270| glucan 14-alpha-maltohexaosidase Bacillus cereus MM3

gi|228615225| glucan 14-alpha-maltohexaosidase Bacillus cereus AH621 gi|2642326| alpha amylase Geobacillus stearothermophilus

gi|507056452| alpha-amylase Bacillus cereus

gi|51 10137571 alpha-amylase Bacteroides thetaiotaomicron

gi|1351934| alpha-amylase Geobacillus stearothermophilus

gi|504109272| alpha-amylase Klebsiella pneumoniae

gi|652433783| alpha-amylase Exiguobacterium sibiricum

gi|228773147| glucan 14-alpha-maltohexaosidase Bacillus thuringiensis serovar tochigiensis BGSC 4Y1

gi|652438445| alpha-amylase Exiguobacterium undae

gi|506403824| alpha-amylase B Halothermothrix orenii H 18

gi|782762336| cytoplasmic alpha-amylase Klebsiella pneumoniae

gi|518247975| hypothetical protein Anoxybacillus kamchatkensis gi|763044452| alpha-amylase Bacillus licheniformis S 16

gi|647494625| alpha-amylase Bacillus licheniformis

gi|304346690| glucan 14-alpha-maltohexaosidase Paenibacillus curdlanolyticus YK9 gi| 1 13762| Maltohexaose-producing amylase Bacillus sp. 707

Protein sequence analysis

Signal peptide cleavage site was predicted using the Signal P4.1 server (httpJwww.cbs. dtu.dk/services/SignalP/). Conserved domain searches were performed using the Pfam server (http://pfam.sanger.ac.uk/). Pairwise sequence comparison between the functional regions identified by the Pfam server and the Lactobacillus reuteri 121 GTFB and Exiguobacterium sibiricum 255-15 GTFC sequences were performed using Jalview (Waterhouse et al., 2009). Alternatively, multiple amino acid sequence alignments were generated with the ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2) and visualized by using Jalview (Waterhouse et al., 2009). Cloning of the gtfD gene from A. chroococcum

The DNA fragment coding for the full length GTFD protein (GenBank entry AJE22990.1, amino acids 29-780) was amplified from A. chroococcum NCIMB 8003 chromosomal DNA with Phusion DNA polymerase (Finnzyme) and cloned into a modified pET15b vector using ligation-independent cloning (LIC). For LIC cloning the following primer pairs with 5' extensions (in bold) were used for PCR amplification: Forward CAGGGACCCGGTGCACCGGCCCCCACGGCGCTCG and Reverse CGAGGAGAAGCCCGGTTACTCCTGGGCCTGGAGGTCCGGAACCC. Basically, the Kpnl-digested vector and gtfD PCR product were isolated from gel and then treated with T4 DNA polymerase (New England Biolabs) in the presence of dTTP and dATP, respectively. The two reaction products were mixed together in a 1:4 molar ratio, and the mixture was used to transform Escherichia coli DH5a cells (Phabagen). This resulted in a gtfD construct containing an N-terminal His6-tag cleavable by a 3C protease. The constructed expression vector pET15b/gf D was transformed into host E. coli BL21 Star (DE3). The gene sequence was verified by nucleotide sequencing (LGC genomics, Berlin, Germany).

Protein expression and purification - A. chroococcum

E. coli BL21 Star (DE3) harboring the gtfD gene of A. chroococcum NCIMB 8003 was cultured in Luria Broth medium supplemented with ampicillin (100 μg ml "1 ) at 37°C and 230 rpm. When the cultures reached OD600 ~ 0.6, the inducer isopropyl- -D-l-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and cultivation was continued for 24 h at 16°C. The cells were harvested by centrifugation (10,000 g, 20 min) and subsequently lysed with B-PER protein extraction reagent (Thermo Scientific, Pierce). After centrifugation (15,000 g, 20 min) the cell-free extracts with soluble GTFD protein were subjected to Ni-IMAC chromatography. Following washing with 20 mM Tris-HCI (pH 8.0) and 1 mM CaC the proteins were eluted with 200 mM imidazole in the same buffer. The GTFD containing fractions were concentrated and exchanged into 20 mM Tris-HCI buffer 1 mM CaC (pH 8.0) in a stirred ultrafiltration unit (Amicon, Beverly, MA) on a 30,000 molecular mass cut-off membrane. Purity was checked by SDS-PAGE, and protein concentrations were determined using a Nanodrop 2000 spectrophotometer (Isogen Life Science, De Meern, The Netherlands). Cloning of the P. beijingensis GTFD gene

The 2241-bp DNA fragment coding for the full-length GTFD enzyme without its putative signal peptide- encoding sequence (amino acids 31 to 776) was amplified by PCR using Phusion DNA polymerase (Finnzyme, Helsinki, Finland) and the P. beijingensis chromosomal DNA (DSM 24997) as the template. The PCR primers used for amplifying the GTFD gene incorporated 5' extensions (in bold) to facilitate the ligation-independent (LIC) cloning and were: PbF (5

CAGGGACCCGGTG CG G A A AG C AATG CG A A AG G 3') and PbR (5

CG AG G AG AAG CCCG GTTA ATTG CT AAACCGTCTT AATG CTTTATTC 3'). The GTFD PCR product was cloned into a modified pET15b vector by ligation-independent cloning (LIC), resulting in a GTFD construct containing an N-terminal His6-tag cleavable by a 3C protease. The constructed expression vector pET15/PbGTFD was transformed into host E. coli BL21 Star (DE3). The construct was confirmed by sequencing (GATC, Cologne, Germany).

Recombinant P. beijingensis GTFD protein production in E. coli and purification

Escherichia coli BL21 Star (DE3) carrying pET15/PbGTFD was grown in 500-ml LB medium containing 100 μg ml "1 ampicillin in a rotary shaker (37°C, 220 rev min "1 ) to an optical density at 600 nm of 0.4- 0.6. Expression of recombinant GTFD was induced by adding isopropyl- -d-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM, and cultivation was continued at 16 °C for 20 h. Cells were harvested by centrifugation (10,000 g x 20 min) and then disrupted with B-PER lysis reagent (Thermo Scientific, Pierce). After centrifugation (15,000 g x 20 min), the soluble GTFD protein was purified from the cell-free extract by His-tag affinity chromatography using Ni 2+" nitrilotriacetate (Ni-NTA) as column material (Sigma-Aldrich). After washing the column with 25 mM Tris-HCI (pH 8.0), 1 mM CaC , bound proteins were eluted with 200 mM imidazole in the same buffer and the imidazole was removed by use of a stirred ultrafiltration unit (Amicon, Beverly, MA) with a 30,000 molecular weight cut off. Purity and homogeneity of the purified protein was analyzed by SDS-PAGE and the amount of protein in the enzyme solutions was routinely determined with a (Nanodrop 2000 spectrophotometer (Isogen Life Science, De Meern, The Netherlands).

Enzyme assays The initial total activity of GTFD enzyme of A. chroococcum NCIMB 8003 was determined by the amylose-iodine method (Bai et al., 2015b) using 0.125% (w/v) amylose V (AVEBE, Foxhol, The Netherlands) or 0.125% (w/v) potato starch (Sigma-Aldrich) as substrates. This assay measures the decrease in absorbance of the a-glucan-iodine complex resulting from transglycosylation and/or hydrolytic activity. Enzymatic assays were performed with 28 μg/ml of enzyme in 25 mM sodium phosphate buffer (pH 6.5) at 50 °C. One unit of activity corresponds to the amount of enzyme converting 1 mg of substrate per min. The optimal pH and temperature were determined over the pH range of 4.5-9.5 and a temperature range of 30-75°C, using amylose V as substrate. Sodium acetate buffer (25 mM) was used at pH 4.5 - 6.0, MOPS buffer (25 mM) at pH 6.0 - 7.0, Tris-HCI buffer (25 mM) at pH 7.0 - 8.0, and glycine-NaOH at pH 9.0 - 9.5. For thermostability studies, the enzyme (0.5 mg/ml) was incubated in the absence of substrate for 10 min at different temperatures from 50 to 95 °C, and then immediately cooled to 4 °C. The residual activity was measured as described above.

The initial activity of the purified P. beijingensis GTFD (PbGTFD) enzyme was determined using 0.125% (w v 1 ) amylose V (AVEBE, Foxhol, The Netherlands) as substrate by the iodine-staining assay. This method monitors the decrease in absorbance at 660 nm in time of the α-glucan-iodine complex resulting from transglycosylation and/or hydrolytic activity. Enzymatic assays were carried out with 12 μg ml 1 of enzyme in 25 mM sodium phosphate buffer (pH 7.0) containing 1 mM CaC at 50°C. One unit of activity is defined as the amount of enzyme converting 1 mg of substrate per min. The optimal pH and temperature were determined over the pH range of 4.5-10.0 and a temperature range of 35-60°C. Sodium citrate buffer (25 mM) was used for pH between 4.5 and 7.0, Sodium phosphate buffer (25 mM) for pH between 7.0 and 8.0, Tris-HCI buffer (25 mM) for pH between 8.0 and 9.0, and sodium bicarbonate buffer for pH between 8.0 and 9.0.

Substrate/product analysis with GTFD

Purified GTFD enzyme (40 μg ml "1 ) of A chroococcum NCBI 8003 was incubated separately with 25 mM sucrose (Acros), nigerose (Sigma-Aldrich), panose (Sigma-Aldrich), isomaltose (Sigma-Aldrich), isomaltotriose (Sigma-Aldrich), isomaltopentaose (Carbosynth), β-cyclodextrins (Sigma-Aldrich), malto-oligosaccharides (MOS) with different degrees of polymerization (G2 to G7), and 0.6% (w/v) amylose V (AVEBE, Foxhol, The Netherlands), potato starch (Sigma-Aldrich) and amylopectin (Sigma- Aldrich). All reactions were performed in 25 mM sodium phosphate buffer, pH 6.5 with 1 mM CaC at 37 °C for 24 h. The same conditions were used for the analysis of the oligosaccharides formed in time with maltohexaose and amylose V, but in this case 20 μg ml 1 of GTFD enzyme was used. For acceptor substrate studies, GTFD (40 μg ml "1 ) was incubated in 25 mM sodium phosphate buffer, pH 6.5, containing 1 mM CaC , with the acceptor substrates maltose or isomaltose (25 mM) in the presence of 0.35% (w/v) amylose V (donor substrate). The reaction mixtures were incubated at 37 °C for 24 h. In all cases, the progress of the reactions was followed by high-performance-anion-exchange chromatography (HPAEC) and/or thin-layer chromatography (TLC).

The recombinant P. beijingensis GTFD enzyme (40 μg ml "1 ) was incubated separately with 25 mM sucrose (Acros), nigerose (Sigma-Aldrich), panose (Sigma-Aldrich), isomaltose (Sigma-Aldrich), isomaltotriose (Sigma-Aldrich), isomaltopentaose (Carbosynth), malto-oligosaccharides (MOS) with degrees of polymerization (DP) 2-7, and 0.6% (w/v) amylose V (AVEBE, Foxhol, The Netherlands), and amylopectin (Sigma-Aldrich). All incubations were performed in 25 mM sodium phosphate buffer (pH 7.0) with 1 mM CaC at 37 °C for 24 h. Reactions were stopped by heating the samples to 100 °C for 8 min. The progress of the reactions was assessed by thin-layer chromatography (TLC) and/or high- performance-anion-exchange chromatography (HPAEC). Thin Layer Chromatography and High Performance Anion Exchange Chromatography with pulsed amperometric detection analysis

Samples were spotted in 1-cm lines on TLC sheets (Merck Silica Gel 60 F254, 20x20 cm), and after drying, the plates were run for 6 h in n-butanol:acetic acid:water, 2:1:1, v/v. The bands were visualized with orcinol/sulfuric acid staining and compared with a simultaneous run of a mixture of glucose and MOS (DP2 to DP7).

Product mixtures from incubations with GTFD were analyzed by HPAEC on an ICS-3000 workstation (Dionex, Amsterdam, The Netherlands), equipped with an ICS-3000 ED pulsed amperometric detection system (PAD). The oligosaccharides were separated on a CarboPac PA-1 column (Dionex; 250 x 4 mm) by using a linear gradient of 10-240 mM sodium acetate in 100 mM NaOH at a 1 ml/min flow rate. The identity of the peaks was assigned using commercial oligosaccharide standards. Production and analysis of the products from amylose incubation with GTFD

Purified A. chroococcum and P. beijingensis GTFD enzymes (0.2 mg) were separately incubated with amylose V under the conditions described in "Substrate/product analysis with GTFD". After incubation for 24 h at 37 °C, the reactions were stopped by transfer to 95 °C for 6 min. The HMM polymer produced by the A. chroococcum GTFD enzymewas isolated by size-exclusion chromatography on a Bio-Gel P-2 column (2.5 x 50 cm; Bio-Rad) using 10 mM NH 4 HC0 3 as eluent at a flow rate of 48 ml/h. The HMM and LMM polysaccharide fractions generated by P. beijingensis GTFD activity on amylose by size-exclusion chromatography on a Superdex S-200 (10 x 300 mm; GE-Healthcare) using 25 mM ammonium bicarbonate as eluent at a flow rate of 0.5 ml min-1. (i) Methylation analysis

Methylation analysis of the isolated polysaccharides was performed as described before (van Leeuwen et al., 2008b) by per-methylation of the polysaccharides using CH3I and solid NaOH in DMSO, followed by acid hydrolysis with TFA. Partially methylated monosaccharides were reduced with NaBD 4 . The resulting partially methylated alditols were per-acetylated using pyridine:acetic anhydride (1:1 v/v) at 120 °C. Partially-methylated alditol acetates (PMAAs) were analysed by GLC-EI-MS and GLC-FID as described (Van Leeuwen et al., 2008b).

(ii) HPSEC analysis

The molecular mass distribution of the products mixtures were determined by HPSEC-MALS-RI as described previously (Bai et al., 2015b). Briefly, samples were dissolved at a concentration of 3.5 mg ml 1 in DMSO-LiBr (0.05 M) and analyzed by high-performance size-exclusion chromatography coupled on-line with a multi angle laser light scattering detector (SLD 7000 PSS, Mainz), a viscometer (ETA-2010 PSS, Mainz) and a differential refractive index detector (G1362A 1260 RID Agilent Technologies). The separation was done by three PFG-SEC columns with porosities of 100, 300 and 4000 A, coupled with a PFG guard column using DMSO-LiBr (0.05 M) as eluent at a flow rate of 0.5 ml min "1 . The system was calibrated and validated using a standard pullulan kit (PSS, Mainz, Germany) with M w ranging from 342 to 805 000 Da. The multiangle laser light scattering signal was used to determine the molecular masses of the amylose and the HMM polymers generated by the A. chroococcum and P. beijingensis GTFD enzymes with a refractive index increment value (dn/dc) of 0.072. The molecular mass of the P. beijingensis LMM polymer was determined by universal calibration method. WinGPC Unity software (PSS, Mainz) was used for data processing. Measurements were performed in duplicate. (iii) NMR spectroscopy

One-dimensional 1 H nuclear magnetic resonance (NMR) spectra of the product mixtures and the isolated polysaccharide were recorded in D2O on a Varian Inova 500 spectrometer (NMR Center, University of Groningen) at a probe temperature of 298K and processed with MestReNova 5.3 (Mestrelabs Research SL, Santiago de Compostella, Spain). Samples were exchanged twice in D2O (99.9 atom% D, Cambridge Isotope Laboratories, Inc., Andover, MA) with intermediate lyophilization, and then dissolved in 0.6 mL of D2O. Chemical shifts were expressed in ppm and calibrated by internal standard acetone (δ 2.225 ppm). The percentage of different linkages was estimated by integration of the respective signal peak areas. Enzymatic treatment of GTFD product

Reuteran GTFA polymer, IMMP GTFB polymer, P. beijingensis GTFD HMM and LMM polysaccharides and A. chroococcum GTFD polymer (5 mg/ml) were subjected to enzymatic digestions using excess a- amylase (Aspergillus oryzae a-amylase; Megazyme), dextranase (Chaetomium erraticum; Sigma- Aldrich), or pullulanase Ml (Klebsiella planticola; Megazyme). Enzymatic hydrolysis of each polymer was carried out in 50 mM sodium acetate buffer pH 5.0 for 48 h at 37 °C. The degree of hydrolysis was analyzed by TLC and/or HPAEC analysis. Starch, dextran and pullulan, were used as positive controls for the a-amylase, dextranase and pullulan treatments, respectively, resulting in complete hydrolysis under these conditions.

Results and discussion Example 1: Identification of a novel GH70 protein encoded by Azotobacter chroococcum

Recently the identification of a novel GH70 subfamily represented by the Exiguobacterium sibiricum 255-15 GTFC 4,6-a-GT (designated GTFC enzyme) has been reported (Gangoiti et al., 2015). Aiming to find new enzymes active on maltodextrins and/or starch, but displaying different product specificities, a BLASTp search was carried out using the E. sibiricum GTFC protein as query sequence. As described before (Gangoiti et al., 2015), this allows identification of GTFC homologs in various Exiguobacterium and Bacillus strains, sharing more than 75 and 54% of identity with the E. sibiricum GTFC protein, respectively (Table 2). This new BLASTp search also resulted in identification of an additional GTFC homolog (78% coverage, 39% identity with the E. sibiricum GTFC) present in the recently elucidated genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412), annotated as a dextransucrase (Robson et al., 2015). The next hits obtained using BLASTp were (putative) GTFB-like 4,6-a- glucanotransferases followed by (putative) family GH70 glucansucrases and family GH13 proteins. The evolutionary relationships among representative GH70 and GH13 family members identified by BLASTp are depicted in Fig. 2. As reported before, GTFC-like proteins cluster separately from glucansucrase- and GTFB-type of enzymes, in between family GH13 and GH70 proteins (Gangoiti et al., 2015). Phylogenetically, the hypothetical dextransucrase encoded by A chroococcum is most closely related to GTFC type of enzymes. However, it only shares 39 to 42% amino acid sequence identity with the other GTFC type of enzymes, a fact reflected in its own long branch in the tree.

Table 2: GTFC- and GTFB-like sequences identified via a BLASTp search using the Exiquobacterium sibiricum 255-15 GTFC protein as query. GTFC-like sequences are indicated. Azotobqcter chroococcum NCIMB 8003 GTFD enzyme is shown in bold.

GTFC-

NCBI protein names Organism Coverage Identity Length Accession

like?

Dextransucrase Exiguobacterium sibiricum 255-15 100% 100% 893 WP_012371512.1 Yes

Exiguobacterium undae DSM Yes

Dextransucrase 100% 94% 893 WP_028105602.1

14481

Dextransucrase Exiguobacterium antarcticum B7 100% 93% 893 WP_014971370.1 Yes

Exiguobacterium antarcticum Yes

Dextransucrase 100% 93% 893 WP_026830256.1

DSM 14480

Dextransucrase Exiguobacterium sibiricum 7-3 100% 92% 893 WP_026827371 .1 Yes

Exiguobacterium acetylicum DSM Yes

Dextransucrase 100% 80% 892 WP_029342707.1

20416

Dextransucrase Exiguobacterium sp. RIT341 100% 75% 892 WP_035410561 .1 Yes

Dextransucrase Bacillus kribbensis DSM 17871 98% 56% 904 WP_035322188.1 Yes

Dextransucrase Bacillus coagulans XZL4 81 % 58% 755 WP_035317646.1 Yes

Dextransucrase Bacillus coagulans H-1 79% 59% 730 WP_042872287.1 Yes

Dextransucrase Bacillus coagulans 2-6 86% 55% 954 AEH52441 .1 Yes alpha amylase, catalytic Bacillus coagulans DSM 1 = Yes

86% 54% 965 AJH 79253.1 domain protein ATCC 7050

Azotobacter chroococcum

Dextransucrase 78% 39% 780 AJE22990.1

NCIMB 8003

Lactobacillus fermentum ATCC

Dextransucrase 72% 35% 1014 WP_003683900.1

14931

Dextransucrase Lactobacillus fermentum 72% 35% 1478 WP_046948074.1

Lactobacillus fermentum 28-3-

Dextransucrase 72% 35% 986 WP_004563243.1

CHN

Lactobacillus delbrueckii subsp.

dextransucrase, partial 71 % 34% 966 EOD02243.1

jakobsenii ZN7a-9 = DSM 26046

glycosyl hydrolase family 70 Pediococcus pentosaceus IE-3 71 % 34% 989 WP_036673144.1

Lactobacillus delbrueckii subsp.

glycosyl hydrolase family 70 71 % 34% 954 WP_035171046.1

/aci/s CRL581

Lactobacillus delbrueckii subsp.

glycosyl hydrolase family 70 delbrueckii DSM 20074 = JCM 71 % 34% 922 WP_044880492.1

1012

cell wall binding repeat Lactobacillus delbrueckii subsp.

71 % 34% 957 WP_003613937.1 protein bulgaricus PB2003/044-T3-4

Lactobacillus delbrueckii subsp.

glycosyl hydrolase family 70 71 % 34% 1210 WP_035182758.1

lactis DSM 20072

Lactobacillus delbrueckii subsp.

Dextransucrase 71 % 34% 1294 ADQ61508.1

bulgaricus ND02

putative glucansucrase Lactobacillus reuteri 78% 30% 1620 AAU08003.2 hypothetical protein Lactobacillus reuteri 78% 30% 885 WP_042746090.1

Lactobacillus acidipiscis KCTC

hypothetical protein 70% 35% 908 WP_035631372.1

13900

putative glucansucrase Lactobacillus reuteri 78% 29% 1383 ABP88725.1

Lactobacillus sanfranciscensis

Dextransucrase 64% 34% 866 WP_041818260.1

TMW 1.1304

inactive glucansucrase Lactobacillus reuteri 121 80% 29% 1619 AAU08014.2 hypothetical protein Lactobacillus plantarum 74% 34% 1 136 WP_045353679.1 hypothetical protein Lactobacillus plantarum 16 77% 29% 1414 WP_041 161886.1 inactive glucansucrase Lactobacillus salivarius GJ-24 68% 34% 1626 EGM52218.1

Dextransucrase Lactobacillus reuteri DSM 20016 75% 30% 1363 ABQ83597.1

Dextransucrase Lactobacillus reuteri JCM 1 1 12 75% 30% 1488 WP_003668618.1

Dextransucrase Lactobacillus reuteri mlc3 75% 29% 1488 WP_019251413.1

This putative dextransucrase sequence of A. chroococcum is classified within family GH70 in the Carbohydrate-Active Enzymes (CAZy) database. Up to date, all GH70 members were encoded by members of the low GC phyla of Firmicutes, Gram-positive bacteria belonging exclusively to the orders Lactobacillales (GSs and GTFB-like 4,6-a-GTs) and Bacillales (GTFC-like 4,6-a-GTs). A. chroococcum NCIMB 8003 is an aerobic free-living member of the gamma-proteobacteria with the capacity to form dessication-resistant cysts. This Gram-negative bacterium is considered a model microorganism in the study of nitrogen fixation and hydrogen metabolism ( obson et al., 1984; Robson 1986). A. chroococcum has been recognized as a producer of extracellular exopolysaccharides (Lawson & Stacey, 1954; Cote & Krull, 1988). In view of its origin and its low sequence identity with other family GH70 proteins, we decided to carry out a detailed characterization of this novel enzyme and designated it as GTFD.

A. chroococcum GTFD protein primary sequence analysis

The complete amino acid sequence of GTFD from A. chroococcum encodes a polypeptide of 780 amino acids. This protein harbors a conserved Gram-negative signal peptide with a predicted signal peptidase cleavage site between amino acids 28 and 29, consistent with an extracellular location of this enzyme. Analysis of this GTFD sequence using the Pfam server revealed that two segments of the sequence (residues 82 - 255 and residues 416 - 780) are associated with family GH70. Sequence comparisons with E. sibiricum GTFC and L. reuteri GTFB revealed that A chroococcum GTFD has the (non-permuted) domain organization observed for the E. sibiricum GTFC-like enzymes, and differs from GTFB and glucansucrases by a circular permutation of conserved regions I -IV. Thus, from the N-terminus to the C-terminus the A. chroococcum GTFD polypeptide chain successively contributes to domains An, Bn, IV, Be, Ac and C, completely lacking domain V (Fig. 1). The A. chroococcum GTFD lacks the Ig-like 2 domains identified in the C-terminal part of the Exiguobacterium GTFC homologs (Gangoiti et al., 2015). Glucansucrases and GTFB-like 4,6-a-glucanotransferases present a long variable domain of 200 ~ 700 residues at N-terminal region that has been proposed to play a role in cell wall attachment (Bath et al., 2005). This variable domain is also not present in A. chroococcum GTFD. However, the mature A. chroococcum GTFD protein (without its signal peptide) has ~ 50 amino acid residues of unknown function at its N-terminus that are not found in the E. sibiricum GTFC enzyme (Fig. 1). The four homology regions (l-IV), considered as signature motifs in both families GH13 and GH70 were identified in the A chroococcum GTFD enzyme (Figure 3). The seven residues that are fully conserved in the GH70 family (van Hijum et al., 2006; Gangoiti et al., 2015) are also present in the A chroococcum GTFD. This includes the three (putative) catalytic residues Asp471, Glu505 and Asp572 (A chroococcum GTFD numbering). Six of these residues also are highly conserved within GH13 enzymes with the exception of Gln208 (GTFD, A chroococcum numbering), which is replaced by a His in GH13 enzymes (Hisl40, BSTA B. stearothermophilus a-amylase numbering). Motifs l-IV of A chroococcum GTFD are very similar to the motifs found in (putative) GTFC and GTFB 4,6-a-glucanotransferases, as revealed by the large number of amino acids conserved among these protein sequences. In previous studies, sequence alignments revealed that some residues known to be important for the activity and product specificity in glucansucrases were different in GTFB- and GTFC-like 4,6-a-glucanotransferases (Leemhuis et al., 2013; Gangoiti et al., 2015). Most notable are the replacement of the conserved residue W1065 in glucansucrases by a Tyr residue in the 4,6-a-glucanotransferases, and the differences in conserved region IV at positions 1137, 1140 and 1141 (GTF180 L reuteri 180 numbering). In the A chroococcum GTFD the conserved residue W1065 is replaced by a Tyr residue, and the amino acid residues at positions 1137 and 1141 are Gin and Asn, respectively, similar to other 4,6-a- glucanotransferases. In contrast, the conserved Q1140 is replaced by a His residue in A chroococcum GTFD, instead of the Lys residue present in most 4,6-a-glucanotransferases. Previous mutational studies in GTF180 glucansucrase demonstrated that residue Q1140 influences the glycosidic linkage reaction specificity (van Leeuwen et al., 2009). Besides, residue Q1140 together with residues N1411 and D1458, "block" the active site beyond subsite -1, preventing the binding of longer oligosaccharides. Due to this feature glucansucrases only transfer a single glucose moiety per reaction cycle, but not oligosaccharides (Vujicic-Zagar et al., 2010). On the other hand, the sequence order of conserved regions in A chroococcum GTFD is l-ll-lll-IV reflecting its non-permuted domain organization, similar to the E. sibiricum GTFC 4,6-a-glucanotransferase and differing from the permuted order ll-lll-IV-l characteristic for glucansucrases and GTFB homologues (Fig. 1). A chroococcum GTFD thus appears to be a novel GH13-GH70 evolutionary intermediate with a domain architecture resembling GH13 enzymes, but displaying a new enzymatic reaction specificity (see below). Purification and biochemical properties of the chroococcum GTFD

The gene encoding A. chroococcum GTFD enzyme without the putative signal sequence was cloned in- frame with an N-terminal His-tag and expressed in E. coli (DE3) BL21 star. Most of the GTFD protein was produced in soluble form and could be purified to homogeneity by metal-chelate chromatography (Fig. 4). SDS-PAGE analysis of purified enzyme revealed a single protein band with an apparent molecular weight of ~90 kDa which fits the theoretical value deduced from the sequence (86.4 kDa).

The effects of pH and temperature on A. chroococcum GTFD activity were determined using amylose V as substrate. The enzyme showed its maximum activity at 60 °C and at pH 6.5 and retained more than 70% of this activity over a pH range from 5.5 to 7.0 (Fig. 5AB). Besides, the enzyme was stable up to 60 °C for at least 10 min in 20 mM Tris-HCI buffer pH 8.0 containing 1 mM CaCI 2 (Fig. 5C). Its thermostability was higher than that of the L. reuteri 121 a-4,6-GT GTFB and GS GTFA, which are stable at temperatures up to 45°C and 50°C, respectively (Kralj et al., 2004; Bai et al., 2015b). The specific total activity values of GTFD on 0.125% (w/v) amylose and starch in sodium phosphate buffer, pH 6.5 containing 1 mM CaC and at 50 °C were 6.6 ± 0.05 and 6.0 ± 0.2 U/mg of protein, respectively. These values are significantly higher than the ones reported before for L. reuteri 121 GTFB (2.8 U/mg) and E. sibiricum 255-15 GTFC (2.2 U/mg) using amylose V as substrate (at their optimal conditions) (Bai et al., 2015b; Gangoiti et al., 2015).

Substrate and product specificity of the Azotobacter chroococcum GTFD

The substrate specificity of GTFD was studied by incubating the enzyme with different oligosaccharides and polysaccharides at 37 °C for 24 h. The enzyme was inactive on sucrose, panose, nigerose, β- cyclodextrins, and isomalto-oligosaccharides with DP2, DP3, and DP5. However, GTFD showed clear hydrolase/transglycosylase activity with malto-oligosaccharides (MOS) of DP3 to DP7 and formed a range of shorter and longer oligosaccharides (Fig. 6). Also polymer accumulation was detected in the case of G4 (a polymer of four glucose monomers) and larger MOS . However, GTFD was unable to use maltose as glucose donor substrate. When exploring the activity of GTFD enzyme with amylose V, potato starch, and amylopectin, some lower molecular mass products appeared on the TLC, reflecting its hydrolase/disproportionating activity on these polymers. Especially maltose accumulated from the various substrates (Fig. 6).

X H NM analysis of the product mixture obtained with GTFD using amylose V and starch as substrates showed two broad signals in the anomeric region (δ = ~5.40-5.35 and ~4.97) corresponding to the a(l->4) and the newly synthesized a(l->6) linkages (Fig. 7). The spectra also revealed the presence of signals corresponding to free glucose units (Get H-l, δ 5.225; H-l, δ 4.637) and 4-substituted reducing-end glucose residues (Rot H-l, δ 5.225; R H-l, δ 4.652). The molar ratio of the a(l->4) linked, a(l->6)-linked and reducing glucose residues for amylose and starch reaction products were 70:28:2 and 80:18:2, respectively. Comparison of these X H NMR spectra with those of the IMMPs generated by GTFB (Leemhuis et al., 2014) from amylose revealed the presence of extra signals strongly overlapping in the a(l->4) anomeric region in the case of the GTFD reaction mixtures. The different built-up of this broad signal at δ ~ 5.40-5.35, indicated that a different product was formed.

The molecular mass distribution of the products generated by GTFD from amylose V was determined by HPSEC-MALS-RI analysis (Fig. 8). The starting amylose V substrate eluted as a single peak at 22 ml, with an average M w of 174 χ 10 3 Da. After incubation of the amylose V with the GTFD enzyme the peak corresponding to the amylose V substrate disappeared and two other main peaks were generated, at 19 and 33 ml. The peak eluting at 19 ml corresponded to a high molecular mass polymer with an average M w of 13 χ 10 s Da, whereas the second peak eluting at 33 ml corresponded to maltose and other low molecular mass oligosaccharides. The elution profile of the products synthesized by GTFD from amylose V was different to the one obtained after incubating this substrate with the L. reuteri 121 GTFB 4,6-a-glucanotransferase. In the case of GTFB, the peak corresponding to the IMMPs (27.6 ml) eluted at a higher elution volume than the one corresponding to the starting amylose V substrate (22 ml), and had an average M w of 15 χ 10 3 Da. These data revealed that GTFD produced a high molecular mass polymer with a M w value about 80 times greater than that of the starting amylose V substrate. This polymer is also much larger than the IMMPs and the IMMOs generated by the L. reuteri 121 GTFB and the E. sibiricum GTFC (Gangoiti et al., 2015), respectively, from amylose.

Characterization of the polymer synthesized by the A. chroococcum GTFD from amylose V

For further analysis the amylose V derived high molecular mass polymer was isolated by size-exclusion chromatography on Biogel P-2 column by collecting the void-volume fraction. ID X H NMR analysis of this polymer revealed that 68 and 32% of the glucosyl units are forming a(l->4) and a(l->6) linkages, respectively, indicating a slight increase in the percentage of a(l->6) linked glucose residues over those in the reaction mixture. The X H NMR spectrum of this GTFD polymer (Fig. 9) showed high similarity to that of the reuteran synthesized by L. reuteri 121 and L. reuteri 35-5 GTFA GSs from sucrose (Kralj et al., 2002; van Leeuwen et al., 2008a). In the ID X H NMR spectrum the structural-reporter-group signal a for 6-substituted Glc residues shows a lower relative intensity for the GTFD product than for the GTFA product. Peak b, indicative for terminal residues, has a higher relative intensity. In the 2D 1 H- 1 H TOCSY spectrum the cross-peak with a is only observed in the a(l->4) anomeric track, indicating that 6-substituted Glc residues only occur in a ->6)-a-D-Glcp-(l->4)-sequence. There is no evidence for two or more consecutive a(l->6)-linked Glc residues. Moreover, signals d and e in the 2D ^C^H HSQC spectrum, corresponding with H-6a and H-6b of 6-substituted residues, have 1 H chemical shift values of δ 3.97 and 3.78, respectively. In the a(l->6)-anomeric track of the 2D 1 H- 1 H TOCSY spectrum no cross peaks at these values are observed. Peak c in the 2D ^C^H HQSC spectrum is indicative for 4- substituted residues (Van Leeuwen et al. 2008a). The resemblance of both a-glucans was confirmed by methylation analysis (Table 3). Methylation analysis of the GTFD high molecular mass product revealed the presence of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted glucopyranose residues in molar percentages of 19, 45, 18 and 18%. This shows a higher ratio of branching than the GTFA reuteran product, i.e. 18% rather than 11%, which fits with the observations in the ID Χ Η NM spectrum (Fig. 9) (van Leeuwen et al., 2008a). As above, the average molecular mass of the polysaccharide produced by A chroococcum GTFD was determined to be 13 χ 10 s Da, whereas that of the reuteran produced by GTFA GS from sucrose was 45 χ 10 s Da (Kralj et al., 2005). Thus, the high molecular mass product of GTFD showed a higher amount of branches, a lower amount of alternating a(l->4) and a(l->6) linkages, and a decreased molecular mass compared to the reuteran synthesized by GTFA from sucrose.

Table 3: Structural analysis of the polymer produced by Azotobacter chroococcum NCIMB 8003 GTFD enzyme from amylose V.

a The data represent the ratios of integration of the surface areas of the a(l->6) linkage signal at 4.97 ppm and the a(l->4) linkage signal at 5.36 ppm in the 1 H NMR spectra of the polysaccharides (see Fig. 4). b The average molecular mass of polysaccharide was determined in duplicate. c Taken from Kralj et al., 2005. Oligosaccharides formed in time from maltohexaose and amylose V by the A. chroococcum GTFD enzyme

In order to get information about the formation of products in the progress of time, both maltohexaose and amylose V were incubated with GTFD, and samples for HPAEC analysis were taken at different time points (Fig. 10). From maltohexaose, GTFD produces in addition to G2, an oligosaccharide of unknown structure with a high DP (peak eluting at around 40 min) as the first main reaction products (Fig. 10A). The excess of G2, compared to G4 suggests that GTFD catalyses a maltotetraosyl-transfer reaction yielding the peak of unknown structure. The simultaneous analysis of a mixture of MOS of DPs from 2 to 30, showed that the retention time of this peak did not match exactly with that of maltodecaose, which eluted slightly later, indicating a structure with DP10 and at least one a(l->6) linkage. Later in time, MOS of lower DPs than that of the starting donor, but also small peaks corresponding to oligosaccharides of higher DPs, are detected as a result of the GTFD main disproportionating activity. The unknown product initially formed decreased in the late stage of the reaction indicating that GTFD can use this oligosaccharide as donor and/or acceptor substrate. From amylose V, GTFD releases G2, G3, G4, G5 and G6 after 10 min of reaction as side products of its hydrolase/transglycosidase activity (Fig. 10B). Most notably, glucose is not detected at the beginning of the reaction when both maltohexaose and amylose are used as substrates. Overall, these data suggest that the A. chroococcum GTFD enzyme presents more than one donor substrate binding subsite, similar to other amylolytic enzymes, such as amylases, cyclodextrin glycosyl transferases and amylomaltases (MacGregor et al., 2001; Uitdehaag et al., 2000; Naessens et al., 2005). Thus, this GTFD does not preferentially transfer glucosyl units one by one, but MOS of low DP instead. The E. sibiricum GTFC also showed an additional endo-a(l->4)-glycosidase activity, combined with the main a(l->6) transfer activity of single glucosyl units to form IMMOs (Gangoiti et al., 2015). In accordance with this mechanism of polymerization, the A chroococcum GTFD produces an a-glucan with a α(1->6)/α(1->4) alternating structure and a(l->6) branching points.

A. chroococcum GTFD acceptor substrate reaction studies

To gain a better understanding of the mode of action of the A. chroococcum GTFD, the acceptor substrate specificity of this enzyme was studied and compared to that of L. reuteri GTFB (Fig. 11AB). When amylose V was incubated with GTFD and maltose as acceptor substrate, larger amounts of maltotriose and maltotetraose were detected than in the incubation with amylose V alone. A small amount of panose was also identified by HPAEC, probably formed by coupling of glucose with an a(l->6) linkage to the non-reducing end of maltose (minor activity). In case of GTFB, among other oligosaccharides with higher DPs, peaks at the positions of panose and maltotriose were detected. These results indicate that GTFD and GTFB have the ability to synthesize a(l->4) linkages and a(l->6) linkages when an acceptor substrate of low DP such as maltose is present. Isomaltose was a poor acceptor substrate for GTFD. HPAEC analysis showed that in the presence of isomaltose, GTFD does not form large amounts of oligosaccharides, and only small amounts of maltose and an unknown product were identified (Fig. 11A). In contrast, GTFB efficiently uses isomaltose as acceptor substrate yielding large amounts of oligosaccharides, corresponding to a isomaltose extended with a series of glucose units (isomaltotriose, isomaltotetraose and isomaltopentaose) (Fig. 11B). This result is in agreement with the presence of consecutive a(l->6) linkages in the GTFB products, and their absence in GTFD products. Enzymatic hydrolysis of the A. chroococcum GTFD polymer

To further explore the nature of the A. chroococcum GTFD polymer and to compare it with the polymers synthesized by L. reuteri 121 GTFA GS (reuteran) and GTFB 4,6-a-GT (IMMP), these 3 a- glucans were treated with a high-dose of a-amylase, dextranase and pullulanase enzymes (Fig. 12). Subsequent TLC analysis revealed that the GTFD polymer, reuteran and IMMP were resistant to the endo-a(l->4)-hydrolase action of a-amylase, whereas starch was completely hydrolysed. Only trace amounts of maltose were detected by TLC in the case of the GTFD polymer and reuteran digestion (Fig. 12A). The GTFD polymer and reuteran were resistant to endo-a(l->6)-hydrolase activity of dextranase, again confirming the absence of consecutive a(l->6) linkages in both polymers. IMMP and dextran, however, were completely degraded by the action of dextranase (Fig. 12B). In the case of pullulanase that cleaves the alternating a(l->6) linkages in pullulan and branched polysaccharides, pullulan was included as a positive control. IMMP was resistant to pullulanase treatment reflecting the presence of consecutive a(l->6) glycosidic bonds only in its structure. In contrast, pullulanase efficiently hydrolysed reuteran and the GTFD polymer, which is in agreement with the presence of alternating α(1->6)/α(1->4), and a(l->4,6) branching points in these polymers (Fig. 12C). It can therefore be seen that the polymer synthesized by L. reuteri 121 GTFB 4,6-a-GT (IMMP), described in EP2248907, contains consecutive a(l->6) glycosidic bonds and does not contain alternating a(l->6) linkages. HPAEC analysis enabled the identification of the reaction products formed from GTFD polymer after pullulanase digestion, and clearly demonstrated that the GTFD polymer is built-up from maltose, maltotriose, and maltotetraose elements linked by a(l->6) linkages (Fig. 13). The same oligosaccharides were obtained by pullulanase Ml hydrolysis of reuteran (van Leeuwen et al., 2008a). However, in case of the GTFD polymer a small additional peak corresponding to maltopentaose was also detected. Combining all data from NM spectroscopy and methylation analysis and pullulanase digestion, composite structures were formulated (Fig. 14), containing all structural elements observed for the GTFD product in comparison with the GTFA product (Van Leeuwen et al. 2008a). The most notable differences between the GTFA and GTFD products are the higher amount of branching for the GTFD product and the lower amount of a(l->6)-linkages. This results in a composite structure containing less alternating a (l->4)/ a( l->6) sequences, and more linear a(l->4) sequences. As indicated in Figure 14, the A chroococcum GTFD product comprises structures having chains of a(l->4) linked D-glucose units interspersed with a(l->6) glucosidic linkages.

Example 2: In vitro digestibility of gelatinized wheat starch treated with A. chroococcum GTFD Generation of the A. chroococcum GTFD a-glucan from wheat starch

A 0.6% suspension was prepared with wheat starch (Sigma S5127) in water. The suspension was heated to 90 °C for 10 min to gelatinize the starch and subsequently cooled to 37 °C. Three hundred and thirty three μί of 50 mM CaC solution were added to the gelatinized starch suspension followed by 283 μί of A. chroococcum GTFD (AcGTFD) enzyme solution (667 μg of enzyme / 100 mg starch). Starch suspension was incubated for 24h at 37 °C. After incubation, the treated suspension was heated at 95 °C for 6 min to inactivate the AcGTFD enzyme. Finally, the treated starch suspension was freeze-dried to obtain a powder of the AcGTFD generated material.

Preparation of digestive enzymes for assay

Pancreatin from porcine pancreas (Sigma P7545) and intestinal acetone powders from rat (Sigma 11630) were extracted in lOmM PBS solution (pH 6.8) at a 40 mg enzyme powders per mL concentration. Enzyme suspensions were vortex-mixed and sonicated in iced water for 7 min. Sonicated suspensions were then centrifuged at 10,000 x g for 30 min at 4 °C. Supernatants were collected and protein content as well as enzyme activities were measured.

Protein content measurements in digestive enzyme preparations

Protein was quantified in the supernatants obtained from the enzyme extractions with the BCA (Bicinchoninic Acid) kit for protein determination (Sigma BCA1-1KT). Supernatants were diluted 10 times and protein contents were measured after reacting with BCA reagent for 30 min at 37 °C in a spectrophotometer at 562 nm wavelength. A standard curve was prepared with bovine serum albumin standards for the calculation of total protein content. Measurements of activity of digestive enzymes

A 1% solution of soluble starch from potato (Sigma S2004) was prepared in lOmM PBS solution (pH 6.8). One hundred microliters of 1% soluble starch solution were added to 100 μΙ of pancreatin supernatant or rat intestinal acetone powders supernatant. Starch and enzyme mixtures were incubated at 37 °C for 10 min with constant stirring. Mixtures were subsequently boiled for 10 min to stop the enzymatic reaction. After allowing the mixtures to cool down, sample tubes were centrifuged at 10,000 x g for 10 min and glucose was measured via a colorimetric assay with the use of the Autokit Glucose (439-90901, Wako Diagnostics). One unit of enzyme activity was defined as the amount of protein required to hydrolyze 1 μg of glucose from soluble starch. In vitro digestibility measurements

A 1% solution of the AcGTFD generated material was prepared in lOmM PBS (pH 6.8). A 300 μΙ aliquot of the solution was pre-heated to 37 °C, along with the enzyme solutions, for 5 min. One hundred units/mg of the AcGTFD product of each digestive enzyme were added to the sample, standard and blank tube, in triplicate. Sample tubes were vortex-mixed and incubated for 20, 60, and 120 min at 37 °C with constant stirring. After each time point, a 0.5 mL aliquot of sample was transferred into a tube containing 1.5 mL of 90% aqueous ethanol. Sample aliquots in ethanol were stored at 4 °C until ready for glucose quantification. Sample aliquots were centrifuged at 10,000 x g for 10 min and glucose was measured via a colorimetric assay with the use of the Autokit Glucose (439-90901, Wako Diagnostics).

1. Results Treatment of gelatinized wheat starch with AcGTFD resulted in a material with a significantly lower rate and extent of in vitro digestibility. As can be observed from Figure 27, AcGTFD treatment (B) reduced the total digestibility of the gelatinized wheat starch by 30% after 120 min of incubation with porcine pancreatin and rat intestinal powders. Moreover, the content of rapidly digestible starch, represented by the % hydrolyzed sample after 20 min of incubation, was significantly reduced by the AcGTFD treatment.

Example 3: Identification of a novel GH70 protein encoded by Paenibacillus beijingensis

A protein homologous to the A. chroococcum GTFD was identified in the genome of Paenibacillus beijingensis DSM 24997 by BLASTp searches within the NCBI and IMG-E platforms. A. chroococcum GTFD showed 48% identity in amino acid sequence to a hypothetical GH70 enzyme encoded by Paenibacillus beijingensis DSM 24997 (Genbank accession WP_052702730.1). Primary sequence analysis of the P. beijingensis GTFD enzyme

The identified P. beijingensis GTFD protein sequence consists of 776 amino acids and contains a putative secretion signal peptidase cleavage site between amino acids 30 and 31, in accordance with the extracellular location of GH70 enzymes. The domain organization of the P. beijingensis GTFD resembles that of E. sibiricum GTFC and A. chroococcum GTFD enzymes, regarded as structurally evolutionary intermediates between GH13 and GH70 families (Fig. 2). Consequently, this enzyme displays a GH13-like domain arrangement with a non-permuted catalytic (β/α)8 barrel, but possess an extra domain IV inserted in domain B. Similar to E. sibiricum GTFC and A. chroococcum GTFD, this enzyme lacks the variable N-terminal domain and the domain V typically found in GH70 GSs and GTFB homologues. Also, the Ig2-like domains identified in the C-terminal part of some GTFC-like proteins, appeared to be absent from P. beijingensis GTFD, as observed in the case of the A. chroococcum GTFD.

On the basis of sequence alignments the four conserved regions of clan GH-H were identified in P. beijingensis GTFD and compared with those corresponding to other GH70 proteins. In accordance with its non-permuted domain organization, the order of these four conserved regions in P. beijingensis

GTFD and A. chroococcum GTFD is l-ll-lll-IV, instead of the permuted order ll-lll-IV-l characteristic of

GH70 glucansucrases and GTFB-like 4,6-a-GTases. The seven amino acid residues that are fully conserved in motifs I to IV of all GH70 family members are also found in both GTFD-like proteins (Fig.

15). Among these seven residues, the nucleophile, the general acid/base and the transition state stabilizer of the catalytic triad were identified as Asp409, Glu442 and Asp512 in P. beijingensis GTFD respectively (using P. beijingensis GTFD numbering).

Purification and biochemical properties of the P. beijingensis GTFD enzyme

Recombinant P. beijingensis GTFD without its peptide signal sequence (amino acids 31 - 776) was expressed in soluble form at high levels and purified to homogeneity from E. coli BL21 star (DE3) by His-tag affinity chromatography yielding 50 mg of pure protein per liter of culture. SDS-PAGE analysis of the pure enzyme revealed the appearance of a single ~ 80-kDa protein band (Fig. 16), consistent with the predicted molecular mass deduced from its amino acid sequence (85 kDa). The effects of pH and temperature on the enzyme activity were determined by the amylose-iodine assay (Fig. 17). The GTFD enzyme of P. beijingensis displayed its maximum activity at pH 7.0 and 50°C. A higher optimum temperature value was reported for the A. chroococcum GTFD enzyme (65 °C), whereas no significant differences in the optimal pH value existed between both GTFD enzymes. In contrast, the GTFB 4,6-a- GTases characterized from Lactobacillus strains have been reported to show significantly more acidic optimum pH values of 4.5 and 5 reflecting their adaptation to the gastrointestinal tract. The specific total activity value of the P. beijingensis GTFD in 25 mM sodium phosphate buffer, pH 7.0, containing 1 mM CaC , and at 50 °C was 6.3 ± 0.17 U mg "1 , and is similar to that of A. chroococcum GTFD (at pH 6.5 and 50°C), namely 6.6 ± 0.05 U mg "1 . Thus, both GTFD enzymes exhibited remarkably higher total specific values than those determined for the L. reuteri GTFB and the E. sibiricum GTFC 4,6-a-GTase, whose specific activity values were 2.8 U mg 1 and 2.2 U mg "1 , respectively (at 40 and pH 5 and 6, respectively) [J. Gangoiti, T. Pijning, L. Dijkhuizen (2015)].

Substrate and product specificity

The substrate specificity of the P. beijingensis GTFD (Fig. 18) was studied by incubating the enzyme with different carbohydrate substrates at 37 °C for 24 h, and compared with that of the A chroococcum GTFD enzyme (Fig. 6). The P. beijingensis GTFD enzyme was inactive on sucrose, panose, nigerose, and isomalto-oligosaccharides with DP2, DP3, and DP5 (Data not shown), similar to the A. chroococcum GTFD and other 4,6-a-GTases. Instead, the P. beijingensis GTFD enzyme catalyzed the conversion of malto-oligosaccharides (MOS) of DP 3 to 7 showing both hydrolysis and transglycosylase (disproportionation) activity (Fig. 18). Indeed, incubation of P. beijingensis GTFD with MOS of DP 3 to 7 revealed the formation of lower- and higher-molecular-mass products. Besides, with G4 and larger MOS as substrates, polymeric material was also clearly detected remaining at the origin of the TLC plates. However, the P. beijingensis GTFD failed to act on maltose. Similar substrate specificity was observed with A. chroococcum GTFD (Fig. 6). The main difference between both GTFD enzymes was observed when amylose V and amylopectin were used as substrates. As reported before, the A. chroococcum GTFD enzyme accumulated G2 and some low molecular mass oligosaccharides from these polymer substrates, reflecting its hydrolase/disproportionating activity. In contrast, these low molecular mass products were not clearly detectable by TLC when exploring the activity of P. beijingensis GTFD on amylose and amylopectin. ^NMR analysis of the product mixture generated from amylose V revealed the presence of two broad anomeric signals corresponding to the α(1->4) (δ ~ 5.40-5.35) and the newly formed a(l->6) linkages (δ ~ 4.97) (Fig. 19). This X H NMR spectrum resembled that of the products derived from amylose V by A. chroococcum GTFD treatment (Fig. 7A) suggesting that both GTFD enzymes have the same product specificity. The spectra also showed the presence of small signals corresponding to free glucose units (Got H-l, δ 5.225; H-l, δ 4.637) and 4-substituted reducing end glucose residues (Rot H-l, δ 5.225; R H-l, δ 4.652). These signals were much smaller in the case of the P. beijingensis GTFD product mixture reflecting that only trace amounts of glucose, maltose and other small oligosaccharides are present in this product, as previously observed by TLC analysis. The molar ratio of the a(l->4)-linked, a(l->6)-linked and reducing glucose residues for both reactions were nearly identical, and were 72:26:2 for the A. chroococcum GTFD and 75:25:<1, for the P. beijingensis GTFD. Methylation analysis of the product mixture synthesized by the P. beijingensis GTFD from amylose V revealed the presence of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted glucopyranose residues in a molar percentage of 18, 54, 8 and 20%, in accordance with the linkage ratios determined by 1 HNM . This result confirmed that the P. beijingensis GTFD acts as a 4,6-a-glucanotransferase cleaving a(l->4) linkages and synthesising a branched a-glucan consisting of a(l->4) and a(l->6) linkages.

Comparison of the products generated from amylose by the P. beijingensis and A. chroococcum GTFD enzymes by HPSEC with multi detection, revealed differences in their molecular mass distribution (Fig. 20). After incubating amylose V with the A. chroococcum GTFD, the single peak corresponding to amylose with a small molecular mass (approximately 200 x 10 3 Da) disappeared and two new peaks were formed: A peak eluting at ~ 19 ml corresponding to a high molecular mass (HMM) polymer with an average M w of 13 x 10 s , and a second peak eluting at ~ 34 ml corresponding to maltose and other small oligosaccharides. By contrast, the HPSEC profile of products synthesized by P. beijingensis GTFD from amylose V revealed the presence of two main polymer populations, whereas maltose and other small oligosaccharides were not significantly accumulated. Besides an early peak eluting at ~ 19 ml and corresponding to a HMM polymer with a M w of 27 x 10 s Da, a second broad peak eluting at ~29 ml and corresponding to a low molecular mass (LMM) polymer with a M w 19 x 10 3 Da was detected. The synthesis of HMM and LMM products may be the result of two distinct processive and non-processive elongation mechanisms in P. beijingensis GTFD. Based on the refractive index response, the P. beijingensis GTFD HMM polymer represented only a small percentage (less than 20%) of the total product, the LMM mass polymer being the main product of the reaction.

Characterization of the high- and low-molecular mass polymers produced by the P. beijingensis GTFD enzyme from amylose V

For a more detailed characterization the HMM and LMM polymers generated from amylose V by the P. beijingensis GTFD were isolated by size-exclusion chromatography analysis on Sephadex S-200 and subjected to 1D/2D (1)H/(13)C NMR spectroscopy. Fig. 21 presents the very similar X H NMR spectra obtained for both polysaccharides, showing a linkage ratio α(1->4):α(1->6) = 70:30 for the HMM polymer and a linkage ratio α(1->4):α(1->6) = 76:23 for the LMM polymer, indicating a slight increase in the percentage of a(l->6)-linked glucose residues in the HMM polymer. 2D NMR data of the P. beijingensis GTFD a-glucans match those of the reuteran type of polymers generated by A. chroococcum GTFD and L. reuteri 121 GTFA glucansucrase from amylose and sucrose, respectively (Fig. 22). Most notably, the typical chemical shift values corresponding to successive a(l->6) linkages were not identified in the 2D NM spectra of P. beijingensis GTFD HMM (Fig. 22) and LMM (not shown) products. The reuteran-like structure of the P. beijingensis GTFD products was further confirmed by methylation analysis, revealing the presence of terminal, 4-substiuted, 6-substituted and 4,6- disubstituted glucopyranosyl units (Table 4). The HMM P. beijingensis GTFD product contains approximately similar amounts of a(l->6) linkages than the reuteran-like polymer synthesized by A chroococcum GTFD (Table 3), but slightly differs in the amount of 6-substituted and 4,6-disubstituted glucopyranosyl residues. This results in a reuteran-like polymer with a slightly lower amount of alternating α(1->4)/α(1->6) glycosidic linkages (i.e. 14% rather than 18%) and a higher amount of branches (21% rather than 18%). Compared to the HMM P. beijingensis product, the LMM P. beijingensis GTFD product presents lower amounts of a(l->6) linkages in linear orientation reflected by the reduced amount of 6-substituted glucopyranosyl units (8% rather than 14%).

Table 4: Structural characterization of the HMM and LMM polymers synthesized by the P. beijingensis GTFD enzyme from amylose V.

3 The data represent the ratios of integration of the surface areas of the a(l->6) linkage signal at 4.97 ppm and the a(l->4) linkage signal at 5.36 ppm in the X H NMR spectra of the polysaccharides (see Fig. 4). b The average molecular mass of polysaccharide was determined in duplicate.

To gain more insight into the carbohydrate structures of the HMM and LMM P. beijingensis GTFD products, and to compare them with the A. chroococcum GTFD reuteran-like polymer and L. reuteri 121 GTFB IMMP, these a-glucans were incubated for 48 h with different hydrolytic enzymes: a- amylase, dextranase and pullulanase Ml (Fig. 23). Examination of the hydrolysis products showed that HMM P. beijingensis GTFD and A. chroococcum GTFD polymers were partly resistant to the endo-a- 1,4-hydrolase activity of the a-amylase. In both cases, only trace amounts of HMM oligosaccharides were detected after 48 h of α-amylase digestion. The P. beijingensis LMM product, however, appeared to be more susceptible to α-amylase digestion, as revealed by the decreased intensity of the spot corresponding to the polymeric material and the accumulation of HMM oligosaccharides. This result correlates well with the decreased molecular mass and lower amount of a(l->6) linkages of the LMM P. beijingensis GTFD product, compared to its HMM counterpart. As reported before, the IMMP GTFB product was also resistant to the action of the a-amylase, whereas the amylose substrate was completely degraded under the same conditions. Both P. beijingensis GTFD products and the A. chroococcum GTFD were resistant to the endo-a-1,6- hydrolase activity of dextranase, reflecting the absence of consecutive a(l->6) linkages in these polymers. In contrast, IMMP and dextran, which contain a linear backbone of a(l->6)-linked D-glucopyranosyl repeating units were efficiently hydrolyzed by the action of dextranase. Pullulanase specifically hydrolyses the a(l->6) linkages of pullulan, amylopectin, and other 4,6-branched polysaccharides. After treatment with pullulanase, the P. beijingensis GTFD and the A. chroococcum GTFD products were degraded into smaller oligosaccharides, reflecting the presence of alternating α(1->6)/α(1->4), and a(l->4,6) branching points in these polymers. IMMP was not hydrolyzed by the action of the pullulanase, which is in agreement with the presence of linear a(l->6) chains in its structure. More details of the precise structures of the P. beijingensis GTFD products were obtained by analysis of the oligosaccharides formed upon incubation of the HMM and LMM polymers with pullulanase Ml by HPAEC (Fig. 24). Incubation of the HMM P. beijingensis GTFD polymer with pullulanase yielded a mixture of MOS up to DP 11 (Fig. 24A), whereas in the case of the LMM P. beijingensis GTFD polymer additional peaks corresponding to MOSs up to DP14 were also identified (Fig. 24B). The digestion of the A. chroococcum GTFD polymer resulted in the formation of MOS of DP 2 to 5 (Fig. 24C) and confirmed that this a-glucan consists of maltose, maltotriose, maltotetraose and maltopentaose units connected via single a(l->6) bonds in linear or branched orientations. The identification of MOSs with higher DPs in the case of the HMM and LMM P. beijingensis GTFD products leads to structures containing longer linear a(l->4) sequences. The significant occurrence of MOSs of DP from 6 to 10 in the LMM P. beijingensis GTFD polysaccharide explains the observed partial digestion of this polymer by the action of α-amylase (Fig. 23A), which cleaves a(l->4)-linked glucans in an endo-type fashion. Composite models.

Using the data obtained by methylation analysis, NM spectroscopy and enzymatic digestion studies composite models could be constructed, reflecting all major structural elements observed for the HMM and LMM P. beijingensis GTFD products (Fig. 25). Comparing with the A. chroococcum GTFD product the linear a(l->4)-linked sequences are longer for HMM (up to DP6 in de model) and even longer for LMM (up to DP 7 in the model). Although longer linear DPs of consecutive a(l->4) are observed in the pullulanase digestion analysis (DP11 for HMM and DP14 for LMM), the amounts are too low to be reflected in the composite model. For the A. chroococcum GTFD product the pullulanase digestion showed only up to DP5 linear a(l->4)-linked sequences.

Oligosaccharides formed in time by the P. beijingensis GTFD enzyme from maltoheptaose

To gain a better understanding of the reaction mechanism of the P. beijingensis and A. chroococcum GTFD enzymes, both enzymes were incubated with maltoheptaose (G7), and the oligosaccharides formed in time were analyzed by HPAEC (Fig. 26). Incubation of G7 (slightly contaminated with G6 and G5) with the P. beijingensis GTFD enzyme yielded Gl, G2 and two peaks corresponding to compounds of unknown structure with a higher DP eluting at 53.5 and 55.5 min at the early stage of the reaction (Fig. 26A). A small peak corresponding to G3 was also identified, whereas the amounts of G5 and G6 remained low. HPAEC analysis of MOS standards of DPs from 2 to 30 revealed that these two peaks of unidentified structure eluted slightly earlier than maltododecaose (G12) and maltotridecaose (G13), suggesting structures with DP of 12 and 13 and at least one a(l->6) linkage. The deficit observed in the G5 and G6 released, together with the formation of Gl and G2 indicates that the P. beijingensis GTFD enzyme catalyzes the transfer of maltopentaosyl- and maltohexaosyl- moieties to a G7 acceptor substrate, yielding the two unknown peaks (peaks eluting at 53.5 and 55.5 min). After 24 h, the unknown oligosaccharides initially formed by the P. beijingensis GTFD enzyme disappeared suggesting that these compounds can be subsequently used as donor and/or acceptor substrates. When exploring the activity of A chroococcum GTFD, G2, G3 and two unknown compounds with high DP eluting at 51.4 and 53.5 min, were detected as the first clear products formed from G7 (Fig. 26B). The appearance of G2 and the peak eluting at 53.5 min, which was also observed in the case of P. beijingensis GTFD, indicates that the A. chroococcum GTFD also has the ability to catalyze a maltopentaosyl-transfer reaction from G7. The excess of G3, compared to G4, together with the identification of a peak eluting at 51.4 min, suggests that A. chroococcum GTFD is also able to cleave off a maltotetraosyl unit and transfer it to a MOS acceptor molecule. Most notably, the release of Gl as a side product of the maltohexaosyl-unit transfer reaction was not seen for the A. chroococcum GTFD during the early stage of the reaction. In agreement with this mode of action, the A. chroococcum GTFD activity on amylose results in the synthesis of a reuteran-like polymer built-up from MOS up to DP 5 linked by a(l->6) linkages. The preference for the transfer of longer glucan chains by the P. beijingensis GTFD enzyme is also reflected by the presence of longer linear a(l->4) sequences in the structure of its reuteran-like products. Overall these results indicate that the architecture of the active site of these GTFD type of enzymes may present more than one donor binding subsite, similar to other starch-converting enzymes of the evolutionary related GH13 and GH77 families (16-18). As a result, these GTFD enzymes have the ability to transfer MOS units, differing from GSs that strictly transfer a single glucose unit per reaction cycle. Differences in the number of donor substrate binding subsites may explain the differences observed in the length of the chains transferred by the P. beijingensis and the A. chroococcum GTFD enzymes.

Example 4: In vitro digestibility of amylose treated with A. chroococcum GTFD or P. beijingensis GTFD

GTFD a-glucans were generated as described above (see section "Production and analysis of the products from amylose incubation with GTFD"). Preparation of digestive enzymes for assay

Pancreatin from porcine pancreas (Sigma P7545) and intestinal acetone powders from rat (Sigma 11630) were extracted in lOmM PBS solution (pH 6.8) at a 40 mg enzyme powders per mL concentration. Enzyme suspensions were vortex-mixed and sonicated in iced water for 7 min. Sonicated suspensions were then centrifuged at 10,000 x g for 30 min at 4 °C and supernatants were collected separately.

Protein content measurements in digestive enzyme preparations

Protein was quantified in the supernatants obtained from the enzyme extractions with the BCA (Bicinchoninic Acid) kit for protein determination (Sigma BCA1-1KT). Supernatants were diluted 10 times and protein contents were measured after reacting with BCA reagent for 30 min at 37 °C in a spectrophotometer at 562 nm wavelength. A standard curve was prepared with bovine serum albumin standards for the calculation of total protein content.

Measurements of activity of digestive enzymes

A 1% solution of soluble starch from potato (Sigma S2004) was prepared in lOmM PBS solution (pH 6.8). One hundred microliters of 1% soluble starch solution were added to 100 μΙ of pancreatin supernatant or rat intestinal acetone powders supernatant separately. Starch and enzyme mixtures were incubated at 37 °C for 10 min with constant stirring. Mixtures were subsequently heated at 100 °C for 10 min to stop the enzymatic reaction. After allowing the mixtures to cool down, sample tubes were centrifuged at 10,000 x g for 10 min and glucose was measured via a colorimetric assay with the use of the Autokit Glucose (439-90901, Wako Diagnostics). One unit of enzyme activity was defined as the amount of protein required to hydrolyze 1 μg of glucose from soluble starch. In vitro digestibility measurements

1% (w/v) solutions of PbGTFD-HMM, PbGTFD-LMM, AcGTFD-HMM, Amylose V, and gelatinized wheat starch were incubated with 100 units of porcine pancreatin and rat intestinal powder extracts in lOmM PBS (pH 6.8) for 20, 60, and 120 min at 37 °C with constant stirring in a total volume of 1.15 ml. After each time point, samples were subsequently heated at 100 °C for 10 min to stop the enzymatic reaction. After allowing the mixtures to cool down, sample tubes were centrifuged at 10,000 x g for 10 min and glucose was measured via a colorimetric assay with the use of the Autokit Glucose (439-90901, Wako Diagnostics). Results

The in vitro digestibility of the α-glucan products obtained from Amylose V incubation with PbGTFD and AcGTFD enzymes was considerably reduced in comparison to gelatinized wheat starch in both rate and extent of digestion. As can be observed from Fig. 28, the enzyme products from Amylose V incubations had a total digestibility of only 10%, 22% and 30% for AcGTFD-HMM, PbGTFD-HMM and Pb-GTFD-LMM, respectively, after 120 min of incubation with porcine pancreatin and rat intestinal powder extracts. Although the initial rates (at 20 min time point) were comparable among the 3 enzyme products, it is evident that both high-molecular mass a-glucans had lower digestibility than the P. beijingensis GTFD low-molecular mass product. All the α-glucan products had a lower digestibility than Amylose V (not plotted).

Overall Conclusions

In this study we report the characterization of the GTFD enzyme of A. chroococcum NCIMB 8003, providing the first example of a family GH70 enzyme in a Gram-negative bacterium. Regarding its amino acid sequence and domain organization, this enzyme is closely related to the E. sibiricum 255- 15 GTFC, the first characterized member of a recently identified novel GH70 subfamily found in non- lactic acid bacteria (Gangoiti et al., 2015). GTFC type of enzymes are considered evolutionary intermediates between families GH13 and GH70, displaying 4,6-a-glucanotransferase activity with malto-oligosaccharides and amylose/starch, as previously found for the GTFB type of enzymes of lactobacilli. The E. sibiricum GTFC enzyme activity results in synthesis of isomalto/malto- oligosaccharides which consist of linear a(l->6)-glucan chains attached to the nonreducing ends of MOS or starch fragments. In contrast, the A. chroococcum GTFD enzyme is unable to synthesize consecutive a(l->6) linkages and converts amylose V into an α-glucan with alternating a(l->4) and a(l->6) glucosidic bonds, and with a(l->4,6) branching points. The GTFD polymer produced from amylose V thus is more similar to the reuteran produced from sucrose by the L. reuteri GTFA glucansucrase (Kralj et al., 2004; van Leeuwen et al., 2008a; Dobruchowska et al., 2013).

The A. chroococcum GTFD is the first enzyme reported so far able to synthesize a reuteran-like polymer from amylose and/or starch. This GTFD enzyme is different from other starch-converting enzymes such as the family GH13 and GH57 branching enzymes that only introduce a(l->6) branching points (Grimaud et al., 2014; Palomo et al., 2011) or the family GH15 dextrin dextrinases that synthesize consecutive a(l->6) linkages (Naessens et al., 2005). Functionally, the A. chroococcum GTFD also differed from L. reuteri 121 GTFB and E. sibiricum GTFC in its mechanism of polymerization as it preferentially transfers an oligosaccharide per reaction cycle, visible in Fig. 10, instead of a single glucose unit. In view of this feature the active site organization in A. chroococcum GTFD is predicted to present more than one donor substrate binding subsite, similar to the GH13 a-amylases. Overall, the A. chroococcum GTFD enzyme is a unique evolutionary intermediate between family GH13 and GH70 enzymes (Fig. 2) that considerably expands our knowledge about the natural diversity of family GH70 members. The A. chroococcum GTFD enzyme represents a powerful tool for the conversion of the starch present in food matrices into a health promoting food ingredient. Indeed, reuteran has been described as a potentially valuable food ingredient being regarded as a dietary fiber. Due to its highly branched structure, reuteran resists enzymatic degradation in the upper gastrointestinal tract and ends up in the large intestine where it can be fully fermented by the colonic microflora. In addition, it appears that reuteran enhances satiety in humans or animals (Ekhart et al., 2006), and can be used as a bread improver (Plijter et al., 2009). The in vivo role of this A. chroococcum GTFD reuteran-like polymer is unknown. It has been proposed that EPS provides protection to cells against desiccation and predation by protozoans or phage attack (Flemming & Wingender, 2010). Early studies reported that A. chroococcum strains NCIMB 8003 and N L B-14341 produce at least two exocellular polysaccharides, one which resembles alginate and a heteropolysaccharide (Lawson & Stacey, 1954; Cote et al., 1988). Following growth of A. chroococcum NRRL B-14341 on starch the same exopolysaccharides were isolated (Cote et al., 1988). Studies in A. vinelandii have demonstrated that alginates are essential for cyst formation (Nunez et al., 1999), and play a role in the protection of the Cvsensitive nitrogenase responsible of nitrogen fixation (Sabra et al., 2000). The A. chroococcum GTFD may have a related physiological function.

We also report on the characterization of the GTFD enzyme of P. beijingensis DSM 24997, a further enzyme able to synthesize a reuteran-like polymer from amylose and/or starch. Whereas the A. chroococcum GTFD activity on amylose results in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GTFD: a high-molecular mass polymer with an average Mw of 27 MDa and a low- molecular mass polymer with an average Mw of 19000 Da. Besides, P. beijingensis GTFD is able to transfer longer MOS units than the A. chroooccum counterpart yielding reuteran polymers containing longer linear a(l->4) sequences. From in vitro digestibility studies, all these polymers show a lower digestibility by digestive enzymes, providing strong support for the application of these enzymes for the reduction of glycemic Index of starchy products. References

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