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

Article pubs.acs.org/Biomac

Structure and Property Engineering of α-D-Glucans Synthesized by Dextransucrase Mutants Romain Irague,†,‡,§ Agnès Rolland-Sabaté,∥ Laurence Tarquis,†,‡,§ Jean Louis Doublier,∥ Claire Moulis,†,‡,§ Pierre Monsan,†,‡,§ Magali Remaud-Siméon,†,‡,§ Gabrielle Potocki-Véronèse,†,‡,§ and Alain Buléon*,∥ †

INSA, UPS, INP, LISBP, Université de Toulouse, 135 Avenue de Rangueil, 31077 Toulouse, France UMR5504, CNRS, 31400 Toulouse, France § UMR792, Ingénierie des Systèmes Biologiques et des Procédés, INRA, 31400 Toulouse, France ∥ UR1268 Biopolymères Interactions Assemblages, INRA, 44300 Nantes, France ‡

ABSTRACT: Seven dextran types, displaying from 3 to 20% α(1→3) glycosidic linkages, were synthesized in vitro from sucrose by mutants of dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, obtained by combinatorial engineering. The structural and physicochemical properties of these original biopolymers were characterized. When asymmetrical flow field flow fractionation coupled with multiangle laser light scattering was used, it was determined that weight average molar masses and radii of gyration ranged from 0.76 to 6.02 × 108 g·mol−1 and from 55 to 206 nm, respectively. The νG values reveal that dextrans Gcn6 and Gcn7, which contain 15 and 20% α(1→3) linkages, are highly branched and contain long ramifications, while Gcn1 is rather linear with only 3% α(1→3) linkages. Others display intermediate molecular structures. Rheological investigation shows that all of these polymers present a classical non-Newtonian pseudoplastic behavior. However, Gcn_DvΔ4N, Gcn2, Gcn3, and Gcn7 form weak gels, while others display a viscoelastic behavior that is typical of entangled polymer solutions. Finally, glass transition temperature Tg was measured by differential scanning calorimetry. Interestingly, the Tg of Gcn1 and Gcn5 are equal to 19.0 and 29.8 °C, respectively. Because of this low Tg, these two original dextrans are able to form rubber and flexible films at ambient temperature without any plasticizer addition. The mechanical parameters determined for Gcn1 films from tensile tests are very promising in comparison to the films obtained with other polysaccharides extracted from plants, algae or microbial fermentation. These results lead the way to using these dextrans as innovative biosourced materials.

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INTRODUCTION Biopolymers such as polysaccharides are widely used as thickening, stabilizing and gelling agents. They are generally extracted from plants (e.g., starch, pectin, cellulose) or algae (e.g., agar, carrageenan), but some of them are also produced by microbial fermentations (xanthan, gellan, β-glucans, bacterial cellulose, pullulan, etc.). Polysaccharides are also of interest for the production of biodegradable materials including films that could be used for packaging.1 This recent field of application addresses environmental problems related to the partial or total persistence of synthetic films such as polyethylene terephthalate (PET), polyvinylchloride (PVC), polyethylene (PE), and polypropylene (PP).2 In particular, the ability of plant and algae polysaccharides to form films has been explored in recent years.3−6 Microbial exopolysaccharides such as pullulan, kefiran and gellan were also considered for this application.7−10 In this context, α-glucans synthesized from sucrose by glucansucrases (GS) from lactic acid bacteria of the genus Leuconostoc, Streptococcus, Lactobacillus, Exigobacterium, and Weissella could offer an alternative source of biopolymers.11,12 Glucansucrases (GS) are transglucosidases classified into family 70 of the glycoside-hydrolases,13 that are able to polymerize the © 2011 American Chemical Society

D-glucosyl units of sucrose to form high molar mass homopolysaccharides. Depending on the reaction conditions, these versatile biocatalysts are able to synthesize a wide range of α-glucans and oligosaccharides that vary in terms of size and glycosidic linkage type. Because they use sucrose, a renewable resource, as substrate, these enzymes are very suitable tools for glycodiversification.11 However, the polymers they synthesize are still only marginally used, representing a small fraction of the biopolymers currently commercialized. To date, only dextrans synthesized by the glucansucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F are commercialized, mainly for analytical and therapeutic applications. The native polymer directly produced by the bacterial strain is of very high molar mass (up to 106 g.mol−1) and composed of 95 and 5% of α(1→6) and α(1→3) linkages, respectively. It is considered as a novel food ingredient by the European Union since 2001. It was in fact shown that incorporation of 5% of this dextran in bakery products improved softness, crumb texture, and loaf

Received: October 17, 2011 Revised: November 17, 2011 Published: November 18, 2011 187

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volume.14 However, diversification of applications of the large panel of α-glucans that are produced by GS is limited today by the lack of data related to their physicochemical properties. In this paper, we describe the investigation of the structural and physicochemical properties of seven high molar mass α-glucans with original structures, in order to evaluate their applicative potential as biosourced materials. These dextrans, which mostly contain α(1→6) glucosyl linkages and between 3 and 20% α(1→3) linkages, were synthesized from sucrose by seven mutants of the DSR-S enzyme that were obtained by combinatorial engineering to modify the linkage specificity of the parental enzyme.15 To do this, asymmetrical flow field flow fractionation coupled with multiangle laser light scattering (AFFFF-MALLS) was used in order to determine the molar mass distribution of these new polymers, and to characterize their conformation in solution. Rheological behavior and glass transition temperature were also investigated, as well as their ability to form innovative films.

SB-802.5 columns in series. Products were eluted using a 0.45 M NaNO3 and 1% (v/v) ethylene glycol solution as eluent at a flow rate of 0.3 mL·min−1.22 The column oven temperature was set at 70 °C. Glucose, fructose, and leucrose concentrations were determined by HPLC, on a Dionex system equipped with an Aminex HPX-87C carbohydrate column (300 × 7.7 mm; Bio-Rad, Hercules, CA, U.S.A.), using ultrapure water as eluent at 0.6 mL·min−1. The temperature of the column oven was set at 80 °C. The percentages of glucosyl moieties incorporated into free glucose (%Gglucose) and leucrose (%Gleucrose) were calculated as follows:

■

where [sucroset0] corresponds to the initial substrate concentration (in mM) and [glucosetf ] and [leucrosetf ] to the final concentration of glucose and leucrose (in mM) at the end of the reaction. The percentages of glucosyl moieties incorporated into high molar mass (HMW) α-glucans (molar mass higher than 107 g·mol−1) were determined as follows:

[glucosetf ]

%Gglucose =

[sucroset 0] × 180/342

× 100

(1)

and

%G leucrose =

MATERIALS AND METHODS

Production of Parental and Mutant Enzymes. E. coli BL21 AI cells carrying a pBAD plasmid encoding the Thio-DSR-S vardel Δ4NHis or the tagged variants were grown for 24 h at 20 °C in flasks containing ZYM-5052 medium supplemented with ampicillin (100 μg.mL−1) and 0.1% (w/v) arabinose. Cells were centrifuged (4,500 g, 15 min, 4 °C) and the pellets were resuspended to a final OD600 nm of 80 in 50 mM sodium acetate buffer (pH 5.2) containing 0.05 g·L−1 of CaCl2, before being sonicated and centrifuged (15000 g, 30−60 min, 4 °C). Supernatants (cell free extracts) were harvested for polymer production. Glucansucrase Activity Assays. One unit of GS activity corresponds to the amount of enzyme that catalyzes the formation of 1 μmol of fructose per minute at 25 °C in 50 mM sodium acetate buffer (pH 5.2) containing 0.05 g·L−1 CaCl2 and 292 mM sucrose. The concentration of reducing sugars was determined using the dinitrosalicylic acid method,16 with fructose as the standard. Glucan Synthesis. Cell-free extract preparations of the parental Thio-DSR-S vardel Δ4N-His and variant enzymes were incubated in 50 mM sodium acetate buffer, pH 5.2, supplemented with 0.05 g·L−1 of CaCl2 and 292 mM sucrose, in a final volume of 20 mL. Polymer syntheses were carried out at 25 °C with 1 U·mL−1 of enzymes until total sucrose depletion (8 h of reaction). α-Glucan Purification. After total sucrose depletion, α-glucans were directly recovered from the crude synthesis media by the addition of one volume of prechilled ethanol at 4 °C and centrifuged (20 min, 16000 g, 4 °C). The precipitation procedure was repeated twice with 50% (v/v) prechilled ethanol at 4 °C to remove mono- and oligosaccharides. Finally, polymers were resuspended in pure water (40 mL) and freeze-dried. NMR Spectroscopy. Freeze-dried polymer samples (15 mg) were exchanged twice with 99.9 % atom D2O, freeze-dried and dissolved in 600 μL of D2O. 1D 1H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer using a 5 mm z-gradient TBI probe at 298 K, an acquisition frequency of 500.13 MHz, and a spectral width of 8012.82 Hz. The 1H-signal from D2O was used for automatic lock, and a gradient shimming was performed on each sample. Before Fourier transformation, the FIDs were multiplied by an exponential function with a line broadening of 0.3 Hz. Spectra were processed with a 64 k zero filling, baseline correction and referenced using the TSP-d4 signal at 0 ppm. All NMR data were acquired and processed using TopSpin 2.1 software. The various signals were assigned as described by Seymour et al.,17 van Leeuwen et al.,18−20 and Maina et al.21 The percentages of α(1→3) and α(1→6) linkages in α-glucans were calculated by integrating the corresponding anomeric proton signals.15 HPLC Analysis. α-Glucan yields were calculated from analysis of the enzymatic reaction media by high-performance size-exclusion chromatography (HPSEC) using two Shodex OH-Pack SB-805 and

%Gglucan =

[leucrosetf ] [sucroset 0]

× 100

[glucantf ] [sucroset 0] × 162/342

(2)

× 100

(3)

where [glucantf ] corresponds to the concentration of glucan determined on an HPSEC chromatogram at the end of the reaction and [sucroset0] to that of substrate at initial time. The proportion of glucose incorporated into intermediate molar mass (IMW) products (molar mass ranging from 0.5 × 103 to 5.7 × 103 g·mol−1) was determined as follows:

%G IMW = 100 − %Gglucan − %G leucrose − %Gglucose

(4)

Asymmetrical Flow Field-Flow Fractionation Coupled with Multiangle Laser Light Scattering (AFFFF-MALLS) Analyses. Dextran molar mass, size, and structure were analyzed by AFFFF-MALLS using synthesis reaction media. Samples were diluted 250 times using Millipore water containing 0.2 g·L−1 sodium azide and filtered through 0.45 μm Durapore membranes (Millipore, Bedford, MA, U.S.A.). The solubilization recoveries (Sr) were calculated as follow:

Sr(%) = 100 ×

CCf CCm

(5)

where CCf and CCm correspond to the carbohydrate concentrations after and before filtration, respectively, determined using the sulfuric acid−orcinol colorimetric method.23 The equipment used for AFFFF-MALLS analyses was previously described by Rolland-Sabaté et al.24 The two online detectors comprised a MALLS instrument (Dawn HELEOS) fitted with a K5 flow cell and a He−Ne laser (λ = 658 nm; Wyatt Technology, Santa Barbara, CA, U.S.A.), and an ERC-7515A refractometer (Erma, Tokyo, Japan) operating with white light. The carrier (Millipore water containing 0.2 g·L−1 sodium azide) used was degassed and filtered through a Durapore (0.1 μm) membrane (Millipore). Because the α-glucans studied were particularly large, a flow method using very low crossflow during elution was carried out. Moreover, the injected masses were adjusted (6−8 μg of polymer were injected) to avoid overloading of the AFFFF channel and to limit aggregation phenomena during the focusing step. The crossflow was initially set at 1 mL·min−1 and the channel flow rate at 0.2 mL·min−1 for the sample introduction and the relaxation/ focusing period. The samples were injected at 0.2 mL·min−1 for 300 s. After the injection pump was stopped, the samples were allowed to relax and focus for 60 s. For elution, the flow rate was set at 188

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0.84 mL·min−1, and the crossflow was maintained at 0.1 mL·min−1 for 2274 s. The elution recoveries (Er) were estimated as follows: estimated Er(%) =

CG Csucrose × %G × %Gglucan × d × %Sr

Film Preparation and Tensile Tests. Aqueous solutions of 5% (w/v) α-glucans were prepared as described for rheological measurements. Films were obtained by casting 1 mL of the aqueous solutions on a Teflon plate at 60 °C for 3 h. The films were equilibrated at 57% relative humidity (RH) using a saturated sodium bromide solution for three days at ambient temperature under vacuum. For tensile tests, 5Atype dumbbell-shape specimens in conformity with the ISO-527-2 standard were cut from the films. Film thickness was measured with a hand-held micrometer. Tensile tests were performed with a TST350 instrument (Linkman scientific instruments, Surrey, U.K.). Elongation (ε) and tensile stress (σ) were measured using Linksys32 software. The distance between the supports was 20 mm and the crosshead speed was fixed at 50 μm·s−1. Measurements were performed twice, at 25 °C, until sample failure.

(6)

where CG corresponds to the concentration of eluted glucan [determined by integration of the differential refractive index (DRI) signal], Csucrose to the concentration of sucrose used for the glucan synthesis, %G to the percentage of total glucose units in the reaction mediums, %Gglucan to the percentage of glucose units incorporated into glucans (determined by HPSEC as described above), and d to the dilution factor of the sample. The weight average molar mass M̅ w (g·mol−1) and the z-average radius of gyration R̅ G (nm) were established using ASTRA software from Wyatt Technology (version 5.3.2.13 for PC), as previously described.24 By plotting the scattering intensities versus scattering angle, we verified that the system was operating in the Rayleigh− Gans−Debye dilute solution regime (results not shown). A refractive index increment (dn/dc) of 0.145 mL·g−1, determined for α-glucans was used. The error made on molar mass value using this dn/dc approximation instead of the exact dn/dc determined for the wavelength of the laser (658 nm) was evaluated at 2%. Mi and RGi (molar mass and radius of gyration of the ith slice, respectively) were obtained at each slice of the chromatogram peak using the Berry extrapolation (with a first order polynomial fit) of the light scattered to zero angle:

⎛ Kc ⎞ ⎜ ⎟ = ⎝ R θ ⎠i

⎞ 1 ⎛ 16π 2n2 2 2 ⎜⎜1 + ⎟⎟ θ R sin ( /2) G i Mi ⎝ ⎠ 3λ2

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RESULTS AND DISCUSSION

Dextransucrase DSR-S vardel Δ4N,26 a truncated mutant of the enzyme DSR-S from Leuconostoc mesenteroides NRRL B-512F, is the most efficient glucansucrase known to date. DSR-S vardel Δ4N combinatorial engineering was recently performed to investigate the structure-specificity relationships of this enzyme and to generate a toolbox of novel biocatalysts for the synthesis of novel α-glucan structures.15 A library of 303 clones displaying altered specificity compared to the parental enzyme was obtained. Among them, seven were selected (the single mutants F353T, S512C, F353W, the triple mutants H463R/ T464D/S512T, H463R/T464 V/S512T, D460A/H463S/ T464L, and the quadruple D460M/H463Y/T464M/S512C) for their ability to produce dextrans (designated as Gnc1 to Gcn7) with various amounts of α(1→3) linkages, ranging from 3 to 20% (Figure 1A,B). Their structural and physicochemical properties were investigated using a panel of various techniques. α-Glucan Size and Conformation in Solution. After filtration of the seven α-glucans in their synthesis reaction media, the solubilization recovery rates (Table 1) ranged from 87 to 100%, except for Gcn1 for which the recovery was slightly lower (77%). The elution recovery, which represents the percentage of macromolecules eluted through the AFFFF channel, ranged from 71 to 100%, which confirms that the majority of each sample could be analyzed by the procedure used. The lower elution recovery values obtained for Gcn_DvΔ4N, Gcn4 and Gcn5 may be due to their very high molar mass and size in solution and/or to aggregation phenomena. Consequently, the M̅ w and R̅ G values that were determined for these polymers do not represent the total αglucan population. The light scattering at 90° (LS90) and the refractometric (DRI) responses obtained at different elution volumes indicate that the various α-glucans have different size distributions (Figure 2A−D). Gcn_DvΔ4N, Gcn1, Gcn2, Gcn3, Gcn6, and Gcn7 elute in one LS90 single peak (Figure 2A,B,E−G) and one DRI peak (Figure 2C,D). LS90 peak tailing is observed. This could be due to the presence of a second population of greater size and high molar mass (HMM). Moreover, the evolution of molar mass (Mi) with elution volume presents a change of slope for Gcn6 (Figure 2E) and Gcn7 (Figure 2G), which corresponds to the LS90 peak asymmetry and could be due to the presence of two suspected populations of α-glucans. However, for all of the polymers analyzed except Gcn4 and Gcn5, no asymmetry is observed on the DRI signal, indicating that the proportion of the putative second population is negligible (Figure 2C,D). For Gcn4 and Gcn5, a more pronounced

(7)

where c is the concentration, K is the optical constant, Rθ is the excess Rayleigh ratio of the solute, λ is the wavelength of the incident laser beam, and θ is the angle of observation. The normalization of photodiodes was achieved using a low molar mass P20 pullulan standard. Rheological Measurements. Aqueous solutions of 5% (w/v) α-glucans were prepared by dissolving freeze-dried polysaccharides with distilled water and gently stirring at 25 °C overnight. Rheological properties were analyzed using a strain controlled rheometer (RFS II, Rheometrics Inc., Piscataway, NJ, U.S.A.) with cone−plate geometry (diameter, 5 cm; cone angle, 0.05 rads). Flow and dynamic measurements were performed at 20 °C. Flow curves were determined for shear rates ranging from 10−2 to 102 s−1 and thixotropic loops from 0 s−1 to 102 s−1 and back to 0. Viscoelastic measurements (G′ and G″ as a function of frequency) were performed under dynamic conditions (oscillatory shear) with frequencies ranging from 10−2 to 102 rad·s−1 and a shear strain amplitude of 5%. Determination of Glass Transition Temperature Tg. Freezedried glucans were equilibrated at 57% relative humidity (RH) using a saturated sodium bromide solution for one week at ambient temperature under vacuum. Glass transition temperatures (Tg) were determined with a differential scanning calorimeter Q100 system (TA Instruments, France). The instrument was calibrated with indium. The measurements were made with 2−30 mg sample mass, using hermetically sealed aluminum pans (TA Instruments, Guyancourt, France), heated from 0 to 120 °C at a rate of 3 °C·min−1. Two heating scans separated by a cooling stage at approximately 10 °C·min−1 were used to prevent any signature resulting from previous physical aging. An empty pan was used as a reference. Glass transition temperature was taken as the inflection point of the heat capacity change.25 Water content was determined after DSC measurement by thermogravimetric analysis (TGA). Thermogravimetric Analysis (TGA). The water content was determined using a TGA2050 (T.A. Instruments, New Castle, DE, U.S.A.). It was taken as the mass difference when heating samples up to 130 °C at a rate of 10 °C·min−1 and maintaining them at the final temperature for 40 min. 189

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Figure 1. (A) 1H NMR spectra of the α-glucans synthesized by the seven selected variants of the DSR-S glucansucrase. (B) Enzymatic synthesis yields (±2.5%) and osidic linkage content (±0.2%) of the α-glucans produced by the parental enzyme DSR-S vardel Δ4N and the seven selected mutants. *Percentage of glucosyl units derived from sucrose, incorporated into HMW α-glucans.

Table 1. AFFFF-MALLS Analysis of α-Glucans Produced by the Parental Enzyme DSR-S Vardel Δ4N and its Mutantsa sample

solubilization recovery (%)

estimated elution recovery (%)

M̅ w (× 108 g·mol−1)

R̅ G (nm)

νG

dGapp (g·mol−1·nm−3)

Gcn_DvΔ4N Gcn1 Gcn2 Gcn3 Gcn4 Gcn5 Gcn6 Gcn7

87 77 94 97 97 99 97 100

71 94 n.a. 91 81 83 100 100

4.01 6.02 3.40 2.37 0.99 (4.27)b 1.32 (4.36)c 1.16 0.76

157.5 206.0 133.7 121.0 76.0 (185.0)b 84.0 (190.0)c 67.0 55.0

0.56 0.48 0.60 0.64 0.56 (0.46)b 0.57 (0.50)c 0.43 0.47

28.2 25.4 45.8 35.2 61.6 (21.9)b 58.8 (20.4)c 102.9 119.9

a

M̅ w represents weight average molar masses, (R̅ G) represents z-average radii of gyration, apparent density is designated by dGapp, and the slope of the log−log plot of the radius of gyration vs the molar mass by νG. The experimental uncertainties for M̅ w, R̅ G, νG, and dGapp were 5%. bThese values were determined from the second populations at an elution volume of between 24.5 and 34 mL (Figure 2F). They correspond to the HMM population and represent 29.3% of the total recovered mass. cThese values were determined from the second populations at an elution volume of between 25 and 34 mL (Figure 2G). They correspond to the HMM population and represent 33.7% of the total recovered mass.

values calculated for a sphere, a random coil in a θ solvent, a random coil in a good solvent or a rod conformation are 0.33, 0.50, 0.60 and 1.00, respectively. The νG values obtained for Gcn_DvΔ4N, Gcn2, and Gcn3 (Table 1), as well as for the main populations of Gcn4 (Gcn4_MP) and Gcn5 (Gcn5_MP), are between 0.50 and 0.64 and are representative of random coil conformation observed for linear or quasi-linear chains. Gcn_DvΔ4N, Gcn2, and Gcn3 have a slight proportion of α(1→3) linkages (less than 10%, Figure 1B). This is in agreement with the νG value of 0.56 recently reported for a linear (1→6)-α-Dglucan in 0.1 M aqueous NaCl at 25 °C.27 The random coil conformation could also be observed for quasi-linear chains produced either by short chains connected to a linear backbone or by a few very long chains connected to the backbone. Gcn4 and Gcn5 polymers, for which the main population exhibits νG values of approximately 0.56−0.57 contain more α(1→3) linkages (Figure 1B) and have a lower M̅ w than Gcn_DvΔ4N, Gcn2, and Gcn3. This indicates that these two polymers probably contain short ramifications. Moreover, in these two samples, νG values of the HMM populations (Gcn4_HMM and Gcn5_HMM) are 0.46 and 0.50, respectively. These values are representative of more compact structures that probably correspond to some aggregates, according to Gcn4 and Gcn5 elution recovery and the huge angular dependency of the light scattered signals (results not shown). The νG values determined for Gcn1, Gcn6, and Gcn7 range from 0.43 to 0.48 (Table 1), that

asymmetric shape is also observed for the LS90 response (Figure 2F,G), which is in line with the shoulder at an elution volume of around 25 mL in the DRI responses (Figure 2C,D). Data treatment for Gcn4 and Gcn5 was thus carried out by considering two populations of α-glucans: the main population (MP) and the population with greater size and high molar mass (HMM). M̅ w and R̅ G values are reported in Table 1. Very high M̅ w is found for all α-glucans, with the highest values being 4.01 and 6.02 × 108 g·mol−1 for Gcn_DvΔ4N and Gcn1, respectively. Gcn4 and Gcn5 main populations (Gcn4_MP and Gcn5_MP), as well as Gcn6 and Gcn7, display the smallest M̅ w ranging from 0.76 to 1.32 × 108 g·mol−1. Intermediate values are found for the other glucans. M̅ w of Gcn4 and Gcn5 HMM populations reaches 4.27 and 4.36 × 108 g·mol−1, respectively. R̅ G followed the same trend as M̅ w, with the highest value at 206 nm for Gcn1 and the lowest one at 55 nm for Gcn7. For each sample, the logarithm of the radius of gyration for each slice i of the chromatogram (RGi) was plotted against the logarithm of the corresponding molar mass (Mi) to determine νG values from the equation:

R Gi = K GMi νG

(8)

The νG parameter provides information about the global conformation of macromolecules in solution. The theoretical 190

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Figure 2. (A−D) Elugrams (light scattering at 90° and DRI responses) of α-glucans vs the elution volume. (E−G) Elugrams (normalized light scattering at 90° responses) of α-glucans and their molar masses (Mi) vs the elution volume. (H) Elugrams (normalized light scattering at 90° and DRI responses) of Gcn4 and Gcn5 vs the elution volume. HMM indicate the high molar mass populations observed for Gcn4 and Gcn5, which represent 29.3 and 33.7% of the total recovered mass for Gcn4 and Gcn5, respectively (H).

is, between the theoretical value of a sphere (0.33) and a random coil in a θ solvent (0.50), meaning that their conformation is denser than that of Gcn_DvΔ4N and Gcn2 to Gcn5.

Finally, the determination of apparent particle density (dGapp) is another way to investigate the molecule structure and conformation. Apparent particle density was calculated on the 191

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Figure 3. (A) Apparent viscosity as a function of the shear rate of the 5% (w/v) polymer solutions. (B−D) storage modulus (G′) and loss modulus (G″) as a function of the angular frequency of the 5% (w/v) polymer solutions.

increase with molar mass.28 This is different for the dextrans studied here since the conformations found for the smallest ones account for the densest structures. Because the α(1→3) osidic linkages are usually considered to correspond to branching points in dextrans, this result would indicate (i) that the branching pattern of these dextrans is highly different from commercial ones or (ii) that a certain proportion of the α(1→3) osidic linkages may belong to the main linear chain instead of being only responsible for branching. The 1H and 13 C NMR spectra of Gcn_DvΔ4N and Gcn1 to Gcn7 do not reveal any additional peak compared to the NMR spectra of commercial dextrans, which could be attributed to α(1→3) linkages in the main chain. However, HMW polysaccharide NMR analysis is not sufficiently sensitive to exclude this phenomenon in some of our engineered α-glucans. Flow Behavior. All glucan solutions prepared at 5% (w/v) display a non-Newtonian shear-thinning behavior because the apparent viscosity of the polymer solution decreases with increasing shear rate (Figure 3A). Moreover, the superimposition of the up and down curves demonstrates a nonthixotropic behavior. The Gcn5 flow curve exhibits two distinct regions: (1) a Newtonian plateau region at low shear rates and (2) a shearthinning region at higher shear rates. This is a classical behavior of so-called entangled macromolecular solutions for which the macromolecular motion is restricted by other neighboring chains. At low shear rate, the number of intermolecular contacts remains constant, with the formation of new contacts that counterbalance the disruption of existing ones by shear deformation. The apparent viscosity therefore remains constant. At high shear rate, the greater disruption of existing contacts leads to a decrease of

basis of a uniform density in the particle and based on the following equation for equivalent homogeneous spheres: 3 dGapp = M w /(4π /3)R Gw

(9)

where R Gw is the weight average radius of gyration. Gcn_DvΔ4N, Gcn1, Gcn2, and Gcn3 exhibit the smallest densities (25.4−45.8 g·mol−1·nm−3, Table 1), which is in accordance with the quasi-linear conformation deduced from their νG values. Moreover, by comparing these densities at the same molar mass (because the density increases slightly when the molar mass decreases), it could be concluded that Gcn2 is denser than Gcn3 and Gcn1. Gcn2, which is less branched than Gcn3 (Figure 1B), may then contain a higher proportion of long chain branches compared to Gcn3. Regarding Gcn1, which is weakly branched (3% α(1→3) linkages, Figure 1B), its νG of 0.48 and its small density would be proof that it mainly contains very long branches. The highest densities are obtained for both Gcn6 and Gcn7 (higher than 100 g·mol−1·nm−3). This high density is in line with their low νG (∼0.45, Table 1), thus, confirming the more compact structure of these two glucans. Because these α-glucans also have the highest amount of α(1→3) osidic linkages (Figure 1B) and the lowest M̅ w, the branched conformation of these glucans is probably dense and more complex than that of the other samples. Gcn4_MP and Gcn5_MP reveal a smaller density (around 60 g·mol−1·nm−3, Table 1) than Gcn6 and Gcn7 (for a comparable molar mass), confirming a different and less dense structure. For these novel dextrans, the amount of α(1→3) osidic linkages decreases as their molar mass increases. The amount of long chain branching in commercial dextrans is expected to 192

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apparent viscosity.29 The shear-thinning behavior is also demonstrated by the variations of shear stress with shear rate (data not shown). Gcn4 and Gcn6 display similar flow properties, but the low shear rate Newtonian zone is not reached and would appear to be at much lower shear rates. The other samples (Gcn_DvΔ4N, Gcn2, and Gcn3) display a reverse behavior at low shear rates, with a yield stress. Gcn_DvΔ4N, Gcn2, and Gcn3 show the highest apparent viscosity at low shear rates, with 2−3 times higher orders of magnitude compared to Gcn5. Viscoelastic Behavior. The mechanical spectra of Gcn_DvΔ4N, Gcn2, and Gcn3 solutions show elastic behavior because the storage (elastic) moduli G′ is greater than the loss (viscous) moduli G″ over the range of frequencies studied (Figure 3B). The slight dependency of G′ on the frequency and the relatively large value of tan δ (tan δ = G″/ G′ > 0.3) are typical of so-called weak gels in which a network is formed by junction zones between the polymer chains. In a previous work, dextran synthesized by a crude extract of DSR-S vardel Δ4N and directly analyzed from the enzymatic reaction medium where it was present at a 3% (w/v) concentration, did not show such a gel-like behavior.22 However, in the present study, the Gcn_DvΔ4N sample was purified by ethanol precipitation and freeze-dried before rheological analysis. Polymer concentration differences in the samples analyzed and freeze-drying may thus affect the polymer viscoelastic properties, as already observed by Sabatié et al.30 Regarding Gcn7, G′ and G″ moduli values are significantly lower than those of Gcn_DvΔ4N, Gcn2, and Gcn3, suggesting the presence of a more tenuous network. In contrast, the mechanical spectra of Gcn1, Gcn4, and Gcn5 (Figure 3C) are typical of entangled macromolecular solutions. Both parameters G′ and G″ are strongly frequency-dependent and increase with frequency. At low frequencies, the viscous modulus G″ dominates the elastic modulus G′ (liquid-like behavior). Because the frequency increases, a crossover point is reached (∼ 1 rad·s−1 for Gcn1 and Gcn4), and G′ is then greater at high frequencies (solid-like behavior). The crossover point is not completely reached for Gcn5 solution within the tested frequency range of 10−2 to 102 rad·s−1, but it presumably occurs at higher frequencies. The behavior of Gcn6 is rather unusual, because G′ tends to level off at low frequencies, even if no crossover is reached within the tested frequency range (Figure 3D). This behavior was previously observed for hydrolyzed oat β-glucans and was attributed to possible polymer self-aggregation.31 Moisture Content and Glass Transition. The water content of the different glucans when equilibrated at aw 0.57 is shown in Table 2. The water content of Gcn_DvΔ4N, Gcn2,

Gcn3, Gcn4, Gcn5, and Gcn6 range from 11 to 13%. These values are classically found when commercial dextrans, pullulans, amylopectins, or starches are equilibrated at the same relative humidity.25,32−34 Surprisingly, the water content determined for Gcn1 is about twice as low as for the other polymers, indicating a lower moisture sorption capacity, which is generally attributed to the number of available sites (i.e., hydroxyl groups) for water binding.25 This peculiar behavior could be attributed to polymer−polymer interactions between the linear chains of Gcn1, probably enhanced by freeze-drying packing stress that decreases the accessibility of available hydroxyl groups. Moreover, Gcn_DvΔ4N is found to form weak gels, while Gcn1 viscoelastic behavior is typical of entangled polymer solutions, thus, revealing probable differences in linear chain interaction properties between these two polymers. The glass transition temperatures reported in Table 2 correspond to the temperature at the midpoint of the transition heat capacity between the glassy and rubbery states measured during the second DSC scan (Figure 4). Gcn2, Gcn3, and Gcn6 display comparable Tg values in a range of 51−53 °C. With the same water content, Gcn_DvΔ4N and Gcn4 display a slightly different Tg of 47 and 56 °C, respectively. Gcn1 and Gcn5 display Tg values that are 1.5−2.5 times lower than those of the other polymers. These results are very astonishing with regard to the water content of these polymers. Indeed, water is known to have a plasticizing effect (increase of the molecular mobility in the material and shift of Tg to a lower value) for many carbohydrates such as mono-, di-, and trisaccharides,35 dextran,34 pullulan,33 galactan,36 and starches.25 These data suggest that molecular mobility in Gcn1 and Gcn5 is higher than in the other polymers and that these two macromolecules would present longer ramifications than other dextrans, as already proposed from AFFFF analysis. Therefore, because their Tg is close to the ambient temperature without adding any plasticizers, Gcn1 and Gcn5 present mechanical properties that are particularly suitable for making materials. Mechanical Behavior of the Films. Translucent films were obtained for all of the α-glucans tested (Figure 5A). Films obtained from dextrans with high Tg (Tg ≫ Troom) are rigid and brittle, while films obtained with Gcn1 and Gcn5 (Tg ≅ Troom) are flexible. These observations are in accordance with the DSC results. Below Tg, materials are in the glassy state and mobility is mainly restricted to local chain segments (β relaxation) and hydroxyl groups (γ relaxation), leading to a lower amplitude of motion and a rigid and brittle behavior.37 Around and above Tg, amplitude and frequency of movements increase (α relaxation), leading to flexible, rubber materials.38 Unfortunately, the rigid films are very difficult to handle and crumbled when cut for tensile tests. Gcn5 is also fragile and we did not obtain any dumbbell-shape specimens. For these reasons, mechanical testing was performed only on Gcn1 film. A tensile stress−elongation curve of Gcn1 film is presented in Figure 5B. This polymer displays a ductile behavior since the rupture occurs in the plastic domain, with a Young modulus (E) of 0.019 GPa (calculated from the initial slope of the curve), a tensile strength at break point (σR) of 3.55 MPa, and an elongation at break point (εR) of 193.2%. Low Young modulus and high elongation at break point are characteristic of a film with a high elasticity. However, the strength at break point is low and could be related to the weak ability to entangle that is also revealed by the viscoelastic behavior characteristic of weak gel. Compared to most biopolymers4−10,33,39−41

Table 2. Water Content, Glass Transition Temperature (Tg), and Changes in Heat Capacity at Tga samples

water content (%)

Tg midpoint (°C)

ΔCp (J·g−1·°C−1)

Gcn_DvΔ4N Gcn1 Gcn2 Gcn3 Gcn4 Gcn5 Gcn6 Gcn7

12.74 7.07 12.59 12.86 12.61 10.96 12.69 n.d.

47.04 19.00 51.82 51.29 56.53 29.80 53.24 n.d.

0.571 0.144 0.543 0.606 0.679 0.192 0.567 n.d.

a

Experimental uncertainty: Tg (±1°), water content (±0.2%). n.d.: not determined. 193

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Biomacromolecules

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Figure 4. Differential scanning calorimetry thermograms for polymers (a) Gcn_DvΔ4N, (b) Gcn4, (c) Gcn3, (d) Gcn2, (e) Gcn6, (f) Gcn5, and (g) Gcn1.

series of dextrans studied could make it possible to design materials with specific mechanical behaviors, either by adding a small amount of plasticizer or by mixing dextrans with different Tg. It could also be interesting to use Gcn1 in mixture with other biopolymers that display complementary properties to produce biobased films closer to synthetic polymers.

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CONCLUSION

The structural and physicochemical properties of seven original α-glucans, enzymatically synthesized in one step from sucrose, were investigated. AFFFF-MALLS results made it possible to cluster these polymers into three groups in terms of their molar mass, size, and branching patterns: (i) Gcn1, which displays the highest M̅ w and a quasi-linear conformation, (ii) Gcn6 and Gcn7, the smallest α-glucans, that are probably also the most densely branched, and (iii) the other α-glucans that display intermediate structures. All these α-glucans display non-Newtonian and nonthixotropic behaviors in solution. However, analyses under dynamic conditions suggested that they adopt different conformations. For the first time, glass transition of a series of high molar mass dextrans with different amounts of branches and molar mass was studied. A particularly low Tg was observed for Gcn1 and Gcn5. Gcn1 films display very interesting mechanical properties compared to other biopolymers extracted from plants, algae, or those produced by microbial fermentation. To our knowledge, this is the first time that films have been obtained from dextrans. These results open novel perspectives for dextran applications as biosourced materials and biodegradable films. Finally, further structural investigations will be necessary to accurately characterize the ramification length and organization and the supramolecular organization of the most promising dextrans and to establish reliable correlations between the structure and physical properties of α-glucans. Making films with specific properties must also be pursued, especially through the glass transition optimization, either by using small amounts of plasticizers or by mixing dextrans with different glass transition temperatures.

Figure 5. (A) Films obtained after casting and conditioning at aw 0.57. (B) Tensile stress−elongation curve of Gcn1 films.

(pullulan, bacterial exopolysaccharides, chitosan, starch, carrageenan, kefiran, gelan, soy protein), even when plasticizers are added, the Gcn1 film displays a very low Young modulus and a high deformation capacity. These results are remarkable but remain far from those of synthetic polymers that displayed 3−5 times higher deformation capacities and 6−13 times higher strength at break point.42 However, this is the first time that films have been prepared from dextran. The wide range of Tg determined for the 194

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AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected].

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ACKNOWLEDGMENTS The authors would like to thank Marion de Carvalho and Laurent Chaunier for their technical support and Denis Lourdin for helpful discussion. This work was funded by the French National Institute for Agricultural Research (INRA), the French National Center for Scientific Research (CNRS), and the French National Research Agency (Project ANR-09CP2D-07-03).

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