In Vitro Synthesis of Hyperbranched α-Glucans Using a Biomimetic

Article pubs.acs.org/Biomac

In Vitro Synthesis of Hyperbranched α‑Glucans Using a Biomimetic Enzymatic Toolbox Florent Grimaud,†,‡,§ Christine Lancelon-Pin,∥ Agnès Rolland-Sabaté,⊥ Xavier Roussel,# Sandrine Laguerre,†,‡,§ Anders Viksø-Nielsen,▽ Jean-Luc Putaux,∥ Sophie Guilois,⊥ Alain Buléon,⊥ Christophe D’Hulst,# and Gabrielle Potocki-Véronèse*,†,‡,§ †

Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France UMR792 Ingénierie des Systèmes Biologiques et des Procédés, INRA, F-31400 Toulouse, France § CNRS, UMR5504, F-31400 Toulouse, France ∥ CERMAV-CNRS, BP 53, F-38041 Grenoble cedex 9, France (affiliated with Université Joseph Fourier, member of Institut de Chimie Moléculaire de Grenoble and Institut Carnot PolyNat) ⊥ UR1268 Biopolymères Interactions Assemblages, INRA, F-44300 Nantes, France # UGSF, UMR 8576, Université Lille1, sciences et technologies, Bât. C9, F-59655 Villeneuve d’Ascq, France ▽ Novozymes A/S, Krogshoejvej, 36, DK-2880 Bagsvaerd, Denmark ‡

S Supporting Information *

ABSTRACT: Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis. The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 106 to 4.4 × 107 g.mol−1) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.

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INTRODUCTION In most living organisms, energy and carbon resources are stored in the form of amylopolysaccharides. Starch is the storage polymer in green plants, red algae, and some cyanobacteria.1,2 It is made up of two homopolymers of α-Dglucosyl units, namely amylose and amylopectin. In both polymers, glucosyl units are linked by α(1,4) glycosidic bonds to form linear chains, with α(1,6) branch points. Amylose is mainly linear with less than 1% α(1,6) bonds, whereas amylopectin is a branched polymer with 5−6% α(1,6) linkages. In bacteria, fungi, and animals, glycogen is the storage polymer and is made up of α-D-glucosyl units that are joined by α(1,4) glycosidic linkages, with 7−10% branch points introduced by α(1,6) bonds evenly distributed within the glycogen particles.3,4 A glycogen-type polymer, referred to as phytoglycogen, has also been isolated from several higher plants lacking an isoamylasetype starch debranching enzyme (DBE).5−7 © 2013 American Chemical Society

Starch is industrially used in a wide range of food and nonfood applications. Most applications are based on the modification or degradation of native starch by enzymatic or hydrothermal processing.8 Alternately, modified starch can be obtained via cross-breeding and genetic manipulation of starchproducing plants.9,10 High-amylose starches are valued in the production of biomaterials and can be used as a source of slowly digested carbohydrates, whereas low-amylose starches are valorised as texturizers.11 However, starches produced by plant variants still require additional modifications achieved by enzymatic or physical processing, chemical substitution, and/or cross-linking to obtain optimal functionalities.9,10 Additionally, animal and fungal glycogens are used in the cosmetic industry Received: October 30, 2012 Revised: January 11, 2013 Published: January 11, 2013 438

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as an active ingredient for skin application.12 Glycogen is also used in food and drink formulations as a slowly digestible carbohydrate.13 Finally, oyster glycogen and sugary-1 maize phytoglycogen have been shown to present immunostimulating properties and antitumor activities.14 The biosynthesis of starch and glycogen requires the coordinated action of different enzymes in order to achieve specific polymer structures and physiological functions.15−18 Starch is synthesized in specialized organelles (the plastids) by starch synthases (SSs; EC 2.4.1.21, CAZy family GT5), starch branching enzymes (BEs, EC 2.4.1.18, CAZy family GH13), and starch DBEs (EC 2.4.1.41, CAZy family GH13). SSs catalyze the transfer of the α-D-glucosyl units from the donor glucosyl-nucleotide ADP-Glc to a growing chain composed of α(1,4)-linked glucosyl residues. BEs catalyze the formation of branch points within linear chains by cleaving an α(1,4) linkage and transferring the released chain to the same chain (intrachain branching) or to another linear chain (interchain branching) through the formation of an α(1,6) linkage. DBEs hydrolyze some of the branches previously introduced by BEs, presumably allowing amylopectin crystallization.19,20 Glycogen is mainly synthesized in liver and skeletal muscles or in the cytoplasm of living organisms (archaea, bacteria, and eukarya) by the action of glycogenin (EC 2.4.1.186), glycogen synthases (GSs; EC 2.4.1.21), and glycogen-branching enzymes (GBEs; EC 2.4.1.18). Glycogenin uses UDP-glucose as the glucosyl donor to specifically glucosylate one of its tyrosine residues.21 Self-glucosylation then proceeds through the formation of α(1,4) glucosidic linkages between new glucosyl residues onto the glucosyl residue that is linked to the protein, until a chain of 8 to 12 residues is formed. The resulting oligosaccharide is then used as a primer for GSs and GBEs to catalyze the formation of α(1,4) and α(1,6) glycosidic linkages, respectively. Modifying starch or glycogen structure to yield specific properties remains a challenging task that requires a thorough understanding of the biosynthesis process, including the identification of the function of each biosynthetic enzyme and the understanding of the fine regulations of these actors. For these reasons, in vitro enzyme-based approaches using bacterial enzymes are very attractive to design innovative αglucans with controlled properties. Only two types of elongating enzymes have been used in vitro to synthesize or to modify α-glucans: (1) α(1,4) Glucan phosphorylase (GP, EC 2.4.1.1), which catalyzes the reversible transfer of a glucosyl unit from the nonreducing end of an α(1,4)-glucan chain to inorganic phosphate (Pi) to produce glucose-1-phosphate (G1P). In combination with a BE, GP was used to synthesize glycogenlike particles22−25 and other branched α-glucans from G1P and maltoheptaose as substrates.26,27 (2) Bacterial amylosucrases (ASs; E.C. 2.4.1.4; CAZy family GH13), which are particularly interesting enzymatic tools, since they use only sucrose, a cheaper substrate than ADP-, UDPglucose or G1P, to polymerize glucosyl residues via the formation of α(1,4) linkages.28 Up to now, four ASs, from Neisseria polysaccharea,29 Deinococcus radiodurans,30 Deinococcus geothermalis31 and Alteromonas macleodii32 have been isolated and characterized as recombinant enzymes. The potential of AS from N. polysaccharea has been extensively investigated to develop original glycodendrimers through chain elongation of native glycogen.28

In this study, we have used bacterial AS and BE to mimic in vitro the elongation and branching steps involved in glycogen and starch biosynthesis. This biomimetic system was used to produce branched α-glucans from sucrose as unique substrate, in one step. The influence of the BE/AS activity ratio and sucrose concentration on the product structure was modeled to fully exploit this biomimetic system and to enlarge the panel of available α-glucans.

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EXPERIMENTAL SECTION

Enzyme Sources. The amylosucrase from N. polysaccharea (NpAS, Protein ID CAA09772.1) was produced as a fusion protein glutathione-S-transferase/amylosucrase (GST-NpAS), by recombinant Escherichia coli strain BL21 carrying the pGST-NpAS plasmid. GSTNpAS was further purified by affinity chromatography using glutathione-sepharose 4-B (Amersham-Pharmacia), as previously described.33 To simplify, GST-NpAS will be referred to as NpAS in the following. The BE from Rhodothermus obamensis (RoBE, Protein ID BAB69858.1) was obtained from Novozymes (Bagsvaerd, Denmark). The RoBE encoding gene was cloned and subsequently expressed in Bacillus subtilis. The recombinant enzyme was purified to near homogeneity using ion exchange chromatography, as previously described.34 The RoBE preparation was desalted by using a gel filtration column (PD10, Amersham) with 50 mM Tris-HCl buffer, pH 7.0, as the eluent. Commercial isoamylase (210 U·mg−1) from Pseudomonas sp. were purchased from Megazyme International (Ireland). Proteinase K from Tritirachium album was purchased from Euromedex (Mundolsheim, France). Enzyme Activity Assays. Amylosucrase Activity. The activity of the purified NpAS was measured using the dinitrosalycilic (DNS) acid method.35 All assays were performed at 30 °C in 50 mM Tris-HCl buffer, pH 7.0, by using 50 g·L−1 sucrose and 0.1 g·L−1 glycogen (G8751, Sigma Chemical Co.). Fructose concentration was determined by the DNS method using fructose as standard. One unit of amylosucrase corresponds to the amount of enzyme that catalyzes the production of 1 μmol of fructose per min in the presence of 50 g·L−1 sucrose and 0.1 g·L−1 glycogen. BE Activity. The total activity of the BE was measured using the iodine assay, by monitoring the decrease of absorbance at 660 nm of iodine and linear α(1,4)-glucans complexes. All assays were performed at 30 °C in 50 mM Tris-HCl buffer, pH 7.0, by using 2 g·L−1 amylose type III from potato (A-0512, Sigma Chemical Co.) and 20 μg of BE. At different time intervals, 10 μL aliquots were taken, and the reaction was stopped by addition of 160 μL of iodine reagent (0.1% I2 and 1% KI) and 630 μL of 50 mM Tris-HCl buffer, pH 7.0. One unit of enzyme activity was defined as the decrease of 1.0 unit of absorbance per min at 30 °C. α-Glucan Synthesis. Reactions were performed at 30 °C, in 50 mM Tris-HCl buffer, pH 7.0, containing NpAS, RoBE and sucrose. All reactions were performed with the same amount of NpAS activity (1 U.mL−1). However, various amounts of RoBE (from 0.04 to 6 U.mL−1) and sucrose (from 17.1 to 205.2 g·L−1) were tested in order to determine the impact of the RoBE/NpAS activity ratio (referred to as aRoBE/aNpAS in the following) and sucrose concentration on the structure of the synthesized α-glucans. Reactions media were stirred at 30 °C for 24 h to allow total sucrose consumption. After 24 h, the reaction media were heated during 5 min at 95 °C to inactivate the enzymes. Sodium azide was finally added at a final concentration of 0.02% (w/v). Sucrose Quantification. After 24 h, the complete sucrose consumption in the reaction media was checked by quantifying sucrose using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Aliquots of the reaction media were collected and centrifuged (10 min, 10 000 g). The soluble fraction was diluted to a final concentration of 10 mg·L−1 and analyzed by HPAEC-PAD on a 4 × 250 mm Dionex Carbopac PA100 column. A gradient of sodium acetate (from 6 to 300 mM in 30 min) in 150 mM NaOH was applied at a 1 mL.min−1 flow rate. 439

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Detection was performed using a Dionex ED40 module with a gold working electrode and a Ag/AgCl pH reference. Transmission and Scanning Electron Microscopy (TEM and SEM). The reaction media were submitted to the action of 23 μg.mL−1 proteinase K during 10 min at 37 °C to degrade NpAS and RoBE. The reaction media were dialyzed against distilled water for 8 h in Spectra/ Por dialysis tubing (MWCO 5000) in order to remove residual sugars and salts. Drops of 0.001 wt % glucan suspensions were deposited onto glow-discharged carbon-coated grids and negatively stained with 2 wt % uranyl acetate. The preparations were observed with a Philips CM200 transmission electron microscope operating at 80 kV. The images were recorded on Kodak SO163 films. Size-distribution histograms were determined by measuring the diameter of about 1000 particles from digitized TEM images, using the ImageJ program. Some specimens were allowed to dry onto copper stubs. After coating with Au/Pd, they were observed in secondary electron mode using a Jeol JSM6300 scanning electron microscope operating at 8 kV. α-Glucan Structure Analysis. The structure of the α-glucans produced by the tandem action of NpAS and RoBE was characterized in comparison with that of oyster glycogen (Sigma, Saint Quentin Fallavier, France), and phytoglycogen extracted from sugary-1 maize kernels (kindly provided by INRA, Plant Breeding Department, Saint Martin de Hinx, France), as previously described.36 Chain Length Distribution Analysis. The chain length (CL) distribution was determined by HPAEC-PAD, after debranching the αglucans. Aliquots of the reaction media were collected and diluted to 1 g·L−1 total sugars in 5 mM sodium acetate buffer adjusted to pH 3.5 with glacial acetic acid. α-Glucans were solubilized by heating at 130 °C in an oil bath during 30 min, and slowly cooled to 50 °C in a water bath. The reaction mixture was divided into two equal parts. Part one was treated with 10 units of isoamylase from Pseudomonas sp. (Megazyme) for 24 h at 50 °C to hydrolyze the α(1,6) glucosidic linkages, while part two was only incubated for 24 h at 50 °C. No αglucan retrogradation appeared during all the debranching experiment. To avoid any α-glucan precipitation during the HPAEC-PAD analysis, which was carried out at 30 °C, parts one and two were then diluted 2 times in 1 M NaOH (final concentration), and directly injected in the HPAEC-PAD system previously described. A sodium acetate gradient in 150 mM NaOH was applied as follows: 0−2 min, 0 mM; 2−23 min, 0−225 mM; 23−123 min, 225−450 mM; 123−130 min, 450−450 mM. The α-glucan CL distribution was analyzed before and after debranching. To identify the chains involved in α(1,6) glucosidic linkages, a differential HPAEC-PAD profile was obtained by subtracting the peak area corresponding to the oligosaccharides contained in the reaction media before debranching to those obtained after debranching. The concentration of of each chain in the differential profile was estimated by using the linear relationship between the detector response per mole of α(1,4) chains and its CL. The linear curve coefficients were determined from maltooligosaccharide standards of CL between 2 and 7 and were used for longer compound quantification. Number- and weight-average branch chain length (BCLn and BCL w , respectively) and dispersity (recommendation of the International Union of Pure and Applied Chemistry, IUPAC) (d = BCL w /BCLn ) were calculated as previously described.37 High-Performance Size Exclusion Chromatography (HPSEC). To quantify reaction yields, the reaction media were analyzed by HPSEC.28 Aliquots of the reaction media were diluted in water to 17.1 g·L−1 total sugars. Then, 100 μL of the dilution was solubilized by addition of 25 μL of 5 N NaOH. Samples were then diluted by the addition of 187.5 μL water and neutralized by addition of 312.5 μL of 0.4 N HCl. In all cases, the resulting samples contained 200 mM NaCl. HPSEC analyses were performed on a series of Polysep-GFC-P P6000 and P2000 columns (Phenomenex, Torrance, CA), using a chromatographic system consisting of a P680 HPLC pump (Dionex), an Automated Sample Injector (ASI 100, Dionex), and an RI-101 refractometer (Shodex). Sugars were eluted at 50 °C with 200 mM sodium chloride at 0.5 mL.min−1. Fructose and maltooligosaccharides of CL ranging from 2 to 7 glycosyl units were used as standards. The elution recovery yield was calculated as the ratio between the total areas of the refractive index profiles of the samples collected at the final

and at the initial reaction times. It was checked that for all samples, the elution recovery yield was higher than 90%. α-Glucan Purification. In order to separate the synthesized αglucans from other reaction products (glucose, fructose, sucrose isomers, and maltooligosaccharides), 70% (v/v) ethanol at 4 °C was added to the reaction media. The α-glucan precipitate was isolated by centrifugation (10 min, 10 000 g) and resuspended in 1 volume of Milli-Q water. This step was repeated three times. The resulting purified polymer was resuspended in Milli-Q water and freeze-dried. High-Performance Size Exclusion Chromatography Coupled with Multi-Angle Laser Light Scattering and Quasi-Elastic Light Scattering (HPSEC-MALLS-QELS). Purified α-glucan samples were dissolved in water and kept at room temperature for one night. The solutions were filtered through 0.45 μm Durapore membranes (Waters, Bedford, MA, USA) and directly injected in the HPSECMALLS-QELS system. Sample recovery rates were calculated from the ratio of the initial mass and the mass after filtration determined using the sulfuric acid-orcinol colorimetric method.38 The HPSEC-MALLSQELS equipment was the same as that previously described.39 The HPSEC column used was a Shodex KW 802.5 (8 mm ID × 30 cm) from Showa Denko K.K. (Tokyo, Japan) coupled with its corresponding guard column Shodex KW (6 mm ID × 5 cm) and maintained at 30 °C. A Dawn Heleos MALLS system fitted with a K5 flow cell and a GaAs laser (λ = 658 nm) from Wyatt Technology Corporation (Santa Barbara, CA, USA) and an RID-10A refractometer from Shimadzu (Kyoto, Japan) were used as detectors. Online QELS measurements were performed at 142.5° for a time interval of 7 s using a WyattQELS system (Wyatt Technology Corporation). Before use, the mobile phase (Millipore water containing 0.2 g·L−1 sodium azide) was carefully degassed and filtered through Durapore GV (0.2 μm) membranes from Millipore, and eluted at a flow rate of 0.3 mL.min−1. Sample recovery rates were calculated from the ratio of the mass eluted from the column (integration of the DRI signal) and the injected mass, determined using the sulfuric acid-orcinol colorimetric method.34 Mi, the molar mass of the ith slice, was calculated using the Astra software (Wyatt Technology Corporation, version 5.3.4.20 for Windows) as previously described.39,40 A value of 0.145 mL.g−1 was used as the refractive index increment (dn/dc) for glucans. The normalization of photodiodes was achieved using a low molar mass pullulan standard (P20). The hydrodynamic radius of the ith slice (RHi) was calculated using the Stokes−Einstein relation (RH ≡ kBT/ 6πηDt, where kB is Boltzmann constant, T is the temperature, η is the viscosity of the solvent, and Dt is the translational diffusion coefficient), with the translational diffusion coefficient of the ith slice (Di) obtained from online QELS measurement, as previously described.39 Average values, M̅ w and R̅Hz were calculated using the Astra software by summations taken over the whole peaks as previously described.36,39 The integration edges were adjusted in order to retain the most reliable data. NMR Spectroscopy. Freeze-dried α-glucan samples (10 mg) were exchanged twice with 99.9 atom % D2O, lyophilized and dissolved in deuterated dimethyl sulfoxide (DMSO-d6) 80%/D2O 20%. 1D/1H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer using a 5 mm z-gradient TBI probe at 363 K, an acquisition frequency of 500.13 MHz, and a spectral width of 8012.82 Hz. The 1H-signal from DMSO-d6 was used for automatic lock, and a gradient shimming was performed on each sample. Before Fourier transformation, the free induction decays (FIDs) were multiplied by an exponential function with a line broadening of 0.3 Hz. Spectra were acquired and processed using the TopSpin 2.1 software. The various signals were assigned as previously described.41 The percentages of α(1,4) and α(1,6) linkages in α-glucans were calculated by integrating the corresponding anomeric proton signals. α-Glucan Fractionation. Products synthesized with aRoBE/aNpAS = 2 and 205 g·L−1 sucrose were subdivided by ultrafiltration using Amicon Ultra-15 centrifugal filter devices (100 kDa molecular weight cutoff, Millipore) into two fractions: the filtrate (100 kDa). One milliliter of the reaction was diluted twice in 200 mM NaCl to 102.5 g·L−1 total sugars. Then, 2 mL of the dilution was 440

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debranching and 1H NMR, respectively. The fitted models for the evolution of branch chain length (BCLn ) and BD as functions of Sucr (in g·L−1) and aRoBE/aNpAS are provided in eqs 2 and 3, respectively:

solubilized by addition of 0.5 mL of 5 N NaOH. Samples were then diluted by the addition of 3.75 mL water and neutralized by addition of 6.25 mL of 0.4 N HCl. The resulting sample (12.5 mL) contained 200 mM NaCl. A total of 10 mL was centrifuged (20 min, 5000 g) on the Amicon Ultra-15 centrifugal filter. The filtrate (9.5 mL) was taken while the retentate was washed two times with 10 mL of 200 mM NaCl directly on the Amicon Ultra-15 centrifugal filter (20 min, 5000 g). Finally, the retentate was resuspended in 10 mL of 200 mM NaCl. The resulting fractions were directly analyzed by HPSEC on the series of Polysep-GFC-P P6000 and P2000 columns. The CL distribution of each fraction was analyzed by HPAEC-PAD, after debranching of the α-glucans and the corresponding branching degree (BD) determined by 1H NMR after polymer purification by ethanol precipitation. Experimental Design and Statistical Analysis. Experiments started with a first central composite design42 (CCD 1) used to study the effect of aRoBE/aNpAS and sucrose concentration (Sucr) on two structural parameters of the produced α-glucans: branch chain length (BCLn ) and BD. These two parameters were used to explore the structural diversity of branched α-glucans that were obtained by using our enzymatic system. aRoBE/aNpAS ranged from 0.04 to 2.50, and Sucr ranged from 17.1 to 205.2 g·L−1. The results of this first experimental design showed that benefit could be gained in exploring a broader range of values for aRoBE/aNpAS. Therefore, a second central composite design (CCD 2) was performed with aRoBE/aNpAS ranging from 2 to 6. Each CCD consisted of 13 experiments listed in Supporting Information (SI) Table S1. Statistical modeling was performed using the Minitab software (release 16). The results of each CCD were combined, and a quadratic linear model was fitted to the data for each structural parameter (eq 1): I

Y ̂ = β0 +

× (Sucr × 1000/342.3) + 0.200 × (aRoBE /aNpAS)2 − 0.000017 × (Sucr × 1000/342.3)2 − 0.000945 × (aRoBE /aNpAS) × (Sucr × 1000/342.3)

i=1

i

j

(2)

BD = 2.82 + 2.91 × (aRoBE /aNpAS) − 0.0270 × (Sucr × 1000/342.3) − 0.0861 × (aRoBE /aNpAS)2 + 0.000039 × (Sucr × 1000/342.3)2 − 0.00011 × (aRoBE /aNpAS) × (Sucr × 1000/342.3) (3)

The results of the variance analysis for the models fitted to experimental BCLn and BD are given in SI Table S2. The R2 coefficients represent the quality of the fit. They indicate that the models used for BCLn and BD explain more than 93.6 and 93.3%, respectively, of the variability of the parameters that are related to BCLn and BD. The resulting response surface curves indicated that increasing aRoBE/aNpAS from 0.04 to 6.00 in the reaction resulted in a BCLn decrease from 12.2 to 7, and a BD increase from 0.5% to 13% (Figure 1a,b). The less branched α-glucans, which present a branch chain length of BCLn 12.2 and a BD of 0.5 were obtained under the following conditions: 1 U·mL−1 NpAS, 0.4 U·mL−1 RoBE, and 177.5 g·L−1 sucrose. The most branched α-glucans, presenting a branch chain length of BCLn 7 and a BD of 13%, were obtained under the following conditions: 1 U·mL−1 NpAS, 5.4 U·mL−1 RoBE and 44.5 g·L−1 sucrose. The response surface curves obtained for BD (Figures 1b) suggest that it could be possible to obtain higher BD values by increasing aRoBE/aNpAS. However, experiments with aRoBE/aNpAS = 10 and 19, and 111.2 and 34.2 g·L−1 sucrose, respectively, did not allow reaching BD values higher than 13.2, while the predicted values were 18.3 and 24.5 in these conditions. This indicates that this model cannot be used for aRoBE/aNpAS higher than 6. Taken together, these results show that the branching pattern of the α-glucans synthesized by the tandem action of NpAS and RoBE depends both on the initial sucrose concentration and on the ratio between elongating and branching activities. However, based on these results, it was impossible to know if several polymer structures were cosynthesized during the reactions. α-Glucan Size and Yield. To better understand the influence of aRoBE/aNpAS and Sucr on α-glucan structural parameters and dispersity, the polymer synthesis yield was determined for 10 reaction conditions that correspond to the limits of the designed experimental plan. Five aRoBE/aNpAS values (0, 0.04, 0.4, 2 and 4) and two Sucr values (17.1 g·L−1 and 205.2 g·L−1) were selected. The size distribution of the α-glucans produced in these conditions was first determined by HPSEC (Figure 2). For

I

∑ βi Xi + ∑ βiiXi2 + ∑ ∑ βijXiXj i=1

BCLn = 9.54 − 1.86 × (aRoBE /aNpAS) + 0.0181

(1)

where Ŷ is the predicted response, I is the number of factors (two in this study), β0 is the model constant, βi is the linear coefficient associated to factor Xi, βii is the quadratic coefficient associated to factor Xi, and βij is the interaction coefficient between factors Xi and Xj. The analysis of variance (ANOVA) table for the quadratic linear model was carried out, and each coefficient and their significance level were estimated. The quadratic linear model allowed drawing response surface curves for each structural parameter. Response surface curves were drawn with SigmaPlot software (release 11.0).

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RESULTS Modeling of the Influence of Sucrose Concentration and RoBE/NpAS Activity Ratio on the α-Glucan Branching Pattern. NpAS and RoBE were used to synthesize branched α-glucans in one-pot incubations. First, the reaction conditions were chosen to promote a synergy between NpAS and RoBE. The joint use of NpAS and RoBE required pH and temperature conditions in which each enzyme was sufficiently active to achieve its specific function. The optimal activity conditions have previously been shown to be pH 7/30 °C for NpAS33 and pH 6/65 °C for RoBE.34 Activity assays indicated that RoBE maintained a 10% of activity at pH 7 and 30 °C. However, the branching profiles of amylose modified by RoBE at 30 and 60 °C are highly similar, indicating that RoBE specificity is not altered at 30 °C (SI Figure S1). These conditions were then used for the tandem reaction using NpAS and RoBE. The specific activities of both enzymes at pH 7 and 30 °C were 9 U·mg−1 and 102 U·mg−1 for NpAS29 and RoBE,43 respectively. In addition, we checked that up to 600 mM sucrose and fructose did not affect RoBE activity. Second, experiments were designed to explore the panel of structures that can be synthesized by varying both Sucr and aRoBE/aNpAS. The branch chain length distribution and BD of the reaction products were analyzed by HPAEC-PAD after 441

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aRoBE/aNpAS = 0, only one polymer population was synthesized, with a corresponding elution volume comprised between 32.0 and 37.5 mL. The linear product synthesized from 205.2 g·L−1 sucrose was referred to A600 in a previous study.28 It has a CL w of 39 and a dispersity of 2.3.28 On the basis of these previously published data, we deduced that the elution times delimiting the low molar mass polymers (LMMPs) (32.0 and 37.5 mL) approximately correspond to the elution times of linear α(1,4)linked glucans with CL = 150 and 8, respectively. Chromatograms of α-glucans synthesized from 17.1 or 205.2 g·L−1 sucrose revealed that increasing aRoBE/aNpAS from 0 to 4 resulted in the decrease of the amount of LMMP in favor of a polymer population eluting earlier, referred to as high molar mass polymer (HMMP). The amount of HMMP increased from 0 to 3.0 g·L−1 for 17.1 g·L−1 sucrose and from 0 to 47.8 g·L−1 for 205.2 g·L−1 sucrose. Moreover, the initial sucrose concentration affected HMMP yield and size. The higher the initial sucrose concentration, the higher the size and very likely the M̅ w of HMMP. The increase in HMMP yield was correlated with BD increase. However, as LMMP and HMMP coexist in the reaction media in various proportions, it was not possible to assign an accurate BD to each population. To clarify this point, a fractionation step was carried out using ultrafiltration with a 100 kDa molecular weight cutoff. We selected the products prepared from 205.2 g·L−1 sucrose and with aRoBE/aNpAS = 2. Indeed, in such conditions, HMMP and LMMP were synthesized in sufficient amount to allow their fractionation: 19% of HMMP, 11% of LMMP, and 70% of monosaccharides and oligosaccharides of CL ≤ 7. The HPSEC chromatograms show that the ultrafiltrate only contains LMMP, CL 2−7 oligosaccharides, and monosaccharides, while the retentate, corresponding to the sample fraction that did not pass through the membrane, contains 74% HMMP, 7.6% LMMP, and 17.4% α-glucans of CL ≤ 7 (SI Table S3). Both fractions were purified by ethanol precipitation to separate the synthesized polymers from monosaccharides and CL 2−7 oligosaccharides. Their BD was determined by 1H proton NMR analysis. The BD of the filtrate products was 0, indicating that the amount of α(1,6) linkage contained in the LMMP, if any, was below the detection limit. The BD of the retentate products was 7.6, while it was 5.2 before ultrafiltration. From these data, we concluded that the BD of the total reaction products did not represent that of HMMP. This is particularly true when large amounts of linear or weakly branched LMMP were synthesized, using a low aRoBE/aNpAS. Morphology of the Reaction Products. The morphology of the products synthesized in the conditions summarized in SI Table S4 was characterized by electron microscopy. For the group of experiments carried out from 17.1 g·L−1 sucrose, a sediment was formed in most samples (SI Figure S2a−d) after the suspensions were centrifuged. After dialysis against water, the supernatant and sediment were separated and observed by TEM. In the series of experiments carried out from 17.1 g·L−1 sucrose, the first reaction was performed with NpAS only in the medium. The synthesized product appeared as loose random networks that were similar to those generally observed for retrograded amylose (Figure 3a).28 For aRoBE/aNpAS = 0.04, the supernatant contained 80−100 nm particles with an irregular surface (Figure 3b), whereas the sedimented fraction consisted of fibrous networks containing 100 nm blocks with a lamellar

Figure 1. Branched α-glucans produced from sucrose by the tandem action of NpAS and RoBE: response surface showing the influence of initial sucrose concentration (17.1−205.2 g·L−1) and aRoBE/aNpAS (0.04−6) on the branch chain length (BCLn ) (a) and the BD (%) (b). Experimental points of the central composite designs are indicated by red crosses. Experimental points that were analyzed in more detail (HBP17 and HBP205) are indicated by crossed circles. aRoBE/aNpAS: RoBE/NpAS activity ratio.

Figure 2. Chromatograms (differential refractive index (DRI) response) of products synthesized from 17.1 (a) and 205.2 g·L−1 (b) sucrose by the tandem action of NpAS and RoBE. aRoBE/aNpAS: RoBE/NpAS activity ratio; HMMP: high molar mass polymers; LMMP: low molar mass polymers. CL 2−7 and CL 1 correspond to oligosaccharides and monosaccharides, respectively.

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Figure 3. TEM images of products synthesized from Sucr = 17.1 g·L−1 with aRoBE/aNpAS = 0 (a), 0.04 (b,c), 0.4 (d,e), 2.0 (f,g), and 4.0 (h). (b,d,f,h) Particles observed in the water-soluble fraction of the reaction media; (c,e,g) networks observed in the sedimented fraction. All preparations were negatively stained with uranyl acetate. 443

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structure (Figure 3c). For aRoBE/aNpAS = 0.4, the supernatant contained a polydisperse distribution of particles clearly resembling native glycogen (Figure 3d, SI Figure S3). The corresponding size distribution, determined from the TEM images, is shown in SI Figure S4. The number-average mean radius of the particles is R n(TEM) = 22.5 nm with a standard deviation (std) of 13.4 nm. The sedimented fraction contained arborescent and fibrous aggregates to which 100−150 nm glycogen-like particles were sometimes associated (Figure 3e). The product prepared with aRoBE/aNpAS = 2.0 mostly contained polydisperse glycogen-like particles (Figure 3f and SI Figure S3), with R n(TEM) = 24.7 nm (std = 14.5 nm), but some aggregates containing a mixture of fibrous networks and particles were occasionally observed (Figure 3g). Very similarly, the sample obtained with aRoBE/aNpAS = 4.0 contained smaller glycogen-like particles (R n(TEM) = 15.0 nm, std = 7.6 nm) (Figure 3h and SI Figure S4) and a few arborescent networks (not shown). For the group of experiments carried out from 205.2 g·L−1 sucrose, a precipitate was formed in some samples during the reaction (SI Figure S2e-h). In that case, after dialysis, the supernatant was observed by TEM, and the precipitate was visualized by SEM. The precipitates obtained with aRoBE/aNpAS = 0 and 0.04 contained compact aggregates of ovoidal particles with a lamellar structure (SI Figures S5a and S5b). No α-glucan particle was visible in the supernatant of the reactions carried out with aRoBE/aNpAS = 0.04 and 0.4. For aRoBE/aNpAS = 0.4, the precipitate consisted of aggregates of ∼200 nm particles (SI Figure 4c). With aRoBE/aNpAS = 2.0, a precipitate was formed, but the supernatant had a slight bluish opalescence. The SEM image in SI Figure 5d shows that the precipitate consisted of aggregates of 1−3 μm spheroidal particles. The TEM images of the supernatant content revealed polydisperse glycogen-like particles, with R n(TEM) = 38.4 nm and std = 14.9 nm (Figure 4a and SI Figure S4). With aRoBE/aNpAS = 4.0, the sample contained glycogen-like particles (R n(TEM) = 25.5 nm, std = 16.0 nm) (Figure 4b and SI Figure S4). Structural Characterization of the Hyperbranched αGlucans. To investigate the structure of the HMMP products that were produced by the synergetic action of RoBE and NpAS in more detail, we selected two reaction conditions that allowed us to maximize HMMP yield. The two hyperbranched α-glucans that were synthesized using a high aRoBE/aNpAS (1 U·mL−1 NpAS and 4 U·mL−1 RoBE) from two extreme sucrose concentrations (17.1 and 205.2 g·L−1, respectively) were analyzed in more details. Both α-glucans were purified by ethanol precipitation and analyzed by various techniques in order to compare their structure with that of native hyperbranched α-glucans (i.e., oyster glycogen and phytoglycogen). They will be referred to as HBP17 and HBP205 in the following. The 1H NMR analysis of oyster glycogen, phytoglycogen, HBP17, and HBP205 indicated that the α-glucans synthesized in vitro were more branched (10−12%) than the glycogens extracted from maize (6.8%) and oyster (7.5%) (SI Table S5 and Figure S6). In addition, the purified α-glucans were enzymatically debranched with isoamylase before analysis by HPAEC-PAD. The CL distribution of HBP17 and HBP205 showed a maximum CL of 7, like for oyster glycogen and phytoglycogen. However, compared to these native macromolecules, HBP17

Figure 4. TEM images of the particles observed in the water-soluble fraction of reaction media corresponding to Sucr = 205.2 g·L−1 and RoBE/NpAS activity ratio = 2.0 (a) and 4.0 (b). All preparations were negatively stained with uranyl acetate.

and HBP205 contained a lower amount of chains with CL 15− 30, and the CL distributions appeared to be monomodal and narrower (Figure 5). The molar mass, size, conformation, and molecular density of HBP17 and HBP205 were determined by HPSEC-MALLSQELS. The solubilization recovery of glycogen, phytoglycogen, HBP17, and HBP205 ranged between 97 and 99%, and elution recoveries were between 93 and 100% (SI Table S5). These high values indicate that the solubilization and fractionation procedures allowed us to analyze the large majority of the molecules contained in the samples. Oyster glycogen, phytoglycogen, HBP17, and HBP205 chromatograms (Figure 6) show that the latter two exhibit broader size distributions than oyster glycogen and phytoglycogen. The M̅ w values of oyster glycogen and phytoglycogen were 5.7 × 106 g·mol−1 and 1.77 × 107 g·mol−1, respectively. For HBP205, M̅ w (4.39 × 107 g·mol−1) was 12 times higher than that of HBP17 (3.7 × 106 g·mol−1). However, the z-average hydrodynamic radius of HBP205 (RHz = 41.9 nm) was found to be about twice higher than that of HBP17 (20 nm) and oyster glycogen (20.7 nm), and 1.4 times higher than that of phytoglycogen (31.5 nm). RHz values are closely related to the weight-average radius values (R w(TEM) ) determined by TEM analysis (SI Table S5). By plotting the hydrodynamic radius versus the molar mass, information about the global conformation of macromolecules in solution can be obtained from the νH parameter in the equation RH = KH(Mw)νH, where KH is a constant. The νH parameter theoretical values are 0.33, 0.50−0.60, and 1.00 for a sphere, a random coil in good and θ solvent conditions, and a rod, respectively. The νH values of native glycogens and synthesized molecules are close to the 0.33 theoretical value 444

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Figure 5. Chain length distribution of oyster glycogen (a), phytoglycogen (b), HBP17 (c), and HBP205 (d) after debranching and HPAEC-PAD analysis. The concentration of each chain is expressed with respect to 1 mg of total product.

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DISCUSSION Influence of the Reaction Conditions on Polymer Structure and Yield. First, unsurprisingly, the increase of aRoBE/aNpAS resulted in the increase of BD and the decrease of the branch CL. However, different populations of α-glucans were simultaneously produced. To simplify the description of the phenomena, we have arbitrarily outlined two populations of α-glucans (LMMP and HMMP): (i) The LMMP population corresponds to linear or very weakly branched chains. For the experiments with Sucr = 17.1, these molecules would form the large fibrous aggregates that were observed in TEM images (Figure 3), through an association/crystallization process that is undetermined yet and will not be discussed in this paper. For the experiments with Sucr = 205.2 g·L−1, these chains would rapidly precipitate and form the thick aggregates observed by SEM (SI Figure S5). (ii) The HMMP population corresponds to hyperbranched particles, with a shape similar to that of native animal or plant glycogens, as observed by TEM (Figure 3 and SI Figure S3). Second, with the increase of aRoBE/aNpAS, the HMMP yield increases to the detriment of the LMMP one. The size distribution of the particles tends to narrow, and their average diameter decreases. Indeed, increasing aRoBE/aNpAS allows one to increase the number of branched molecules. Consequently, in high aRoBE/aNpAS conditions, linear chains that were used as substrate by RoBE are distributed among a higher number of branched molecules and, as a consequence, the hyperbranched particles are smaller. However, increasing aRoBE/aNpAS does not allow one to indefinitely decrease the branch CL of HMMP. Indeed, it was not possible to reach branch CL lower than 6.9, even by increasing the RoBE/NpAS ratio to 19. This is due to the RoBE substrate specificity. It was previously established that the minimum CL of donor glucan for RoBE was 10, and that linear glucan segments with CL ≥ 12 were the preferred substrates.43 The synthesis of mixtures of two polymer populations with highly different structures is probably due to the high affinity of NpAS and RoBE for branched amylopolysaccharides. Indeed, NpAS prefers to elongate the external branches of glycogen,45 and branched molecules are also preferred acceptors for

Figure 6. Chromatograms (DRI responses) (a) and molar mass distributions (b) of native glycogens and hyperbranched products synthesized in vitro: oyster glycogen (□), phytoglycogen (△), HBP17 and HBP205 (thick gray and black lines, respectively).

calculated for a spherical molecule (SI Table S5), indicating a spheroidal conformation for all specimens. The apparent molecular density calculated from the relation ρHapp = M̅ w /(4π/3)RHz3 ranges from 133.3 (HBP17) to 194.0 g·mol−1·nm−3 (oyster glycogen). These ρHapp values are very high compared to those of amylopectin and commercial dextrans of comparable molar mass (7 to 15 g·mol−1·m−3).39,44 445

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RoBE.43 With our system, it is thus likely that once α-glucans with a sufficient number of branching points are produced, NpAS and RoBE act preferentially on these molecules rather than elongating and branching linear chains. That is probably why, even when using low aRoBE/aNpAS, the one-pot incubation of RoBE and NpAS did not allow us to produce intermediate structures with branch CL >12, as found in amylopectin for instance. The in vitro synthesis of such molecules with long branches may thus be attempted either in one-pot synthesis by adding a debranching activity in the NpAS/RoBE reaction medium, or by performing iterative syntheses based on alternating chain elongation by NpAS and branching by RoBE. Such molecules would indeed present an interest for complexing bioactive molecules for instance. In contrast, the hyperbranched polymers described in this paper would rather be interesting for the preparation of highly concentrated solutions with low viscosity, which could have high potential for medical applications. Enzymatic systems have already been described in the literature to synthesize in vitro branched α-glucans. Among them, the so-called SP-GP-BE method was based on the use of sucrose phosphorylase (SP), glucan phosphorylase (GP), and BE as catalysts, as well as sucrose and maltotetraose as substrates to synthesize glycogen-like particles.23,46 More recently, the IAM-BE-AM method was developed, in which the branched linkages of starch were first hydrolyzed by isoamylase (IAM) from Pseudomonas amyloderamosa to produce short amylose chains that were assembled into glycogen-like molecules by the BE from Aquifex aeolicus and amylomaltase (AM) from Thermus aquaticus.24 Both with the IAM-BE-AM and SP-GP-BE methods, hyperbranched molecules with BD and branch CL values similar to those of the αglucans synthesized with our RoBE-NpAS system were achieved. However, in all systems mentioned above, it was not known if different polymers were simultaneously produced during the reaction (i.e., hyperbranched α-glucans, amylose-like α-glucans, and α-glucans with intermediate structure). Yet, the described preparation of hyperbranched α-glucans with the IAM-BE-AM and SP-GP-BE methods generally includes a centrifugation step to remove insoluble materials.14 Are Synthetic Hyperbranched α-Glucans Similar to Native Glycogen? Glycogen is an α-glucan mainly built upon α(1,4) linkages and has a regularly branched structure with α(1,6) linkages every 8−12 glucose residues corresponding to branching points.47 The CL of the linear chains is about 10.2,7 This highly dense branched organization47,48 occurs in the form of particles with a diameter ranging from 20 to 50 nm (socalled β-particles).49,50 Glycogen particles are water-soluble, and, therefore, glucose is readily accessible to rapid mobilization through the enzymes of glycogen catabolism.51 The biomimetic system we have designed allows one to synthesize hyperbranched α-glucans (for instance, HBP17 and HBP205) mostly consisting of water-soluble particles. Their morphology and conformation in solution are similar to those of glycogen and phytoglycogen. Nevertheless, there were some differences between the synthetic and native polymers: the CL distribution was narrower, the BD was higher and the M̅ w distributions were broader than those of oyster glycogen and phytoglycogen. The most significant differences are the particle size and M̅ w which can reach, in the HBP205 conditions, 4.4 × 107 g·mol−1.

Such molecules with high M̅ w values have also been synthesized by using the IAM-BE-AM method, in which a BE from Aquifex aeolicus was used.52 These data show that by adjusting the reaction parameters (in particular substrate concentration), in vitro systems allow one to synthesize hyperbranched molecules with higher M̅ w than those achieved in vivo. The in vivo mechanism of glycogen synthesis significantly differs from those occurring in vitro. Indeed, glycogen metabolism occurs in specialized cellular compartments where membranes infer physical constraints which could contribute to limit the particle size. In cells, glycogen metabolism is also tightly regulated by cascade signaling and allosteric enzyme regulation53 in order to answer to the metabolic demands. The successive action of enzymes involved during synthesis and degradation may thus limit the M̅ w of natural glycogen. In addition, we do not know if the biomimetic system we set up allowed us to reach a limit particle size. Experiments are in progress to address this question. The results may help us to better understand the phenomena involved in glycogen biosynthesis and to clarify the relationships between the molecular architecture, steric hindrance, and morphology of the hyperbranched macromolecules.

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CONCLUSION Our in vitro system based on the tandem use of the N. polysaccharea AS and R. obamensis BE in the presence of sucrose, allows one to synthesize, in a single step, mixtures of hyperbranched α-glucans and of linear or very weakly branched chains. An experimental plan has been used to explore the panel of molecular structures that can be obtained by using this biomimetic toolbox. The synthesis of mixtures of polymer populations with different molar mass, BD, and aggregation properties depended on initial aRoBE/aNpAS and sucrose concentration. Although glycogen-like particles were observed, we did not have any evidence of the synthesis of amylopectinlike macromolecules, i.e., with heterogeneous branching patterns. However, as DBEs are known to play a crucial role in the in vivo synthesis of amylopectin, the addition of such enzymes in our system should be tested to achieve such complex molecular architectures.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental design results, variance analysis of the fitted models, concentration of the reaction products obtained after size fractionation, macromolecular characteristics of native glycogens and synthesized α-glucans, temperature dependency of RoBE product specificity, micrographs of the series of products synthesized in various conditions, TEM analysis of native glycogens, size distribution histograms of the glycogenlike particles, SEM analysis of the precipitates obtained in reaction media, and 1H NMR spectra of native glycogens and synthetic hyperbranched products. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; fax: +33 (0)561 559 400. Notes

The authors declare no competing financial interest. 446

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ACKNOWLEDGMENTS We thank Agence Nationale de la Recherche for financial support (Grant Number ANR-09-CP2D-07-01), and Nelly Monties and Sandrine Morel for technical assistance.

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