Optimization of Isomaltooligosaccharide Size Distribution by Acceptor

Article pubs.acs.org/JAFC

Optimization of Isomaltooligosaccharide Size Distribution by Acceptor Reaction of Weissella confusa Dextransucrase and Characterization of Novel α‑(1→2)-Branched Isomaltooligosaccharides Qiao Shi,*,† Yaxi Hou,† Minna Juvonen,† Paï vi Tuomainen,† Ilkka Kajala,§ Shraddha Shukla,# Arun Goyal,# Hannu Maaheimo,§ Kati Katina,† and Maija Tenkanen† †

Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, FI-00014 University of Helsinki, Finland VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, FI-02044 VTT, Finland # Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India §

S Supporting Information *

ABSTRACT: Long-chain isomaltooligosaccharides (IMOs) are promising prebiotics. IMOs were produced by a Weissella confusa dextransucrase via maltose acceptor reaction. The inputs of substrates (i.e., sucrose and maltose, 0.15−1 M) and dextransucrase (1−10 U/g sucrose) were used to control IMO yield and profile. According to response surface modeling, 1 M sucrose and 0.5 M maltose were optimal for the synthesis of longer IMOs, whereas the dextransucrase dosage showed no significant effect. In addition to the principal linear IMOs, a homologous series of minor IMOs were also produced from maltose. As identified by MSn and NMR spectroscopy, the minor trisaccharide contained an α-(1→2)-linked glucosyl residue on the reducing residue of maltose and thus was α-D-glucopyranosyl-(1→2)-[α-D-glucopyranosyl-(1→4)]-D-glucopyranose (centose). The higher members of the series were probably formed by the attachment of a single unit branch to linear IMOs. This is the first report of such α-(1→2)-branched IMOs produced from maltose by a dextransucrase. KEYWORDS: isomaltooligosaccharides, dextransucrase, maltose acceptor reaction, response surface modeling, α-(1→2)-linkage, centose

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INTRODUCTION Isomaltooligosaccharides (IMOs) are glucooligosaccharides (GLOS) containing predominantly α-(1→6) linkages. They have served as functional dietary carbohydrates for decades in Asia and have attracted growing interest in other parts of the world.1−3 Because of the high demand for IMOs, numerous enzymatic routes for their synthesis and purification have been developed.1,4−6 Industrial IMO production includes enzymatic hydrolysis of starch into high-maltose syrup, followed by the action of α-transglucosidase, which catalyzes the transfer of the nonreducing glucosyl residue of maltose to the 6-OH group of glucose (released by hydrolysis), the nonreducing glucosyl unit of maltose, or any GLOS present in the solution.1 Isomaltose, panose, and isomaltotriose are the main components in the IMOs produced from hydrolyzed starch; however, the composition of commercial IMOs, especially the proportion of digestible carbohydrates, differs substantially, as does their functionality as prebiotics.7 The degree of polymerization (DP) is believed to have a significant impact on the digestibility of IMOs, with IMOs with a higher DP preferred for longer persistence in the colon.8 Commercial IMOs of DP 2.7 were found to be digested in the small intestine of rats, whereas IMOs of DP 3.3 were nondigestible.9 Thus, an alternative approach that can control the product DP is desired for the production of prebiotic IMOs. Various lactic acid bacteria from genera Leuconostoc, Lactobacillus, Streptococcus, and Weissella produce dextransu© 2016 American Chemical Society

crases (EC 2.4.1.5), which belong to glycoside hydrolase family 70. Dextransucrases catalyze the synthesis of dextrans from glucopyranosyl residues of sucrose, whereas fructose is released freely. Dextrans are extracellular polysaccharides containing mainly consecutive α-(1→6) linkages and fewer α-(1→2), α(1→3), or α-(1→4) branch linkages.10 In the presence of suitable hydroxyl group-containing acceptors, such as lowmolecular-weight carbohydrates, dextransucrases also catalyze so-called acceptor reactions, where the glucopyranosyl residue of sucrose is diverted from dextran formation and transferred onto the acceptor, forming GLOS.10 Acceptor reactions of dextransucrases have been exploited to synthesize prebiotic GLOS.1,11−14 The type of glucosidic linkage formed in the product depends on the acceptor substrate and the specificity of the enzyme. Maltose is the most studied acceptor due to its high effectiveness. For dextransucrases predominantly synthesizing α-(1→6) linkages, the maltose acceptor products are a homologous series of IMOs.15 The initial product, panose (α-DGlcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp), can be elongated by successive attachment of glucosyl residues to 6-OH of the nonreducing end glucose, forming linear (panose-series) IMOs. Received: Revised: Accepted: Published: 3276

March 23, 2016 April 6, 2016 April 6, 2016 April 6, 2016 DOI: 10.1021/acs.jafc.6b01356 J. Agric. Food Chem. 2016, 64, 3276−3286

Article

Journal of Agricultural and Food Chemistry

Table 1. Central Composite Experimental Design and Response Data for Modeling the Effects of Reaction Conditions on IMO Synthesis runa

maltose (mol/L)

sucrose (mol/L)

E392-rDSR (U/g sucrose)

panose (g/L)

LIM4b (g/L)

LIM5 (g/L)

LIM6 (g/L)

total IMOc (g/L)

consumed maltose (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0.15 1 0.15 1 0.15 1 0.15 1 0.15 1 0.575 0.575 0.575 0.575 0.575 0.575 0.575 0.575

0.15 0.15 1 1 0.15 0.15 1 1 0.575 0.575 0.15 1 0.575 0.575 0.575 0.575 0.575 0.575

1 1 1 1 10 10 10 10 5.5 5.5 5.5 5.5 1 10 5.5 5.5 5.5 5.5

10.39d 19.33 3.89 120.10 13.45 47.87 3.86 120.55 5.96 91.66 37.91 45.70 48.53 43.40 47.62 49.39 47.50 49.00

17.24 5.03 22.33 105.46 22.90 23.38 13.18 138.99 19.30 98.77 31.57 98.17 79.17 62.11 79.08 83.00 81.13 84.61

7.69 39.67 31.20 15.63 2.58 25.98 80.15 33.83 37.78 6.58 97.11 40.06 45.95 56.26 55.05 58.39 58.82

1.46 31.35 3.90 4.50 31.19 19.00 27.96 5.92 0.64 46.89 8.82 14.28 17.00 15.82 18.23 17.83

45.69 25.89 162.84 287.16 70.01 82.98 204.43 408.68 154.46 265.03 89.98 365.89 199.51 191.76 229.36 240.15 236.31 243.86

36.71 20.55 86.25 38.15 37.03 28.50 94.19 50.19 84.20 40.37 23.02 64.74 39.77 59.17 49.84 46.69 49.96 49.26

a

One reaction mixture was prepared for each run. Runs 15−18 are four replicates. bLIM4, LIM5, and LIM6 denote the linear isomaltooligosaccharides of DP4−6. The values represent apparent concentrations analyzed using panose as a standard. cTotal IMOs represents all isomaltooligosaccharide products including DP >6 members of principal linear series and members of minor branched series. dSamples were analyzed twice, and the average values are presented. A hyphen (−) denotes that the value was below the lower limit of quantification. stirred cell (Millipore, Witten, Germany), followed by dilution in a 20 mM sodium acetate buffer, pH 5.4. The preparation showed a single enzyme band corresponding to E392-rDSR in SDS-PAGE analysis.22 The enzyme activity was determined by the Nelson−Somogyi assay,24 where 1 unit is the amount of enzyme that catalyzes the formation of 1 μmol of reducing sugar in 1 min in a 20 mM sodium acetate buffer (pH 5.4) containing 2 mM CaCl2 and 146 mM sucrose. Experimental Design for IMO Synthesis. The effects of three factors, namely, the concentrations of sucrose (Sigma, St. Louis, MO, USA), maltose (Merck, Darmstadt, Germany), and E392-rDSR, on the synthesis of maltose acceptor products were examined. Preliminary experiments were carried out under three sucrose/maltose ratios (2:1, 1:1, and 1:2) in two concentration ranges anchored at 0.15 and 0.5 M, respectively. Two enzyme dosage levels in relation to substrate sucrose (1 and 10 U/g sucrose) were used for each pair of sucrose and maltose. The reactions were conducted in 1 mL of 20 mM sodium acetate buffer, pH 5.4, containing 2 mM CaCl2 at 30 °C for 24 h. The reactions were terminated by boiling in water for 10 min. After the preliminary experiments, the following ranges were selected for independent variables in central composite design: sucrose (0.15−1 M), maltose (0.15−1 M), and dextransucrase (1−10 U/g sucrose). The design consisted of 18 experiments, including 4 replicates at the center point of design to allow estimation of the replicate error (Table 1). The reactions were conducted the same way as in preliminary experiments. The yield of IMO of DP3, DP4, DP5, and DP6, respectively, the total IMO yield, and the percent of consumed maltose were each modeled using a quadratic regression model, which takes into account the effects of a variable alone, the effects of the interactions between two variables, and the quadratic effects of variables alone. Modde 9.0 (Umetrics AB, Umeå, Sweden) was used to analyze the results by a multiple regression method (MLR or PLS) and to generate contour plots. The fit of the model to the experimental data was indicated by the coefficient of determination, R2, which describes the extent of the variance in a modeled response variable that can be explained by the model. The coefficient of prediction, Q2, measures how well a response is predicted for a new experimental condition. Q2 is determined on the basis of the prediction of the residual sum of squares. A Q2 no less than 0.5 indicates the model has good predictive ability. In a good model, the

The dextransucrase acceptor reactions produce maltosebased IMOs and dextran concomitantly. Their relative yields are dependent on the concentrations of sucrose and maltose and the dextransucrase dosage.16,17 The chain-length distribution of IMOs can be controlled by varying the sucrose/maltose ratio. With an increase in the ratio, the DP of IMO products is increased.16,18,19 However, these studies selected limited points of substrate concentrations, making it difficult to predict the IMO product profile under different conditions. Thus, the aim of this work was to use experimental design and response surface modeling to study in detail how substrate concentrations and enzyme dosage affect the yield of individual IMOs of different DPs. The IMO synthesis was performed with the recombinant dextransucrase (E392-rDSR) from an efficient dextran producer (Weissella confusa VTT E-90392) in food matrices.20,21 The gene encoding of dextransucrase has recently been expressed in Lactococcus lactis.22 In addition to linear IMO series, unknown minor products were noted for E392-rDSR in high-performance anion-exchange chromatography analysis. Unknown products from maltose were also observed for W. conf usa Cab3 dextransucrase (Cab3-DSR) in our preceding work.23 The synthesis of minor maltose products was found common to E392-rDSR, Cab3-DSR, and a commercial Leuconostoc mesenteroides dextransucrase, on the basis of the similar chromatographic profiles of their product mixtures. Thus, this study also aimed to characterize these minor products, as they are the coproducts of the target IMOs from dextransucrase reactions.

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MATERIALS AND METHODS

Preparation of E392-rDSR. The W. conf usa VTT E-90392 dextransucrase gene was previously expressed in L. lactis NZ9800.22 The culture supernatant was concentrated by ultrafiltration with a Prep/Scale tangential flow filter (TFF; 6 ft2, cutoff 10 kDa) (Millipore, Bedford, MA, USA) and then with a 100 kDa cutoff Amicon 8400 3277

DOI: 10.1021/acs.jafc.6b01356 J. Agric. Food Chem. 2016, 64, 3276−3286

Article


MSn and NMR analysis. The total carbohydrate concentration of selected fractions was determined by phenol−sulfuric acid assay. Commercial dextransucrase from Leuconostoc mesenteroides (Sigma, St. Louis, MO, USA) was also tested for the activity for synthesizing the minor products. The reaction was carried out with 10 U/g sucrose of the commercial enzyme in 20 mM sodium acetate buffer (pH 5.4) containing 0.2 M sucrose and 0.1 M maltose at 30 °C for 24 h (similar to the condition previously used for Cab3-DSR). The product mixture was analyzed by HPAEC-PAD for the presence of the minor products. MSn Analysis of Minor Trisaccharide Product of Maltose Acceptor Reaction. A fraction with a panose/minor trisaccharide ratio of ∼7:3 was used for preliminary characterization of the minor trisaccharide product by electrospray ionization multistage ion trap mass spectrometry (ESI-IT-MSn). Panose was analyzed alongside for comparison. The samples were studied in both negative and positive modes ([M + Cl]− and [M + Li]+, respectively) using an Esquire LC quadrupole ion trap mass spectrometer with an electrospray ion source (Bruker Daltonik GmbH, Bremen, Germany). A total of 10 μL of the fraction (with a total carbohydrate concentration of 0.5 mg/mL) was diluted in 200 μL of methanol/water/formic acid (50:49:1, v/v/v), and 1 μL of 10 mg/mL ammonium chloride or lithium acetate was added for adduct ion formation ([M + Cl]− and [M + Li]+, respectively). The samples were introduced to the ion source with a direct infusion syringe pump at a flow rate of 5 μL/min. Nitrogen gas was used as both nebulizing and drying gas. The ionization parameters were as follows: drying gas temperature, 325 °C; drying gas flow, 4 L/min; capillary voltage, ±3200 V; and nebulizer pressure, 10 psi. The ion trap parameters were automatically adjusted by the mass of the analyte. The fragmentation amplitude range was 0.60−0.65 V. NMR Analysis of Minor Trisaccharide Product of Maltose Acceptor Reaction. A fraction containing nearly equimolar amounts of panose and the minor trisaccharide and a pure sample of panose were analyzed in parallel by 1D 1H and 2D (multiplicity-edited HSQC, DQFCOSY, TOCSY, and HMBC) NMR experiments. The fraction selected was freeze-dried and redissolved in D2O (MagniSolv) (Merck KGaA) to a carbohydrate concentration of 0.6 μmol/mL. The samples were analyzed in 5 mm Wilmad Ultra-Imperial NMR tubes (SigmaAldrich) on a 600 MHz Avance III NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a QCI cryoprobe. The measurements were performed at 11 °C because at this temperature the residual water signal overlapped with one anomeric proton signal of panose and did not affect the signals of the minor product of interest. In the 1D 1H experiments, the residual water signal was suppressed by a 4 s volume selective presaturation (so-called 1D NOESY presaturation) using the Bruker pulse program noesygppr1d. In the TOCSY experiments, a DIPSI2 spin lock and a mixing time of 180 ms were used. The HMBC experiments were optimized for 8 Hz (62.5 ms) long-range coupling constants. In the two-dimensional experiments, matrices of 2K × 256 (2K × 512 in HMBC) data points were collected and zero filled once in F1. A π/2-shifted sine bell weighting function was applied in both dimensions prior to the Fourier transformation. Topspin v.3.2 (Bruker) software was used for processing and analyzing the NMR spectra. The chemical shifts of 1 H (δH) and 13C (δC) were referenced to acetone (δH 2.225 and δC 31.55, respectively). Mechanistic Study on the Synthesis of the Minor Series. To understand how higher DP minor products are synthesized, pure panose and the fraction containing equal molarity of panose and minor trisaccharide (centose, the pure compound was not available) were tested as acceptors. A sucrose/acceptor ratio of ∼4:1 was used to compare the effectiveness of several acceptors for E392-rDSR in our preceding work.27 This ratio was thus kept here, using 1 mM acceptor, 5 mM sucrose, and 5.5 U/g sucrose of E392-rDSR in reactions conducted at room temperature for 24 h. The reaction mixtures were analyzed qualitatively by HPAEC-PAD for enzyme selectivity toward centose and panose as acceptors and their product patterns.

values of both R2 and Q2 are high and not separated by more than 0.2−0.3. Analysis of Monosaccharides, Oligosaccharides, and Dextran. High-performance anion-exchange chromatography (HPAECPAD) was used to quantitate monosaccharides and oligosaccharides in the reaction mixtures. The instrument was equipped with a 250 × 4 mm i.d., 8.5 μm, CarboPac PA-100 column (Dionex, Sunnyvale, CA, USA), a Decade detector (Antec, Leyden, The Netherlands), a Waters 717 autosampler, and two Waters 515 pumps, as described previously.25 The elution was performed with 75 mM NaOH (8 min), followed by a gradient to 100 mM NaOAc in 75 mM NaOH (27 min) with a flow rate of 1 mL/min. For the qualitative analysis of the profiles of acceptor product fractions from W. confusa dextransucrases, the same system was run by another elution method, starting with 100 mM NaOH (15 min), followed by a gradient to 120 mM NaOAc in 100 mM NaOH (20 min) with a flow rate of 1 mL/min. Prior to the analysis, samples were filtered through a 10 kDa Amicon Ultra-0.5 centrifugal filter (Millipore, Billerica, MA, USA) or with 0.45 μm membrane (Pall Corp., Port Washington, NY, USA). Glucose, fructose, maltose (Merck, Darmstadt, Germany), sucrose (BDH Chemicals Ltd., Poole, UK), leucrose (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and panose (TCI Europe N.V., Zwijndrecht, Belgium) were used as standards. The DP4−6 members of principal product series that are panose homologues are referred to as linear isomaltotetrasaccharide (LIM4), isomaltopentasaccharide (LIM5), and so on. Each linear IMO was quantitated on the basis of the peak area using panose (10−400 μg/ mL) as a standard as no reference compounds were available. To evaluate the effect of DP on the response factor (slope of standard curve) in HPAEC-PAD detection, a similar quantitative analysis of maltotetraose, maltopentaose, and maltohexaose (Sigma-Aldrich, St. Louis, MO, USA) of known amounts was carried out with maltotriose (Sigma-Aldrich) as a standard. The response factors of maltotetraose, maltopentaose, and maltohexaose were approximately 100, 94, and 80% of that of maltotriose, respectively. Thus, it is unlikely for LIM4− 6 to be significantly underestimated when quantitated with panose as the standard. Moreover, because the dextransucrase generally produced a lesser amount of higher IMOs, the magnitude of bias was small. The total IMO yield (including small amounts of DP >6 principal linear products and all minor products) was also estimated by integrating the total area of the peaks. The IMO yield (%) was calculated on the basis of glucose equivalents, according to

yield = total IMOs/(0.47 × initial sucrose + initial maltose) Dextran produced in the preliminary maltose acceptor reactions was estimated using a phenol−sulfuric acid assay.26 Dextran was precipitated from the reaction mixtures with 1 volume of ethanol and resuspended in water. This procedure was repeated once. Aqueous phenol solution (5% w/v, 0.5 mL) was mixed with an equal volume of dextran sample, after which 2.5 mL of concentrated sulfuric acid was added. The mixture was incubated at room temperature for 30 min. The absorbance at 480 nm was measured against blank, with glucose (20−200 μg/mL) as a standard. Characterization of Minor Products of Maltose Acceptor Reaction. Unknown minor products were noted in the maltose acceptor reactions of E392-rDSR in HPAEC-PAD analysis. They were also observed for Cab3-DSR, as shown by their similar product profiles under similar synthesis and analysis conditions (data not shown). Thus, the earlier-prepared product fractions from Cab3-DSR23 were used to characterize the common minor products. The synthesis was carried out under a single condition in our preceding work,23 employing 10 U/g sucrose of Cab3-DSR in the presence of 0.3 M sucrose and 0.15 M maltose. The polymeric dextran concomitantly synthesized was removed by ethanol precipitation. IMOs in the aqueous solution were separated according to DP by gel filtration.23 Fractions were collected and analyzed qualitatively for oligosaccharides by HPAEC-PAD. As the minor trisaccharide product eluted slightly earlier than the major product panose in gel filtration, the fractions enriched with the minor trisaccharide were selected for subsequent 3278


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Table 2. Effects of Factors Expressed as Coefficients in Models for IMO Yield (in Respect to Degrees of Polymerization) and Maltose Consumption in Dextransucrase-Catalyzed Acceptor Reaction

panose (g/L) LIM4 (g/L) LIM5 (g/L) LIM6 (g/L) total IMOs (g/L) consumed maltose (%)

coefficient of determination (R2)

regression eqa

response

44.78 + 36.20M + 16.52S + 2.69E + 23.69M × S 78.46 + 27.67M + 27.80S + 3.13E − 20.26M2 − 14.43S2 + 27.58M × S 55.09 + 2.22M + 18.58S + 3.97E − 10.09M2 − 7.49E2 + 4.90M × S + 4.22M × E 18.79 − 5.19M + 9.64S + 1.80E − 4.32E2 − 2.48M × S + 0.88S × E 236.07 + 42.46M + 111.45S + 23.68E − 27.90M2 − 45.86E2 +41.93M × S + 14.09M × E 50.16 − 16.06M + 18.77S + 4.77E + 8.99M2 − 9.42S2 − 8.43M × S

R2 R2 R2 R2 R2

= = = = =

0.95 0.95 0.85 0.85 0.97

R2 = 0.97

coefficient of prediction (Q2) Q2 Q2 Q2 Q2 Q2

= = = = =

0.89 0.82 0.60 0.68 0.87

Q2 = 0.90

a

M, initial maltose concentration (mol/L); S, initial sucrose concentration (mol/L); E, dextransucrase concentration (U/g sucrose); M2, S2, and E2, quadratic effects of maltose concentration, of sucrose concentration, and of dextransucrase concentration, respectively; M × S, M × E, and S × E, interactions of maltose and sucrose concentrations, of maltose and dextransucrase concentrations, and of sucrose and dextransucrase concentrations, respectively.

Figure 1. Contour plots showing the influence of initial sucrose and maltose concentrations in E392-rDSR acceptor reaction on the yields (g/L) of (A) panose, (B) LIM4, (C) LIM5, (D) LIM6, (E) total IMOs, and (F) the consumed maltose (%). The reactions were conducted with an enzyme dosage of 5.5 U/g sucrose for 24 h.

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RESULTS AND DISCUSSION

and the glucosyl moiety from sucrose was predominantly transferred to maltose forming linear IMOs. This complied with earlier observations16 that a dramatic decrease in the weight percent of dextran occurred when maltose and sucrose concentrations were increased above 100 mM. By comparing the chromatograms of reaction mixtures with different initial sucrose/maltose ratios, it was noted that when the sucrose/ maltose ratio dropped from 2:1 to 1:2, the production of panose and LIM4 seemed to increase, whereas that of LIM5

Effects of Sucrose, Maltose, and E392-rDSR on IMO Synthesis. The production of panose and higher linear IMOs (referred to as LIM4, etc.) was studied as a function of sucrose and maltose concentrations and the dextransucrase (E392rDSR) dosage, as their effects were demonstrated previously.16,18,19 According to the preliminary results, dextran synthesis was largely suppressed under the selected conditions, 3279


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tration. The maltose utilization ratio was negatively influenced by the maltose input amount. To our knowledge, this is the first study of the effects of acceptor reaction factors on the IMO product profile by optimization design. Increasing maltose input had varying effects on the production of IMO components. It promoted the production of lower DP while inhibiting that of DP6. This may be due to the fact that maltose binds more favorably to dextransucrase than the elongated IMOs. Thus, when maltose was in excess, it outcompeted the IMO product for the chance to be elongated, and more IMO of lower DP accumulated as a result. Only when the maltose concentration was low enough did the IMOs have a better chance to grow. In another work,17 the synthesis of total IMOs was optimized by response surface methodology using Lc. mesenteroides NRRL B-512F dextransucrase and lower substrate concentration ranges. Similarly, dextran was found to be of much lower concentration than oligosaccharides, and the total IMO yield was maximized when sucrose and maltose concentrations were at their highest (100 and 200 mM, respectively). In the present study, substrate concentration ranges were set higher for a higher IMO product concentration, which is desired for a more cost-effective purification. Several purification methods have been developed to remove unwanted monosaccharides (e.g., glucose and fructose) and disaccharides (e.g., maltose and sucrose) from IMO products, including adsorption separation and selective fermentation by yeasts or bacteria.1,5,28,29 In vitro fermentation of dextransucrase-synthesized IMOs by human fecal bacteria showed that the IMOs with DP5−7 had a relatively high selectivity toward beneficial bacteria compared to the lower DP.30 The IMOs of higher DP also persist longer in the colon and can reach the most distal regions, where most chronic intestinal disorders originate.30 Therefore, a good yield of IMOs with higher DP was targeted in the optimization for a higher prebiotic potential. According to the modeling results, medium maltose (∼0.5 M) and high sucrose (1 M) concentrations would be the preferred conditions. The corresponding run 12 yielded IMOs with abundant LIM4−6 and a reasonable maltose utilization. The condition could be further optimized by fine-tuning the factors toward a higher conversion of substrates and a lower enzyme dosage. Dextransucrase acceptor reactions may be used to adjust the sizes of IMOs for altered functionality, as probiotic bacteria have different preferences for IMOs of various sizes.7,31 Characterization of Minor Products of Maltose Acceptor Reaction. In addition to the principal IMOs, a number of unknown minor products were observed in the maltose reactions of E392-rDSR by HPAEC-PAD analysis. Under similar conditions, the product profile was similar to that of another W. conf usa dextransucrase Cab3-DSR23 (data not shown), indicating that both enzymes synthesize the minor products. This is consistent with the sequence similarity between the two Weissella dextransucrases (Genbank accession no. KP729387 and KJ173611, 93% amino acid identity), the amino acid substitutions of which were shown unlikely to cause drastic differences in catalytic properties.22,32 Moreover, the minor products were also observed for the commercial Lc. mesenteroides dextransucrase (data not shown), suggesting a shared mechanism for the dextransucrases to synthesize the minor products. Because the IMOs from Cab3-DSR have been isolated by gel filtration in our preceding work,23 the resultant fractions were taken for further characterization for the common minor products. HPAEC-PAD analysis showed that

and LIM6 decreased significantly. The trend was similar in both substrate concentration ranges. The dextransucrase dosage appeared to have a smaller effect on the IMO profile. As the preliminary experiments showed that dextran synthesis was largely suppressed in the selected experimental regions, a central composite design covering the whole region was followed for the modeling of IMO synthesis. Eighteen experiments were performed according to the experimental design shown in Table 1. The yield of each linear IMO up to DP6 and the total IMOs and the percent of consumed maltose were examined as responses (Table 1). A number of unknown minor products were also observed, which were not quantitated due to low concentration; however, the content of the minor trisaccharide product was found to be affected in a similar way to panose (data not shown). According to HPAEC-PAD analysis of the carbohydrates present in the 24 h reaction mixtures, the substrates consumed (maltose and the glucosyl moiety of sucrose) corresponded well with the quantitated total IMOs. The IMO yield (%) was calculated on the basis of glucose equivalents, although oligosaccharides containing fructose may also contribute to the total IMOs. The IMO profile varied between the experimental points under different conditions, and the four replicates at the center point of the design showed good reproducibility (Table 1). A quadratic equation was used to model each response by excluding irrelevant terms to obtain the highest coefficients of determination (R2) and prediction (Q2) possible. The relatively high R2 and Q2 values (Table 2) indicated that the models fit well with the experimental data and can be used to predict the behavior of responses within the experimental region. For each response, three two-dimension contour plots with maltose and sucrose concentrations as the two axes were used to illustrate the models, and each plot corresponded to a different enzyme dosage. As enzyme dosage had a minor effect on the responses, contour plots at an enzyme dosage of 5.5 U/g sucrose were chosen to represent the effects of the two main variables in Figure 1. In the present study, dosage was used in relation to sucrose concentration, and the concentration ratio of enzyme to sucrose was demonstrated to mostly affect substrate conversion rate and, to a lesser degree, the IMO profile. The effect of a wide range of dextransucrase concentration over fixed maltose and sucrose concentrations was studied,16 and the amount and number of acceptor products generally increased with increasing amount of enzyme. However, at moderate enzyme dosage similar to that used in the present study, the effects were also insignificant. This is probably due to the long reaction time (24 h) used in both studies, which allows sucrose to be depleted under most conditions and the IMO yield to be maximized. Dextransucrase dosage may affect the IMO production when the reaction process is monitored; however, when only the end points were compared, the effect of enzyme dosage was not reflected. The yield of each linear IMO of DP3−6 and the total IMOs, as well as the percent of consumed maltose, was improved by increasing initial sucrose concentration, whereas initial maltose concentration exhibited different effects on the production of each IMO (Figure 1). Maximal production of panose and LIM4 was attained when maltose and sucrose were both at the highest concentration tested. As they constituted the main IMOs produced, the total IMO yield was influenced in a similar manner. The production of LIM5 and LIM6, however, was favored at medium and the lowest maltose concentration, respectively, while maintaining the highest sucrose concen3280


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Figure 2. HPAEC-PAD profiles of the product mixture of Cab3-DSR maltose acceptor reaction and the selected fractions for DP3−8 isolated by gel filtration. Labeled peaks are as follows: the major IMO products (Pan and LIM4−8), the minor products (BIM3−8), glucose (Glc), fructose (Fru), leucrose (Leu), and maltose (Mal), with the structures of the two products for DP3−5 illustrated. The fractions are in their elution order in the gel filtration.

nomenclature of Domon and Costello.34 The difference in the fragment ion profiles between the mixture and panose was due to the presence of BIM3 in the mixture. In the MS2 spectrum of the [M + Cl]− ion for the mixture (Figure 3A2), the presence of minor product resulted in an increase of cross-ring fragment ions such as m/z 425, 263, and 221 as compared with the pure panose (Figure 3A1). The profile of the fragment ions originating from the minor product resembled that reported for the trisaccharides produced by dextransucrase acceptor reactions with lactose and cellobiose, which contained a 2,4disubstituted reducing residue.27 The MS2 spectra of the [M + Li]+ ion for panose and the mixture (Figure 3B1,B2, respectively) were generally similar. However, the fragment ions at m/z 391 and 451 in the spectrum of the mixture were lower in intensity than those in the spectrum of panose, indicating a lack of such fragment ions from BIM3. When Y2 and C2 ions at m/z 349 were further fragmented at the MS3 stage, a marked increase of cross-ring fragment ion at m/z 229 was observed in the spectrum of the mixture (Figure 3C2) as compared with the corresponding spectrum of panose (Figure 3C1). The cross-ring fragment ion at m/z 229 (loss of 120 Da) was previously reported to be diagnostic of a (1→2) linkage.27 Moreover, cross-ring cleavage ions formed by loss of 6-OH > 3-OH > 4OH,38 the decrease of retention for BIM4 could be explained by the fact that the substitution at the C-2 of the reducing residue of panose prevents the interaction of the most acidic 2OH group with the column material. This effect outweighs the additional retention brought by the new glucosyl residue. According to pattern similarity in their maltose acceptor products, 15 dextransucrases from W. conf usa and the commercial one from Lc. mesenteroides were all known as typical dextransucrases, which produce dextrans with predominantly α-(1→6) linkages and a low degree of α-(1→3) branching. In agreement with a previous study on specificity similarity between typical dextransucrases (E392-rDSR and Lc. mesenteroides NRRL B-512F dextransucrase),27 we found that the specificity to form the α-(1→2)-branched IMOs from maltose is common among these enzymes. In an earlier study of maltose acceptor products of Lc. mesenteroides B-512F dextransucrase, in addition to panose series, a series of minor products were also detected with thin-layer chromatography (TLC). The minor DP3 was claimed to be a branched trisaccharide, α- D -Glcp-(1→4)-[α- D -Glcp-(1→6)]- D -Glcp, which was estimated on the basis of results on maltotriose acceptor products.39 However, the claimed structure was not found in the present study using similar enzymes. We thus suspect that the minor DP3 observed earlier on TLC39 is probably centose as well. A similar α-(1→2) branch linkage was also formed on β-(1→4)-linked disaccharides lactose and cellobiose by E392-rDSR and Lc. mesenteroides B-512F dextransucrase.27,40 Despite differences in conformation between these disaccharides, they could adopt a similar binding mode in which the C-2 of reducing glucosyl residue is oriented toward the active site. We speculate that the higher members of minor IMO series are synthesized through branch formation on an elongated linear IMO, which means that the elongated IMO can still interact with the active site to allow the reducing end to be α-(1→2) glucosylated. The resultant branched IMO can no longer form a regular α-(1→6) linkage at the nonreducing end, probably because they are not accommodated in the enzyme acceptor site. It is interesting to note that although the typical dextransucrases form α-(1→3) branches in dextran, they form an α-(1→2) branch on the reducing end of low-molecularweight acceptors instead. Such α-(1→2)-branched IMOs have not been described from acceptor reaction of a glycoside hydrolase family 70 enzyme. Other types of α-(1→2)-branched IMOs are produced by Lc. mesenteroides NRRL B1299 dextransucrase from maltose. The acceptor products are three homologous IMO series, with the major series constituting linear IMOs. The second and third series have an additional single glucosyl residue α-(1→2) attached on the terminal and the penultimate α-(1→6)-linked residue of the nonreducing end, respectively.41 These IMOs with α-(1→2) linkages are successfully marketed as prebiotics 3284



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due to their low digestibility.1 An engineered enzyme GBDCD2 from Lc. mesenteroides NRRL B-1299 transfers the glucosyl moiety from sucrose onto dextran or GLOS through the formation of α-(1→2) linkages.42 It synthesizes highly α(1→2)-branched GLOS from maltose-based IMOs of DP4 and DP5; however, the branches are formed on α-(1→6)-linked glucosyl residues, not on maltose. A significant amount of IMOs was simultaneously produced with dextran during sourdough fermentation with W. conf usa in the presence of endogenous maltose and added 10% sucrose.20 The HPAECPAD profile of the IMOs produced resembles that observed in the present study, suggesting the presence of minor series of IMOs in W. conf usa sourdough. These series of IMOs could have additional health benefits as the α-(1→2) linkage has shown high resistance to in vitro and in vivo gastrointestinal digestion.42 In addition, the α-(1→2)-linkage-containing kojibiose and two α-(1→2)-branched trisaccharides based on lactose and cellobiose, respectively, all showed in vitro selectivity toward probiotic bacteria.14,43,44 In this study, the effects of sucrose, maltose, and a Weissella dextransucrase (E392-rDSR) on the IMO product profile were studied by response surface modeling to synthesize mainly long-chain IMOs for extended persistence in the colon. The principal products of the maltose acceptor reaction were linear panose series IMOs. A series of unknown minor products was also noted. Quadratic regression models were established for the yield of each linear IMO of DP3−6 as well as the total IMOs and the maltose consumption ratio. The yield for each IMO was maximized with the highest sucrose input concentration but various maltose inputs. The optimal maltose concentration for DP3−4, DP5, and DP6 was at its highest, medium, and lowest level, respectively. The dextransucrase dosage had smaller effects on these responses. The results would help to select the optimal conditions for size-controlled synthesis of IMOs by dextransucrase-catalyzed acceptor reaction. Formation of the minor series of products from maltose seems to be a general property of typical dextransucrases, as manifested by two W. conf usa dextransucrases (E392-rDSR and Cab3-DSR) and a commercial Lc. mesenteroides dextransucrase. The minor oligosaccharides were characterized for the first time, and they were identified to be IMOs with a single unit branch α-(1→2) linked to the reducing end of maltose. The α-(1→2) branch was proposed to be formed on the principal series of linear IMOs. This study expands the knowledge on dextransucrase specificity and thus product composition.

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AUTHOR INFORMATION

Corresponding Author

*(Q.S.) Phone: +358 9 191 58241. Fax: +358 9 191 58475. Email: qiao.shi@helsinki.fi. Funding

This study was supported by the Academy of Finland (Contract 255755) via the joint WISEDextran project and by the Ministry of Science and Technology, New Delhi, India (A.G.). Financial support from the ABS Graduate School (Q.S.) and the Raisio plc Research Foundation (M.J.) is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01356. Experimental data from the preliminary test and central composite design for optimization of synthesis conditions toward longer IMOs, contour plots representing the influence of reaction conditions on IMO production, full 1H and 13C assignments for BIM3 (centose) and overlaid 2D DQFCOSY and TOCSY NMR spectra, correlation between HPAEC retention time and DP for different oligosaccharides, and HPAEC-PAD profile of panose acceptor products of W. conf usa dextransucrase (PDF) 3285


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Optimization of Isomaltooligosaccharide Size Distribution by Acceptor

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