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Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis
Food Chemistry 135 (2012) 1547–1554
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using b-galactosidase from Kluyveromyces lactis Qiuyun Shen a, Ruijin Yang a,b,⇑, Xiao Hua a, Fayin Ye a, He Wang a, Wei Zhao a, Kun Wang a a b
State Key Laboratory of Food Science & Technology and School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jinangsu, China Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jinangsu, China
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Article history: Received 29 January 2012 Received in revised form 7 April 2012 Accepted 29 May 2012 Available online 9 June 2012 Keywords: b-Galactosidase Transgalactosylation Lactose Fructose Lactulose Allo-lactulose NMR
a b s t r a c t The enzymatic transgalactosylation of lactose in the presence of fructose using b-galactosidase from Kluyveromyces lactis (KlbGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(b-D-galactopyranosyl-(1 ? 1)-D-fructose) with two main isomers in D2O was identified to be the major transgalactosylation product while lactulose(b-D-galactopyranosyl-(1 ? 4)-D-fructose) turned out to be the minor one, indicating that KlbGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4 g/l, respectively, at 66.5% lactose conversion (200 g/l initial lactose concentration). Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Lactose(b-D-galactopyranosyl-(1 ? 4)-D-glucopyranose) is a disaccharide found most notably in milk, usually at concentrations approaching 5% (w/v) (Ganzle, Haase, & Jelen, 2008). In 2006, about 870,000 metric tons crystalline lactose were produced worldwide with an expected compound annual growth rate between 3% and 5% until 2012. However, lactose has a reduced field of application, since a significant part of the word population is lactose intolerant, leading to bloating, flatulence, and other gastrointestinal symptoms after lactose consumption (Harju, 1987). Currently, economic and environmental considerations dictate that effective methods should be developed to exploit the renewable raw material lactose. One of the major options for bioconversion of lactose into commercially important products is the utilization of transgalactosylation property of microbial b-galactosidases(b-D-galactohydrolase, EC 3.2.1.23) presented in either free or immobilized form (Irazoqui et al., 2009; Shen et al., 2011; Torres, Goncalves, Jose, & Rodrigues, 2010). Transgalactosylation is commonly observed as a kind of side reaction of lactose hydrolysis catalyzed by b-galactosidase, when ⇑ Corresponding author at: State Key Laboratory of Food Science & Technology, Jiangnan University, 214122, China. Tel./fax: +86 510 85919150. E-mail address: [email protected] (R. Yang). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.115
the galactosyl acceptor is not the hydroxyl group from the water molecule but from another nucleophilic molecule possessing a hydroxyl function (Wallenfels, 1951). This property of microbial bgalactosidase has been utilized for production of numerous different prebiotic galactosyl-containing oligosaccharides varying in their degree of polymerization and glycosidic linkage, mainly di-, tri- and tetrasaccharides. When the enzymatic hydrolysis of lactose is conducted in presence of fructose, the galactosyl moiety can be glycosidically linked with fructose, forming the corresponding disaccharide galactosylfructose (Vaheri & Kaupinnen, 1987). The theoretical possible reactions and products of the biotransformation process from lactose in the presence of fructose using microbial b-galactosidase are shown in Scheme 1 (Barbara et al., 2006; Cardelle-Cobas, Martínez-Villaluenga, Villamiel, Olano, & Corzo, 2008; Martines-Villaluenga et al., 2008; Torres et al., 2010). Any sugar present throughout the reaction can be the nucleophile to accept the transferred galactosyl moiety, yielding a mixture of glucose, galactose, unreacted lactose and fructose, trans-D-galactosylated oligosaccharides indicating that the transgalactosylation catalyzed by b-galactosidase is a very complex reaction. b-Galactosidases from different sources, including Kluyveromyces lactis (K. lactis), the major source of bgalactosidase, have been described as catalysts for the transgalactosylation from lactose to fructose, directing towards the formation
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Scheme 1. Possible reactions and products of lactose transgalactosylation in the presence of fructose catalyzed by b-galactosidase.
of lactulose(b-Vaheri-galactopyranosyl-(1 ? 4)-b-D-fructose) (Kim, Park, & Oh, 2006; Lee, Kim, & Oh, 2004; Mayer et al., 2004; Mayer, Kranz, & Fischer, 2010). However, little information about other transgalactosylation products has been previously reported, probably due to the fact that the physicochemical properties of these formed oligosaccharides were so similar, that the analytical methods used, could not distinguish them or they were not isolated and identified successfully. However, it was found that two main transgalactosylation oligosaccharides were formed during the transgalactosylation from lactose to fructose utilizing KlbGal by HPLC-RI in our study. Since the chemical structures of oligosaccharides (the number or type of hexose moieties, the position and conformation of links between the hexose) may affect their probiotic properties (Delzenne, 2003; Djouzi & Andrieux, 1997), the formed oligosaccharides were chromatographically purified and then characterized by liquid chromatography/mass spectrometry (LC/MS) and extensive nuclear magnetic resonance (NMR) studies including 1D (1H, 13C, and DEPT 135) and 2D (HSQC, and HMBC) NMR experiments. The reason for the main products formation was also discussed. The influence of oligosaccharide structure on prebiotic selectivity has been demonstrated previously, showing that oligosaccharides containing galactosyl has a strong prebiotic character (Sanz, Gibson, & Rastall, 2005). Otherwise, Förster-Fromme observed that enzymatically synthesized lactulose had a slightly higher prebiotic index than lactulose derived by alkaline isomerisation (FörsterFromme et al., 2001). Therefore, this study will reveal the obtention of novel oligosaccharides with potential prebiotic properties. 2. Materials and methods 2.1. Materials b-galactosidase MaxilactÒ 5000 from K. lactis was kindly supported by DSM Co. Ltd., (Beijing). The optimal conditions for this
enzyme were pH 6.6–7.0, temperature 35–40 °C. Lactose (AR) and fructose (AR) were purchased from Sinopharm Chemical Reagent Co. Ltd., (Shanghai) and used without further purification. All the standards for HPLC analysis were purchased from Sigma– AldrichÒ Co., (USA). 2.2. Transgalactosylation reaction A lactose solution of 200 g/l was prepared in 0.1 N sodium phosphate buffer (pH 6.8) containing 20% g/l fructose, and the reaction was initiated by adding KlbGal at a final concentration of 3 Units/ ml. The transgalactosylation reaction was performed at a final volume of 10 ml and 38 °C in an orbital shaker at 200 rpm. Samples (200 ll) were withdrawn at specific time intervals and immediately immersed in boiling water for 5 min to inactivate the enzyme. The samples were stored at 4 °C for subsequent HighPerformance Liquid Chromatograph (HPLC) analysis. Control samples were prepared in the same manner, except that no enzyme was added and no changes in lactose were observed. 2.3. Chromatographic determination of saccharides Transglycosylation reaction samples were analyzed by an HPLC system (Hitachi L-2000, Tokyo, Japan) equipped with a differential refractive index (RI) detector (Hitachi L-2490, Japan) and an amino column (ShodexÒ Asahipak NH2P-50 4E, Japan). Samples and standard solutions were filtered through a nylon Millipore FH (0.22 lm) (Bedford, MA) membrane before injection. The column was eluted at 30 °C with acetonitrile and pure water (75:25, v/v) at a flow rate of 1 ml min1. Data processing was performed using Elite 2000 program suit (Hitachi, Japan). Quantification of lactose, lactulose and the novel disaccharide relied on their respective calibration curves. The regression coefficients of the curves for each standard or the purified novel products were always greater than 0.999.
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Fig. 2. LC/MS spectra of the transgalactosylation products: (a) product 1 and (b) product 2.
2.5. LC/MS analysis LC/MS analysis of oligosaccharides 1 and 2 was obtained using a UPLC BET HILIC column (2.1 100 mm, 1.7 lm, Waters), maintained at 30 °C, using a mobile phase of acetonitrile and water containing 0.1% ammonium hydroxide, at the flow rate of 0.3 ml/min. The volume of sample injected was 0.1 ll. MS detection was performed in electrospray negative ionization (ESI) mode with 3.0 kV applied to the capillary, 300 °C N2 as desolvation gas with the flow rate at 500 l/h. The MS scan was over a range of 100– 800 m/z. 2.6. NMR analysis
Fig. 1. HPLC-RI chromatogram of the bioconversion of 200 mg/ml lactose in the presence of 200 mg/ml fructose in a 50 ml scale using b-galactosidase from K. lactis at pH 6.8, 37 °C, 200 rpm and 4 Units/ml of enzyme after 1 h (Fig. 1a), 3 h (Fig. 1b), 6 h (Fig. 1c) and 24 h (Fig. 1d) of reaction: 1, fructose; 2, galactose + glucose; 3, product 1; 4, product 2; 5, lactose.
2.4. Purification Transglycosylation reaction products were purified by semipreparative HPLC-RI (Hitachi, Japan) using a preparative amino column (NH2P-50 10E, Shodex, Japan). Compounds were separated under isocratic conditions with acetonitrile/water (75:25, v/v) at a flow rate of 3 ml/min and fractions corresponding to each transgalactosylation products were collected. Fractions from 50 runs were pooled, concentrated by rotary evaporator, analyzed by HPLC as previously described and lyophilized for liquid chromatography/ mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) analyses.
The chemical structures of synthesized oligosaccharide 1 and 2 were identified by proton (1H NMR), carbon (13C NMR), distortionless enhancement by polarization transfer (DEPT) 135, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum coherence (HMBC) spectra. NMR spectra were acquired at 293.1 K with a Bruker (Bruker Biospin, Rheinstetten, Germany) Avance III 400 MHz spectrometer equipped with a Bruker 5 mm TXI probehead with Z-gradient. Data acquisition and processing were done with Bruker Topspin 2.1 and ACDLABS 12.0. 1H NMR spectra were acquired using the Bruker zg0 or ag0pr pulse programs using the following setting: frequency = 400.14 MHz, acquisition time = 9 s, number of transients = 16. 13C NMR and DEPT 135 spectra were acquired using the same setting: frequency = 100.62 MHz, acquisition time = 1.4 s, number of transients = 1046. Manual phase correction and automatic polynomial baseline correction were used for both spectra. 1H chemical shifts were referenced to the residual solvent signal at dH 4.79 (D2O) relative to DSS. 2D HSQC and HMBC spectra were acquired using the Bruker pulse programs hsqcetgpsisp2 and hmbcgpndqf respectively, with standard acquisition parameters. The acquired 2D data was Fourier transformed and manually phase corrected. 3. Results The HPLC-RI profiles of oligosaccharides formed during enzymatic lactose conversion by KlbGal are shown in Fig. 1. The reaction was performed at 38 °C using solution of 200 mg/ml lactose and the same concentration of fructose in 0.1 M phosphate buffer, pH 6.8, 3 Units/ml enzyme, and monitored for 24 h. From a comparison of
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Fig. 3. 1D NMR spectra of disaccharide 1: (a) 1H NMR (D2O, 400.14 MHz) and (b) 13C NMR (D2O, 100.62 MHz) spectra. Anomeric region was labeled. Note: G as galactose and F as fructose.
the retention time with standards, peaks 2 was assigned to glucose and galactose, which co-eluted, and peak 1 and peak 5 were assigned to fructose and lactose, respectively. According to the retention time, peaks 3 and 4 might both be disaccharides. As can be seen during hydrolysis of lactose, in D-fructose, glucose and galactose were released, and transfer products 1 and 2 (peaks 3 and 4, respectively) were formed simultaneously. In this high lactose and fructose content solution, galactosyl residues of galactosyl-enzyme intermediates were transferred not only to water but also to fructose or other sugars and galactosyl-containing oligosaccharides (transfer products 1 and 2) were formed (Scheme 1). The concentration of lactose was decreased by hydrolysis and transgalactosylation reactions of KlbGal, with increasing time. Most of the oligosaccharides were produced and the concentration of fructose was decreased due to the its acceptance of galactosyl residues at initial stage of the reaction, and the maximum yields of transfer products 1 and 2 were 15.4 and 47.5 g/l, respectively after 3 h with 66.5% of lactose vanished (Fig. 1b). Once the transfer products reached the maximum, longer time of the reaction brought the decrease of the formed products and the release of fructose (Fig. 1c, d), indicating that the transgalactosylation products may act as substrates of KlbGal and were hydrolyzed or produced some other galacto-oligosaccharides with reaction processing (MartinesVillaluenga et al., 2008). To isolate and identify the transfer products catalyzed by KlbGal in the presence of fructose, the biotransformation of lactose with high concentration in the presence of fructose was performed in a total volume of 50 ml at 38 °C, pH 6.8. Products 1 and 2 were isolated by collecting samples from the separated HPLC peaks using semi-preparative column. LC/MS analysis of the pure compound 1 and 2 both showed ions at m/z 341.1 [MH] and m/z 377.1 [M+Cl], corresponding to hexose disaccharides (Fig. 2). Therefore, disaccharides were the main oligosaccharides formed as a result of transgalactosylation catalyzed by KlbGal. Guerrero et al. studied the enzyme source on the selectivity of lactulose synthesis and also found that enzyme from K. lactis produced mainly GOS-2 (Guerrero, Vera, Huerta, Plou, & Illanes, 2010). Since the prebiotic property of a disaccharide depends upon its chemical structure (nature of monosaccharide and type of glycosidic linkage), an extensive NMR study was further performed to elucidate the structures of the two transfer disaccharides, including 1D (1H, 13C and DEPT 135) and 2D (HQSC and HMBC) NMR experi-
ments. Using an authentic standard and with the standard-addition technique, oligosaccharide 1 (peak 3) was tentatively identified as lactulose (b-D-galactosyl-(1 ? 4)-fructose). Its 1HNMR and 13C NMR spectrums (Fig. 3), which extremely coincided with the published NMR data for lactulose (Mayer et al., 2004), further confirmed that the transgalactosylation product 1 was lactulose with three isomers: (1A) b-D-galactopyranosyl-(1 ? 4)-b-D-fructopyranose (the major isomer), (1B) b-D-galactopyranosyl-(1 ? 4)-b-D-fructofuranose, and (1C) b-D-galactopyranosyl-(1 ? 4)-a-D-fructofuranose. For disaccharide 2, the 1H and 13C chemical shifts were assigned by the interpretation of their 1D NMR (1H, 13C and DEPT 135) spectra (Fig. 4) and 2D NMR (HSQC, HMBC) spectra (Fig. 5) and by further comparison with those of lactulose and to the literatures (Landersjo et al., 2006; Taubert, Konschin, & Sundholm, 2005). The 13C NMR (Fig. 4b) spectrum of disaccharide 2 in D2O displayed two sets of signals, indicating a mixture of isomers 2A and 2B with the population being 3:1 approximately because of the mutarotational equilibrium between pyranose and furanose forms. The major set of resonances, corresponding to the most populated isomer (2A), contained two anomeric carbons at d 103.33 and d 97.58. Accordingly, the 1H NMR spectrum (Fig. 4a) showed only one anomeric signal at d 4.45 and DEPT 135 spectra (Fig. 4c) displayed one anomeric carbon at d 103.33 and three negative signals at d 71.55, d 63.42 and d 61.00, illustrating that the anomeric carbon at d 97.58 was quaternary and the disaccharide 2 was consisted of a fructose and a galactose moiety. HMBC correlations between both H-10 (galactosyl) at d 4.45 and C-1 (fructose) at d 71.55 and between both C-10 (galactosyl) at d 103.33 and H-1a/b (fructose) at d 3.94/3.83, respectively, unambiguously established the 1 ? 1 linkage between the galactose and fructose moieties of disaccharide 2 (Fig. 5b). This result was further supported by the 5–10 ppm low-field shifts observed for C-1 at d 71.55 (fructose) and C-10 at d 103.33 (galactose) (Fig. 4b). The pyranoid b-D-galactosyl moiety was deduced from the chemical shift of anomeric proton (d 4.45) and the coupling constant (3JH-10 , H-20 = 7.71 Hz) (Fig. 4a) and was confirmed by the observed C-40 at d 68.62, C-30 at d 72.49 and C-50 at d 75.25 (Fig. 4b). The positive HMBC correlation between H-6a/b (d 3.71/ 4.05) and C-2 (d 97.56) of the fructose established a b-D-fructopyranosyl moiety in the major isomer 2A (Fig. 5b). From the comparison of NMR data and those proposed for lactulose isomers (Mayer et al., 2004), we determined that the most populated isomer for
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Fig. 4. 1D NMR spectra of disaccharide 2: (a) 1H NMR (D2O, 400.14 MHz), (b) 13C NMR (D2O, 100.62 MHz) and (c) DEPT 135 spectra (D2O, 100.62 MHz). In the DEPT 135 spectrum, the sign of each peak provides the multiplicity of the carbon: positive (upward) for CH and CH3 and negative (downward) for CH2, quaternary carbons do not appear. Note: G as galactose and F as fructose.
disaccharide 2 was b-D-galactopyranosyl-(1 ? 1)-b-D-fructopyranose (Fig. 6). It was also possible to measure C-2 (d 100.55) C-3 (d 80.66) and C-5 (d 76.23) resonances for minor isomer, which were typical of furanose (Fig. 4b). In the same way as shown above for isomer 2A, we determined the minor isomer 2B was b-D-galactopyranosyl-(1 ? 1)-b-D-fructofuranose (Fig. 6).
In conclusion, the two main products were identified as b-Dgalactosyl-(1 ? 4)-fructose (lactulose) and b-D-galactosyl-(1 ? 1)fructose (allo-lactulose), respectively, demonstrating that fructose could be used as a galactosyl acceptor for the enzymatic synthesis of galactosyl-fructoses by KlbGal. Moreover, the formed galactosyl-(1 ? 1)-fructose with two main isomers, b-D-galactopyranosyl-(1 ? 1)-b-D-fructopyranose and b-D-galactopyranosyl-
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Fig. 5. 2D NMR spectra of disaccharide 2: (a) 2D NMR heteronuclear single quantum coherence (HSQC) spectra and (b) 2D heteronuclear multiple quantum coherence (HMBC) spectra. Note: G as galactose and F as fructose, a/b as one of the two protons linked to the same carbon atom.
(1 ? 1)-b-D-fructofuranose, was three times the amount of the formed lactulose, indicating that the KlbGal showed a preference for establishing b-(1 ? 1) linkages and forming disaccharides in the presence of fructose. The enzymatic synthesis process will be further optimized and scaled up to allow for a thorough study of their potential prebiotic properties in our future work.
4. Discussion b-Galactosidases, a type of glycosidase, in nature act as hydrolases. However, interestingly, they can catalyze the transfer of a
galactose moiety from a donor to a carbohydrate molecule under special conditions and have already been used for the synthesis of several oligosaccharides containing the galactose moiety. Carbohydrate molecules represent multiple hydroxyl groups, thus transgalactosylation catalyzed by b-galactosidase are not regionspecific but stereospecific and a mixture of regionisomers is probably obtained. The regionisomers produced depends upon the source of the enzyme, the acceptor and the reaction conditions (Irazoqui et al., 2009; Tricone, Tramice, Giordano, & Andreotti, 2008; Yoon & Ajisaka, 1996). The regioselectivity of KlbGal has been investigated using various acceptors and primary hydroxyl groups were glycosylated
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structural feature of this oligosaccharide will be useful for the study of its fermentation mechanism, and also for the research of structure-function relationship of oligosaccharides.
Acknowledgements The authors gratefully acknowledge the financial support provided by the ‘Five-twelfth’ National Science and Technology Support Program (2011BAD23B03), the Natural Science Foundation of Jiangsu Province (BK2011149) and the Innovative Research Program for Graduate Students of Jiangsu Province (CXZZ11_0487).
References
Fig. 6. Structures of the major isomers of disaccharide 2: (A) b-D-galactopyranosyl(1 ? 1)-b-D-fructopyranose, and (B) b-D-galactopyranosyl-(1 ? 1)-b-D-fructofuranose. Arrows (?) indicate important HMBC correlations.
preferentially to secondary hydroxyl groups in all of the acceptors studied. In the preparation of GOS, KlbGal produced predominantly b-(1 ? 6) oligosaccharides (60 -galactosyl-lactose and b-D-galactosyl-(1 ? 6)-D-glucose) (Asp, Burvall, Dahlqvist, Hallgren, & Lundblad, 1980). Otherwise, KlbGal afford N-acetyl-allolactosamine (bGal-(1 ? 6)-GlcNAc) as the major transglycosylation product and bGal-(1 ? 4)-GlcNAc as the minor one between lactose and GlcNAc (Bridiau & Maugard, 0000). The enzymatic transgalactosylation during lactulose hydrolysis was also studied using the KlbGal, and two main trisaccharides were produced, which exhibited a galactose unit linked to C-6 of the galactose moiety of lactulose, while the other one presented a galactose unit linked to C-1 of the fructose moiety of lactulose (Martines-Villaluenga et al., 2008). Since hydroxyl groups linked to C-1 and C-2 of fructose are primary, the abundant products produced by transgalactosylation from lactose to fructose using KlbGal are expected to be b-D-galactosyl-(1 ? 1)-D-fructose and b-D-galactosyl-(1 ? 6)-D-fructose according to the regioselectivity of KlbGal. However, there was no peak corresponding to (1 ? 6)-galactosyl-fructose in the HPLC-RI spectra because of the fact that the abundant isomer existed in fructose aqueous solution is b-D-pyranose tautomer with the primary hydroxyl group linked to C-6 being endocyclic due to its condensation reaction with carbonyl group (Flood, Johns, White & Crystal, 1996; Polacek & Kaatze, 2001). Lee et al. observed that when lactose was used as a galactose donor and fructose as an acceptor, not only lactulose but also other galacto-oligosaccharides produced by b-galactosidase in permeabilized cells of K. lactis, but the byproducts were not isolated and identified (Lee et al., 2004). Even though synthesis of allo-lactulose (b-D-galactosyl-(1 ? 1)-D-fructose) using the reversed hydrolysis activity of b-galactosidase from E. coli and transgalactosylation activity of b-galactosidase from Bifidobacterium adolescentis DSM 20083 has been previously mentioned (Ajisaka, Nishida, & Fujimoto, 1987; Förster-Fromme et al., 2001), this is the first time that a complete identification and a structural characterization of this product have been carried out. The detailed elucidation of the
Ajisaka, K., Nishida, H., & Fujimoto, H. (1987). Use of an activated carbon column for the synthesis of disaccharides by use of a reversed hydrolysis activity of bgalactosidase. Biotechnology Letters, 9, 387–392. Asp, N. G., Burvall, A., Dahlqvist, A., Hallgren, P., & Lundblad, A. (1980). Oligosaccharide formation during hydrolysis of lactose with Saccharomyces lactis lactase (Maxilact): Part 2-oligosaccharide structures. Food Chemistry, 5, 147–153. Barbara, S., Thu-ha, N., Marlene, S., Klaus, D. K., Werner, L., & Dietmar, H. (2006). Production of prebiotic galacto-oligosaccharides from lactose using bgalactosidases from Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 54, 4999–5006. Bridiau, N., & Maugard, T. A. (2011). Comparative Study of the Regioselectivity of the b-Galactosidases from Kluyveromyces lactis and Bacillus circulans in the Enzymatic Synthesis of N-Acetyl-lactosamine in Aqueous Media. Biotechnology Progress, 27, 386–394. Cardelle-Cobas, A., Martínez-Villaluenga, C., Villamiel, M., Olano, A., & Corzo, N. (2008). Synthesis of oligosaccharides derived from lactulose and pectinex Ultra SP-L. Journal of Agricultural and Food Chemistry, 56, 3328–3333. Delzenne, N. M. (2003). Oligosaccharides: State of the art. Proceeding of the Nutrition Society, 62, 177–182. Djouzi, Z., & Andrieux, C. (1997). Compared effects of three oligosaccharides on metabolism of intenstinal microflora in rats inoculated with a human faecal flora. British Journal of Nutritional, 78, 313–324. Flood, A. E., Johns, M. R., & White & Crystal, E. T. (1996). Mutarotation of D-fructose in aqueous- ethanolic solutions and its influence on crystallisation. Carbohydrate Research, 288, 45. Förster-Fromme, K., Schuster-Wolff-Bühring, R., Hartwig, A., Holder, A., Schwiertz, A., Bischoff, C. S., et al. (2001). A new enzymatically produced 1-lactulose: A pilot study to test the bifidogenic effects. International Dairy Journal, 21, 940–948. Ganzle, M. G., Haase, G., & Jelen, P. (2008). Lactose: Crystallization, hydrolysis and value-added derivatives. International Dairy Journal, 18(7), 685–694. Guerrero, C., Vera, C., Huerta, L. M., Plou, F., & Illanes, A. (2010). Effect of reaction conditions and enzyme source on the selectivity of lactulose synthesis. Special Abstracts/Journal of Biotechnology, 150S, S402–S403. Harju, M. (1987). Lactose hydrolysis. Bulletin of Internal Dairy Federation, 212, 50–55. Irazoqui, G., Giacomini, C., Batista-Viera, F., Brena, B. M., Cardelle-Cobas, A., Corzo, N., et al. (2009). Characterization of galactosyl derivatives obtained by transgalactosylation of lactose and different polyols using immobilized bgalactosidase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 57, 11302–11307. Kim, Y. S., Park, C. S., & Oh, D. K. (2006). Lactulose production from lactose and fructose by a thermostable b-galactosidase from Sulfolobus solfataricus. Enzyme and Microbial Technology, 39, 903–908. Landersjo, C., Stevensson, B., Eklund, R., Ostervall, J., Sodernan, P., Widmalm, G., et al. (2006). Molecular conformations of a disaccharide investigated using NMR spectroscopy. Journal of Biomolecular NMR, 35, 89–101. Lee, Y. J., Kim, C. S., & Oh, D. K. (2004). Lactulose production by b-galactosidase in permeabilized cells of Kluyveromyces lactis. Applied Microbiology and Biotechnology, 64, 787–793. Martines-Villaluenga, C., Cardelle-Cobas, A., Olano, A., Corzo, N., Villamiel, M., & Jimeno, M. L. (2008). Enzymatic synthesis and identification of two trisaccharides produced from lactulose by transgalactosylation. Journal of Agricultural and Food Chemistry, 56, 557–563. Mayer, J., Conrad, J., Klaiber, I., Lutz-Wahl, S., Beifuss, U., & Fischer, L. (2004). Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose. Journal of Agricultural and Food Chemistry, 52, 6983–6990. Mayer, J., Kranz, B., & Fischer, L. (2010). Continuous production of lactulose by immobilized thermostable b-glycosidase from Pyrococcus furiosus. Journal of Biotechnology, 145, 387–393. Polacek, R., & Kaatze, U. (2001). Chair-chair ring inversion of D-fructose coupled to the mutarotation in aqueous-ethanolic solution. Chemical Physics Letters, 345, 93–99. Sanz, M. L., Gibson, G. R., & Rastall, R. A. (2005). Influence of disaccharide structure on prebiotic selectivity in vitro. Journal of Agricultural and Food Chemistry, 53, 5192–5199.
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Q. Shen et al. / Food Chemistry 135 (2012) 1547–1554
Shen, Q., Yang, R., Hua, X., Ye, F., Zhang, W., & Zhao, W. (2011). Gelatin-templated biomimetic calcification for b-galactosidase immobilization. Process Biochemistry, 46, 1565–1571. Taubert, S., Konschin, H., & Sundholm, D. (2005). Computational studies of 13C NMR chemical shifts of saccharides. Physical Chemistry Chemical Physics, 7, 2561–2569. Torres, D. P. M., Goncalves, M. P. F., Jose, A. T., & Rodrigues, L. R. (2010). Galactooligosaccharides: Production, properties, applications, and significance as prebiotics. Comprehensive reviews in food science and food safety, 9, 438– 454.
Tricone, A., Tramice, A., Giordano, A., & Andreotti, G. (2008). Glycoside hydrolases in Aplysia fasciata: Analysis and applications. Biotechnology & Genetic Engineering Reviews, 25, 129–148. Vaheri, M., & Kaupinnen, V. (1987). The formation of lactulose (4-O-b-galactopyranosylfructose) by b-galactosidase. Acta Pharmaceutica Fennica, 87, 75–83. Wallenfels, K. (1951). Enzymatische synthesis von oligosacchariden aus disacchariden. Naturwissenschaften, 38, 306–307. Yoon, J. H., & Ajisaka, K. (1996). The synthesis of galactopyranosyl derivatives with b-galactosidases of different origins. Carbohydrate Research, 292, 153–163.
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using b-galactosidase from Kluyveromyces lactis Qiuyun Shen a, Ruijin Yang a,b,⇑, Xiao Hua a, Fayin Ye a, He Wang a, Wei Zhao a, Kun Wang a a b
State Key Laboratory of Food Science & Technology and School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jinangsu, China Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jinangsu, China
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Article history: Received 29 January 2012 Received in revised form 7 April 2012 Accepted 29 May 2012 Available online 9 June 2012 Keywords: b-Galactosidase Transgalactosylation Lactose Fructose Lactulose Allo-lactulose NMR
a b s t r a c t The enzymatic transgalactosylation of lactose in the presence of fructose using b-galactosidase from Kluyveromyces lactis (KlbGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(b-D-galactopyranosyl-(1 ? 1)-D-fructose) with two main isomers in D2O was identified to be the major transgalactosylation product while lactulose(b-D-galactopyranosyl-(1 ? 4)-D-fructose) turned out to be the minor one, indicating that KlbGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4 g/l, respectively, at 66.5% lactose conversion (200 g/l initial lactose concentration). Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Lactose(b-D-galactopyranosyl-(1 ? 4)-D-glucopyranose) is a disaccharide found most notably in milk, usually at concentrations approaching 5% (w/v) (Ganzle, Haase, & Jelen, 2008). In 2006, about 870,000 metric tons crystalline lactose were produced worldwide with an expected compound annual growth rate between 3% and 5% until 2012. However, lactose has a reduced field of application, since a significant part of the word population is lactose intolerant, leading to bloating, flatulence, and other gastrointestinal symptoms after lactose consumption (Harju, 1987). Currently, economic and environmental considerations dictate that effective methods should be developed to exploit the renewable raw material lactose. One of the major options for bioconversion of lactose into commercially important products is the utilization of transgalactosylation property of microbial b-galactosidases(b-D-galactohydrolase, EC 3.2.1.23) presented in either free or immobilized form (Irazoqui et al., 2009; Shen et al., 2011; Torres, Goncalves, Jose, & Rodrigues, 2010). Transgalactosylation is commonly observed as a kind of side reaction of lactose hydrolysis catalyzed by b-galactosidase, when ⇑ Corresponding author at: State Key Laboratory of Food Science & Technology, Jiangnan University, 214122, China. Tel./fax: +86 510 85919150. E-mail address: [email protected] (R. Yang). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.115
the galactosyl acceptor is not the hydroxyl group from the water molecule but from another nucleophilic molecule possessing a hydroxyl function (Wallenfels, 1951). This property of microbial bgalactosidase has been utilized for production of numerous different prebiotic galactosyl-containing oligosaccharides varying in their degree of polymerization and glycosidic linkage, mainly di-, tri- and tetrasaccharides. When the enzymatic hydrolysis of lactose is conducted in presence of fructose, the galactosyl moiety can be glycosidically linked with fructose, forming the corresponding disaccharide galactosylfructose (Vaheri & Kaupinnen, 1987). The theoretical possible reactions and products of the biotransformation process from lactose in the presence of fructose using microbial b-galactosidase are shown in Scheme 1 (Barbara et al., 2006; Cardelle-Cobas, Martínez-Villaluenga, Villamiel, Olano, & Corzo, 2008; Martines-Villaluenga et al., 2008; Torres et al., 2010). Any sugar present throughout the reaction can be the nucleophile to accept the transferred galactosyl moiety, yielding a mixture of glucose, galactose, unreacted lactose and fructose, trans-D-galactosylated oligosaccharides indicating that the transgalactosylation catalyzed by b-galactosidase is a very complex reaction. b-Galactosidases from different sources, including Kluyveromyces lactis (K. lactis), the major source of bgalactosidase, have been described as catalysts for the transgalactosylation from lactose to fructose, directing towards the formation
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Scheme 1. Possible reactions and products of lactose transgalactosylation in the presence of fructose catalyzed by b-galactosidase.
of lactulose(b-Vaheri-galactopyranosyl-(1 ? 4)-b-D-fructose) (Kim, Park, & Oh, 2006; Lee, Kim, & Oh, 2004; Mayer et al., 2004; Mayer, Kranz, & Fischer, 2010). However, little information about other transgalactosylation products has been previously reported, probably due to the fact that the physicochemical properties of these formed oligosaccharides were so similar, that the analytical methods used, could not distinguish them or they were not isolated and identified successfully. However, it was found that two main transgalactosylation oligosaccharides were formed during the transgalactosylation from lactose to fructose utilizing KlbGal by HPLC-RI in our study. Since the chemical structures of oligosaccharides (the number or type of hexose moieties, the position and conformation of links between the hexose) may affect their probiotic properties (Delzenne, 2003; Djouzi & Andrieux, 1997), the formed oligosaccharides were chromatographically purified and then characterized by liquid chromatography/mass spectrometry (LC/MS) and extensive nuclear magnetic resonance (NMR) studies including 1D (1H, 13C, and DEPT 135) and 2D (HSQC, and HMBC) NMR experiments. The reason for the main products formation was also discussed. The influence of oligosaccharide structure on prebiotic selectivity has been demonstrated previously, showing that oligosaccharides containing galactosyl has a strong prebiotic character (Sanz, Gibson, & Rastall, 2005). Otherwise, Förster-Fromme observed that enzymatically synthesized lactulose had a slightly higher prebiotic index than lactulose derived by alkaline isomerisation (FörsterFromme et al., 2001). Therefore, this study will reveal the obtention of novel oligosaccharides with potential prebiotic properties. 2. Materials and methods 2.1. Materials b-galactosidase MaxilactÒ 5000 from K. lactis was kindly supported by DSM Co. Ltd., (Beijing). The optimal conditions for this
enzyme were pH 6.6–7.0, temperature 35–40 °C. Lactose (AR) and fructose (AR) were purchased from Sinopharm Chemical Reagent Co. Ltd., (Shanghai) and used without further purification. All the standards for HPLC analysis were purchased from Sigma– AldrichÒ Co., (USA). 2.2. Transgalactosylation reaction A lactose solution of 200 g/l was prepared in 0.1 N sodium phosphate buffer (pH 6.8) containing 20% g/l fructose, and the reaction was initiated by adding KlbGal at a final concentration of 3 Units/ ml. The transgalactosylation reaction was performed at a final volume of 10 ml and 38 °C in an orbital shaker at 200 rpm. Samples (200 ll) were withdrawn at specific time intervals and immediately immersed in boiling water for 5 min to inactivate the enzyme. The samples were stored at 4 °C for subsequent HighPerformance Liquid Chromatograph (HPLC) analysis. Control samples were prepared in the same manner, except that no enzyme was added and no changes in lactose were observed. 2.3. Chromatographic determination of saccharides Transglycosylation reaction samples were analyzed by an HPLC system (Hitachi L-2000, Tokyo, Japan) equipped with a differential refractive index (RI) detector (Hitachi L-2490, Japan) and an amino column (ShodexÒ Asahipak NH2P-50 4E, Japan). Samples and standard solutions were filtered through a nylon Millipore FH (0.22 lm) (Bedford, MA) membrane before injection. The column was eluted at 30 °C with acetonitrile and pure water (75:25, v/v) at a flow rate of 1 ml min1. Data processing was performed using Elite 2000 program suit (Hitachi, Japan). Quantification of lactose, lactulose and the novel disaccharide relied on their respective calibration curves. The regression coefficients of the curves for each standard or the purified novel products were always greater than 0.999.
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Fig. 2. LC/MS spectra of the transgalactosylation products: (a) product 1 and (b) product 2.
2.5. LC/MS analysis LC/MS analysis of oligosaccharides 1 and 2 was obtained using a UPLC BET HILIC column (2.1 100 mm, 1.7 lm, Waters), maintained at 30 °C, using a mobile phase of acetonitrile and water containing 0.1% ammonium hydroxide, at the flow rate of 0.3 ml/min. The volume of sample injected was 0.1 ll. MS detection was performed in electrospray negative ionization (ESI) mode with 3.0 kV applied to the capillary, 300 °C N2 as desolvation gas with the flow rate at 500 l/h. The MS scan was over a range of 100– 800 m/z. 2.6. NMR analysis
Fig. 1. HPLC-RI chromatogram of the bioconversion of 200 mg/ml lactose in the presence of 200 mg/ml fructose in a 50 ml scale using b-galactosidase from K. lactis at pH 6.8, 37 °C, 200 rpm and 4 Units/ml of enzyme after 1 h (Fig. 1a), 3 h (Fig. 1b), 6 h (Fig. 1c) and 24 h (Fig. 1d) of reaction: 1, fructose; 2, galactose + glucose; 3, product 1; 4, product 2; 5, lactose.
2.4. Purification Transglycosylation reaction products were purified by semipreparative HPLC-RI (Hitachi, Japan) using a preparative amino column (NH2P-50 10E, Shodex, Japan). Compounds were separated under isocratic conditions with acetonitrile/water (75:25, v/v) at a flow rate of 3 ml/min and fractions corresponding to each transgalactosylation products were collected. Fractions from 50 runs were pooled, concentrated by rotary evaporator, analyzed by HPLC as previously described and lyophilized for liquid chromatography/ mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) analyses.
The chemical structures of synthesized oligosaccharide 1 and 2 were identified by proton (1H NMR), carbon (13C NMR), distortionless enhancement by polarization transfer (DEPT) 135, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum coherence (HMBC) spectra. NMR spectra were acquired at 293.1 K with a Bruker (Bruker Biospin, Rheinstetten, Germany) Avance III 400 MHz spectrometer equipped with a Bruker 5 mm TXI probehead with Z-gradient. Data acquisition and processing were done with Bruker Topspin 2.1 and ACDLABS 12.0. 1H NMR spectra were acquired using the Bruker zg0 or ag0pr pulse programs using the following setting: frequency = 400.14 MHz, acquisition time = 9 s, number of transients = 16. 13C NMR and DEPT 135 spectra were acquired using the same setting: frequency = 100.62 MHz, acquisition time = 1.4 s, number of transients = 1046. Manual phase correction and automatic polynomial baseline correction were used for both spectra. 1H chemical shifts were referenced to the residual solvent signal at dH 4.79 (D2O) relative to DSS. 2D HSQC and HMBC spectra were acquired using the Bruker pulse programs hsqcetgpsisp2 and hmbcgpndqf respectively, with standard acquisition parameters. The acquired 2D data was Fourier transformed and manually phase corrected. 3. Results The HPLC-RI profiles of oligosaccharides formed during enzymatic lactose conversion by KlbGal are shown in Fig. 1. The reaction was performed at 38 °C using solution of 200 mg/ml lactose and the same concentration of fructose in 0.1 M phosphate buffer, pH 6.8, 3 Units/ml enzyme, and monitored for 24 h. From a comparison of
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Fig. 3. 1D NMR spectra of disaccharide 1: (a) 1H NMR (D2O, 400.14 MHz) and (b) 13C NMR (D2O, 100.62 MHz) spectra. Anomeric region was labeled. Note: G as galactose and F as fructose.
the retention time with standards, peaks 2 was assigned to glucose and galactose, which co-eluted, and peak 1 and peak 5 were assigned to fructose and lactose, respectively. According to the retention time, peaks 3 and 4 might both be disaccharides. As can be seen during hydrolysis of lactose, in D-fructose, glucose and galactose were released, and transfer products 1 and 2 (peaks 3 and 4, respectively) were formed simultaneously. In this high lactose and fructose content solution, galactosyl residues of galactosyl-enzyme intermediates were transferred not only to water but also to fructose or other sugars and galactosyl-containing oligosaccharides (transfer products 1 and 2) were formed (Scheme 1). The concentration of lactose was decreased by hydrolysis and transgalactosylation reactions of KlbGal, with increasing time. Most of the oligosaccharides were produced and the concentration of fructose was decreased due to the its acceptance of galactosyl residues at initial stage of the reaction, and the maximum yields of transfer products 1 and 2 were 15.4 and 47.5 g/l, respectively after 3 h with 66.5% of lactose vanished (Fig. 1b). Once the transfer products reached the maximum, longer time of the reaction brought the decrease of the formed products and the release of fructose (Fig. 1c, d), indicating that the transgalactosylation products may act as substrates of KlbGal and were hydrolyzed or produced some other galacto-oligosaccharides with reaction processing (MartinesVillaluenga et al., 2008). To isolate and identify the transfer products catalyzed by KlbGal in the presence of fructose, the biotransformation of lactose with high concentration in the presence of fructose was performed in a total volume of 50 ml at 38 °C, pH 6.8. Products 1 and 2 were isolated by collecting samples from the separated HPLC peaks using semi-preparative column. LC/MS analysis of the pure compound 1 and 2 both showed ions at m/z 341.1 [MH] and m/z 377.1 [M+Cl], corresponding to hexose disaccharides (Fig. 2). Therefore, disaccharides were the main oligosaccharides formed as a result of transgalactosylation catalyzed by KlbGal. Guerrero et al. studied the enzyme source on the selectivity of lactulose synthesis and also found that enzyme from K. lactis produced mainly GOS-2 (Guerrero, Vera, Huerta, Plou, & Illanes, 2010). Since the prebiotic property of a disaccharide depends upon its chemical structure (nature of monosaccharide and type of glycosidic linkage), an extensive NMR study was further performed to elucidate the structures of the two transfer disaccharides, including 1D (1H, 13C and DEPT 135) and 2D (HQSC and HMBC) NMR experi-
ments. Using an authentic standard and with the standard-addition technique, oligosaccharide 1 (peak 3) was tentatively identified as lactulose (b-D-galactosyl-(1 ? 4)-fructose). Its 1HNMR and 13C NMR spectrums (Fig. 3), which extremely coincided with the published NMR data for lactulose (Mayer et al., 2004), further confirmed that the transgalactosylation product 1 was lactulose with three isomers: (1A) b-D-galactopyranosyl-(1 ? 4)-b-D-fructopyranose (the major isomer), (1B) b-D-galactopyranosyl-(1 ? 4)-b-D-fructofuranose, and (1C) b-D-galactopyranosyl-(1 ? 4)-a-D-fructofuranose. For disaccharide 2, the 1H and 13C chemical shifts were assigned by the interpretation of their 1D NMR (1H, 13C and DEPT 135) spectra (Fig. 4) and 2D NMR (HSQC, HMBC) spectra (Fig. 5) and by further comparison with those of lactulose and to the literatures (Landersjo et al., 2006; Taubert, Konschin, & Sundholm, 2005). The 13C NMR (Fig. 4b) spectrum of disaccharide 2 in D2O displayed two sets of signals, indicating a mixture of isomers 2A and 2B with the population being 3:1 approximately because of the mutarotational equilibrium between pyranose and furanose forms. The major set of resonances, corresponding to the most populated isomer (2A), contained two anomeric carbons at d 103.33 and d 97.58. Accordingly, the 1H NMR spectrum (Fig. 4a) showed only one anomeric signal at d 4.45 and DEPT 135 spectra (Fig. 4c) displayed one anomeric carbon at d 103.33 and three negative signals at d 71.55, d 63.42 and d 61.00, illustrating that the anomeric carbon at d 97.58 was quaternary and the disaccharide 2 was consisted of a fructose and a galactose moiety. HMBC correlations between both H-10 (galactosyl) at d 4.45 and C-1 (fructose) at d 71.55 and between both C-10 (galactosyl) at d 103.33 and H-1a/b (fructose) at d 3.94/3.83, respectively, unambiguously established the 1 ? 1 linkage between the galactose and fructose moieties of disaccharide 2 (Fig. 5b). This result was further supported by the 5–10 ppm low-field shifts observed for C-1 at d 71.55 (fructose) and C-10 at d 103.33 (galactose) (Fig. 4b). The pyranoid b-D-galactosyl moiety was deduced from the chemical shift of anomeric proton (d 4.45) and the coupling constant (3JH-10 , H-20 = 7.71 Hz) (Fig. 4a) and was confirmed by the observed C-40 at d 68.62, C-30 at d 72.49 and C-50 at d 75.25 (Fig. 4b). The positive HMBC correlation between H-6a/b (d 3.71/ 4.05) and C-2 (d 97.56) of the fructose established a b-D-fructopyranosyl moiety in the major isomer 2A (Fig. 5b). From the comparison of NMR data and those proposed for lactulose isomers (Mayer et al., 2004), we determined that the most populated isomer for
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Fig. 4. 1D NMR spectra of disaccharide 2: (a) 1H NMR (D2O, 400.14 MHz), (b) 13C NMR (D2O, 100.62 MHz) and (c) DEPT 135 spectra (D2O, 100.62 MHz). In the DEPT 135 spectrum, the sign of each peak provides the multiplicity of the carbon: positive (upward) for CH and CH3 and negative (downward) for CH2, quaternary carbons do not appear. Note: G as galactose and F as fructose.
disaccharide 2 was b-D-galactopyranosyl-(1 ? 1)-b-D-fructopyranose (Fig. 6). It was also possible to measure C-2 (d 100.55) C-3 (d 80.66) and C-5 (d 76.23) resonances for minor isomer, which were typical of furanose (Fig. 4b). In the same way as shown above for isomer 2A, we determined the minor isomer 2B was b-D-galactopyranosyl-(1 ? 1)-b-D-fructofuranose (Fig. 6).
In conclusion, the two main products were identified as b-Dgalactosyl-(1 ? 4)-fructose (lactulose) and b-D-galactosyl-(1 ? 1)fructose (allo-lactulose), respectively, demonstrating that fructose could be used as a galactosyl acceptor for the enzymatic synthesis of galactosyl-fructoses by KlbGal. Moreover, the formed galactosyl-(1 ? 1)-fructose with two main isomers, b-D-galactopyranosyl-(1 ? 1)-b-D-fructopyranose and b-D-galactopyranosyl-
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Fig. 5. 2D NMR spectra of disaccharide 2: (a) 2D NMR heteronuclear single quantum coherence (HSQC) spectra and (b) 2D heteronuclear multiple quantum coherence (HMBC) spectra. Note: G as galactose and F as fructose, a/b as one of the two protons linked to the same carbon atom.
(1 ? 1)-b-D-fructofuranose, was three times the amount of the formed lactulose, indicating that the KlbGal showed a preference for establishing b-(1 ? 1) linkages and forming disaccharides in the presence of fructose. The enzymatic synthesis process will be further optimized and scaled up to allow for a thorough study of their potential prebiotic properties in our future work.
4. Discussion b-Galactosidases, a type of glycosidase, in nature act as hydrolases. However, interestingly, they can catalyze the transfer of a
galactose moiety from a donor to a carbohydrate molecule under special conditions and have already been used for the synthesis of several oligosaccharides containing the galactose moiety. Carbohydrate molecules represent multiple hydroxyl groups, thus transgalactosylation catalyzed by b-galactosidase are not regionspecific but stereospecific and a mixture of regionisomers is probably obtained. The regionisomers produced depends upon the source of the enzyme, the acceptor and the reaction conditions (Irazoqui et al., 2009; Tricone, Tramice, Giordano, & Andreotti, 2008; Yoon & Ajisaka, 1996). The regioselectivity of KlbGal has been investigated using various acceptors and primary hydroxyl groups were glycosylated
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structural feature of this oligosaccharide will be useful for the study of its fermentation mechanism, and also for the research of structure-function relationship of oligosaccharides.
Acknowledgements The authors gratefully acknowledge the financial support provided by the ‘Five-twelfth’ National Science and Technology Support Program (2011BAD23B03), the Natural Science Foundation of Jiangsu Province (BK2011149) and the Innovative Research Program for Graduate Students of Jiangsu Province (CXZZ11_0487).
References
Fig. 6. Structures of the major isomers of disaccharide 2: (A) b-D-galactopyranosyl(1 ? 1)-b-D-fructopyranose, and (B) b-D-galactopyranosyl-(1 ? 1)-b-D-fructofuranose. Arrows (?) indicate important HMBC correlations.
preferentially to secondary hydroxyl groups in all of the acceptors studied. In the preparation of GOS, KlbGal produced predominantly b-(1 ? 6) oligosaccharides (60 -galactosyl-lactose and b-D-galactosyl-(1 ? 6)-D-glucose) (Asp, Burvall, Dahlqvist, Hallgren, & Lundblad, 1980). Otherwise, KlbGal afford N-acetyl-allolactosamine (bGal-(1 ? 6)-GlcNAc) as the major transglycosylation product and bGal-(1 ? 4)-GlcNAc as the minor one between lactose and GlcNAc (Bridiau & Maugard, 0000). The enzymatic transgalactosylation during lactulose hydrolysis was also studied using the KlbGal, and two main trisaccharides were produced, which exhibited a galactose unit linked to C-6 of the galactose moiety of lactulose, while the other one presented a galactose unit linked to C-1 of the fructose moiety of lactulose (Martines-Villaluenga et al., 2008). Since hydroxyl groups linked to C-1 and C-2 of fructose are primary, the abundant products produced by transgalactosylation from lactose to fructose using KlbGal are expected to be b-D-galactosyl-(1 ? 1)-D-fructose and b-D-galactosyl-(1 ? 6)-D-fructose according to the regioselectivity of KlbGal. However, there was no peak corresponding to (1 ? 6)-galactosyl-fructose in the HPLC-RI spectra because of the fact that the abundant isomer existed in fructose aqueous solution is b-D-pyranose tautomer with the primary hydroxyl group linked to C-6 being endocyclic due to its condensation reaction with carbonyl group (Flood, Johns, White & Crystal, 1996; Polacek & Kaatze, 2001). Lee et al. observed that when lactose was used as a galactose donor and fructose as an acceptor, not only lactulose but also other galacto-oligosaccharides produced by b-galactosidase in permeabilized cells of K. lactis, but the byproducts were not isolated and identified (Lee et al., 2004). Even though synthesis of allo-lactulose (b-D-galactosyl-(1 ? 1)-D-fructose) using the reversed hydrolysis activity of b-galactosidase from E. coli and transgalactosylation activity of b-galactosidase from Bifidobacterium adolescentis DSM 20083 has been previously mentioned (Ajisaka, Nishida, & Fujimoto, 1987; Förster-Fromme et al., 2001), this is the first time that a complete identification and a structural characterization of this product have been carried out. The detailed elucidation of the
Ajisaka, K., Nishida, H., & Fujimoto, H. (1987). Use of an activated carbon column for the synthesis of disaccharides by use of a reversed hydrolysis activity of bgalactosidase. Biotechnology Letters, 9, 387–392. Asp, N. G., Burvall, A., Dahlqvist, A., Hallgren, P., & Lundblad, A. (1980). Oligosaccharide formation during hydrolysis of lactose with Saccharomyces lactis lactase (Maxilact): Part 2-oligosaccharide structures. Food Chemistry, 5, 147–153. Barbara, S., Thu-ha, N., Marlene, S., Klaus, D. K., Werner, L., & Dietmar, H. (2006). Production of prebiotic galacto-oligosaccharides from lactose using bgalactosidases from Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 54, 4999–5006. Bridiau, N., & Maugard, T. A. (2011). Comparative Study of the Regioselectivity of the b-Galactosidases from Kluyveromyces lactis and Bacillus circulans in the Enzymatic Synthesis of N-Acetyl-lactosamine in Aqueous Media. Biotechnology Progress, 27, 386–394. Cardelle-Cobas, A., Martínez-Villaluenga, C., Villamiel, M., Olano, A., & Corzo, N. (2008). Synthesis of oligosaccharides derived from lactulose and pectinex Ultra SP-L. Journal of Agricultural and Food Chemistry, 56, 3328–3333. Delzenne, N. M. (2003). Oligosaccharides: State of the art. Proceeding of the Nutrition Society, 62, 177–182. Djouzi, Z., & Andrieux, C. (1997). Compared effects of three oligosaccharides on metabolism of intenstinal microflora in rats inoculated with a human faecal flora. British Journal of Nutritional, 78, 313–324. Flood, A. E., Johns, M. R., & White & Crystal, E. T. (1996). Mutarotation of D-fructose in aqueous- ethanolic solutions and its influence on crystallisation. Carbohydrate Research, 288, 45. Förster-Fromme, K., Schuster-Wolff-Bühring, R., Hartwig, A., Holder, A., Schwiertz, A., Bischoff, C. S., et al. (2001). A new enzymatically produced 1-lactulose: A pilot study to test the bifidogenic effects. International Dairy Journal, 21, 940–948. Ganzle, M. G., Haase, G., & Jelen, P. (2008). Lactose: Crystallization, hydrolysis and value-added derivatives. International Dairy Journal, 18(7), 685–694. Guerrero, C., Vera, C., Huerta, L. M., Plou, F., & Illanes, A. (2010). Effect of reaction conditions and enzyme source on the selectivity of lactulose synthesis. Special Abstracts/Journal of Biotechnology, 150S, S402–S403. Harju, M. (1987). Lactose hydrolysis. Bulletin of Internal Dairy Federation, 212, 50–55. Irazoqui, G., Giacomini, C., Batista-Viera, F., Brena, B. M., Cardelle-Cobas, A., Corzo, N., et al. (2009). Characterization of galactosyl derivatives obtained by transgalactosylation of lactose and different polyols using immobilized bgalactosidase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 57, 11302–11307. Kim, Y. S., Park, C. S., & Oh, D. K. (2006). Lactulose production from lactose and fructose by a thermostable b-galactosidase from Sulfolobus solfataricus. Enzyme and Microbial Technology, 39, 903–908. Landersjo, C., Stevensson, B., Eklund, R., Ostervall, J., Sodernan, P., Widmalm, G., et al. (2006). Molecular conformations of a disaccharide investigated using NMR spectroscopy. Journal of Biomolecular NMR, 35, 89–101. Lee, Y. J., Kim, C. S., & Oh, D. K. (2004). Lactulose production by b-galactosidase in permeabilized cells of Kluyveromyces lactis. Applied Microbiology and Biotechnology, 64, 787–793. Martines-Villaluenga, C., Cardelle-Cobas, A., Olano, A., Corzo, N., Villamiel, M., & Jimeno, M. L. (2008). Enzymatic synthesis and identification of two trisaccharides produced from lactulose by transgalactosylation. Journal of Agricultural and Food Chemistry, 56, 557–563. Mayer, J., Conrad, J., Klaiber, I., Lutz-Wahl, S., Beifuss, U., & Fischer, L. (2004). Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose. Journal of Agricultural and Food Chemistry, 52, 6983–6990. Mayer, J., Kranz, B., & Fischer, L. (2010). Continuous production of lactulose by immobilized thermostable b-glycosidase from Pyrococcus furiosus. Journal of Biotechnology, 145, 387–393. Polacek, R., & Kaatze, U. (2001). Chair-chair ring inversion of D-fructose coupled to the mutarotation in aqueous-ethanolic solution. Chemical Physics Letters, 345, 93–99. Sanz, M. L., Gibson, G. R., & Rastall, R. A. (2005). Influence of disaccharide structure on prebiotic selectivity in vitro. Journal of Agricultural and Food Chemistry, 53, 5192–5199.
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Q. Shen et al. / Food Chemistry 135 (2012) 1547–1554
Shen, Q., Yang, R., Hua, X., Ye, F., Zhang, W., & Zhao, W. (2011). Gelatin-templated biomimetic calcification for b-galactosidase immobilization. Process Biochemistry, 46, 1565–1571. Taubert, S., Konschin, H., & Sundholm, D. (2005). Computational studies of 13C NMR chemical shifts of saccharides. Physical Chemistry Chemical Physics, 7, 2561–2569. Torres, D. P. M., Goncalves, M. P. F., Jose, A. T., & Rodrigues, L. R. (2010). Galactooligosaccharides: Production, properties, applications, and significance as prebiotics. Comprehensive reviews in food science and food safety, 9, 438– 454.
Tricone, A., Tramice, A., Giordano, A., & Andreotti, G. (2008). Glycoside hydrolases in Aplysia fasciata: Analysis and applications. Biotechnology & Genetic Engineering Reviews, 25, 129–148. Vaheri, M., & Kaupinnen, V. (1987). The formation of lactulose (4-O-b-galactopyranosylfructose) by b-galactosidase. Acta Pharmaceutica Fennica, 87, 75–83. Wallenfels, K. (1951). Enzymatische synthesis von oligosacchariden aus disacchariden. Naturwissenschaften, 38, 306–307. Yoon, J. H., & Ajisaka, K. (1996). The synthesis of galactopyranosyl derivatives with b-galactosidases of different origins. Carbohydrate Research, 292, 153–163.