Carbohydrate Research 382 (2013) 108–112
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Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine using b-fructofuranosidase from Aspergillus oryzae Toshiyuki Nishio a,⇑, Mai Juami a, Toru Wada a, Yuta Sugimoto a, Hiroki Senou a, Wataru Komori a, Chiseko Sakuma b, Takako Hirano a, Wataru Hakamata a, Mitsuru Tashiro c a b c
Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan Department of Chemistry, College of Science and Technology, Meisei University, 2-1-1 Hodokubo, Hino, Tokyo 191-8506, Japan
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Article history: Received 22 July 2013 Received in revised form 3 October 2013 Accepted 7 October 2013 Available online 12 October 2013 Keywords: N-Acetyl-1-kestosamine N-Acetylnystosamine N-Acetylsucrosamine b-Fructofuranosidase Transfructosylation
a b s t r a c t Mycelia of Aspergillus oryzae NBRC100959 contain 2 types of b-fructofuranosidases, transfructosylationcatalyzing enzyme (bFFaseI), and hydrolysis-catalyzing enzyme (bFFaseII). Using bFFaseI extracted from the mycelia of strain NBRC100959, two novel oligosaccharides consisting of GlcNAc and fructose, b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2?1)b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside (N-acetylnystosamine, NysNAc), were synthesized from b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside (N-acetylsucrosamine, SucNAc). We next planned to synthesize 1-KesNAc and NysNAc using A. oryzae mycelia. However, it was thought that the presence of bFFaseII is disadvantageous for the production of these oligosaccharides by bFFaseI, because bFFaseII hydrolyzed 1-KesNAc and NysNAc. We succeeded to produce A. oryzae mycelia containing bFFaseI as the major b-fructofuranosidase, by increasing sucrose concentration in the culture medium. Then, using a dried sample of these A. oryzae mycelia, reaction for the oligosaccharide production was performed. As the results, 190 mg of 1-KesNAc and 60 mg of NysNAc were obtained from 0.6 g of SucNAc. This whole-cell catalysis method facilitates the synthesis of 1-KesNAc and NysNAc because extraction and purification of bFFaseI from mycelia are unnecessary. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Various oligosaccharides that exhibit physiologically useful activities have been developed as functional oligosaccharides, some of which are used as dietary supplements. These oligosaccharides are produced by enzymatic reaction from abundant carbohydrates such as starch, sucrose, and lactose.1–4 Most functional oligosaccharides therefore consist of combinations of only three kinds of monosaccharides: D-glucose, D-fructose, and D-galactose. In order to expand the uses of functional oligosaccharides, we think it is important that other monosaccharides be incorporated. Toward this end, we attempted the enzymatic production of oligosaccharides that incorporate the amino sugar derivative 2-acetamide-2-deoxy-D-glucose (GlcNAc). This monosaccharide can be obtained from the biomass polysaccharide chitin, a b-(1,4) polymer of GlcNAc. Using dried Aspergillus oryzae mycelia containing the transfructosylation-catalyzing enzyme b-fructofuranosidase as a whole-cell catalyst, we recently synthesized b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside ⇑ Corresponding author. Tel./fax: +81 466 84 3951. E-mail address: [email protected] (T. Nishio). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.10.006
(N-acetylsucrosamine, SucNAc) from sucrose and GlcNAc.5 Moreover, we established the methodology for large-scale production of this disaccharide using a packed-bed column reactor with A. oryzae mycelia.6 SucNAc is a disaccharide in which the glucose residue in sucrose is replaced with GlcNAc. We are now investigating the physiological function of this disaccharide. Fructooligosaccharides such as 1-kestose [b-D-fructofuranosyl(2?1)-b- D -fructofuranosyl-(2M1)- a - D -glucopyranoside] and nystose [b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2?1)-bD-fructofuranosyl-(2M1)-a-D-glucopyranoside] are representative functional oligosaccharides, and it has been shown that they enhance bowel health by promoting an increase in the number of enterobacteria, such as the bifidobacteria.7–9 Based upon these studies, we attempted the synthesis of oligosaccharides with structures in which the glucose residues of fructooligosaccharides were replaced with GlcNAc. Several reports have described the production of fructooligosaccharides from sucrose by exploiting the transfructosylation action of b-fructofuranosidases from various strains of Aspergillus.10–15 We therefore attempted enzymatic synthesis of target oligosaccharides from SucNAc using both partially purified A. oryzae transfructosylation-catalyzing b-fructofuranosidase and A. oryzae mycelia containing this
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enzyme. In the present report, the results of these studies are described. 2. Experimental 2.1. Materials GlcNAc and D-fructose were purchased from Funakoshi Corp. (Tokyo, Japan) and Wako Pure Chemical Industries, Ltd (Osaka, Japan), respectively. SucNAc was prepared according to procedures we previously reported.6 All other chemicals were of analytical grade. Charcoal (particle size, 63–300 lm) for column chromatography and diatomaceous earth (Celite 535) were purchased from Wako Pure Chemical Industries, Ltd. The mold cell wall dissolution enzyme Yatalase was obtained from Takara Bio Inc. (Shiga, Japan). Aspergillus oryzae NBRC100959 was obtained from the Biotechnology Section of the National Institute of Technology and Evaluation (Chiba, Japan). 2.2. Analysis of carbohydrates Qualitative and quantitative analyses of carbohydrates were carried out using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. TLC plates (Silica gel 60, 0.25 mm, Merck KGaA, Darmstadt, Germany) were developed twice using a solvent of 1-butanol/pyridine/H2O 8:3:1 (v/v/v) as the mobile phase, and resolved compounds were visualized by spraying the plates with a solution containing phosphomolybdic acid followed by heating.5 HPLC analyses were performed using an LC-10AS pump (Shimadzu Corp., Kyoto, Japan) equipped with a Shodex RI-71 differential refractometer (Showa Denko K.K., Tokyo, Japan) and a COSMOSIL Sugar-D column (u 4.6 250 mm, Nacalai Tesque, Kyoto, Japan). The system was operated under isocratic conditions using a solvent of acetonitrile/H2O 77:23 (v/v) as the mobile phase (flow rate, 0.8 mL min1). The amounts of various oligosaccharides in the reaction mixture were estimated by comparing the area of each oligosaccharide peak with the peak areas of standard samples. SucNAc reaction products were characterized using 1H and 13C NMR spectrometry as well as mass spectrometry (MS). The 1H and 13C NMR spectra were recorded in D2O at 25 °C for N-acetyl-1-kestosamine and 10 °C for N-acetylnystosamine using a Varian NMR system 600 spectrometer (Varian, Palo Alto, CA, USA). The 2D 1H–1H DQF-COSY, 1H–1H TOCSY, 1H–13C HSQC, 1H–13C HSQC-TOCSY, and 1 H–13C HMBC spectra were acquired to make 1H and 13C assignments. The 1H and 13C chemical shifts were relative to sodium 2,2-dimethyl-2-silapentane-5-sulfonate-d6 as an external standard. Mass spectra were recorded using a JMX SX-102A instrument (JEOL Ltd, Tokyo, Japan) under positive-ion fast atom bombardment (FAB) ionization conditions. Optical rotation was measured in H2O at 20 °C using a P-1030 polarimeter (JASCO Corp., Tokyo, Japan). 2.3. Partial purification of b-fructofuranosidase from A. oryzae mycelia Soft granules consisting of Celite 535 and dried A. oryzae NBRC100959 mycelia [mycelia content of granules, 24.5%, (w/w)] containing two types of b-fructofuranosidases were prepared according to procedures we previously reported.5 Although strain NBRC100959 dies out during the process of preparing the dried mycelia, the intracellular b-fructofuranosidases remain in their active form. The mycelial content of the dried granules was calculated by subtracting the weight of Celite 535 used from the weight of the granules. Soft granules containing approximately
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5 g of dried A. oryzae mycelia were suspended in 400 mL of 10 mM sodium phosphate buffer (pH 6.0) containing 0.6 M (NH4)2SO4 and 400 mg of Yatalase, and the suspension was incubated with gentle shaking at 30 °C for 5 h, after which insoluble material was removed from the suspension by suction filtration using no. 5A filter paper (Kiriyama Glass Co., Tokyo, Japan). Proteins in the resulting filtrate were precipitated with (NH4)2SO4 between 30% and 90% saturation and were collected by centrifugation (9100g, 4 °C, 15 min). The b-fructofuranosidase-containing precipitate was dissolved in 10 mL of 10 mM sodium citrate buffer (pH 6.0) and then dialyzed sufficiently against the same buffer to obtain a crude enzyme solution, which was loaded onto a TOYOPEARL SuperQ-650M anion exchange resin (TOSOH Corp., Tokyo, Japan) column (u 2 20 cm) pre-equilibrated with 10 mM sodium citrate buffer (pH 6.0). The b-fructofuranosidases were separated and eluted from the column with a linear gradient from 0 to 0.25 M NaCl in the same buffer. Sucrose hydrolysis activity in the eluate fractions was measured, and fractions containing the different b-fructofuranosidases were collected separately and concentrated using an Amicon Ultra-15 10 K centrifugal filter device (EMD Millipore Corp., Billerica, MA, USA) to obtain solutions of partially purified enzyme. b-Fructofuranosidase hydrolytic activity was assayed using sucrose [0.5% (w/v) in buffer] according to previously reported procedures.5 One unit (U) of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 lmol of sucrose per min under the assay conditions used. 2.4. Production and purification of oligosaccharides In experiment 1 (oligosaccharide production using partially purified enzyme), 4 mL of 20 mM sodium citrate buffer (pH 5.5) containing 4.0 U of partially purified b-fructofuranosidase and 1.2 g of SucNAc was incubated at 30 °C with shaking. In experiment 2 (oligosaccharide production using dried A. oryzae NBRC100959 mycelia), soft granules containing 14 mg of dried mycelia were added to 2 mL of 20 mM sodium citrate buffer (pH 5.5) containing 0.6 g of SucNAc, and the reaction mixture was incubated at 30 °C with shaking. Mycelia were removed from the mixture by suction filtration using no. 5A filter paper to obtain the reaction solution. At specific intervals during the enzymatic reaction in each experiment, the progress of oligosaccharide production was monitored using TLC and HPLC. After 8 h of incubation, the reaction solution was loaded onto a charcoal column (u 3.0 30 cm; solvent, H2O), and the monosaccharides included in the reaction mixture were eluted with water. Oligosaccharides that adhered to the charcoal were eluted with a solvent of 2-PrOH/H2O 3:7 (v/v). Fractions containing the products were collected and concentrated by evaporation, and the target oligosaccharides were further purified by chromatography using an UltraPack NH-40C column [Yamazen Science Inc., Osaka, Japan; u 2.6 30 cm; solvent, 2-PrOH/H2O 7:3 (v/v); flow rate, 8 mL min1]. Fractions containing the oligosaccharide were collected and concentrated by evaporation, and the resulting sample was lyophilized, resulting in a white powder as the final product. 3. Results and discussion 3.1. Oligosaccharide production from SucNAc using bfructofuranosidases extracted from A. oryzae mycelia Previously, we found that the mycelia of A. oryzae NBRC100959 cultivated in liquid medium containing 2% (w/v) sucrose expressed two types of b-fructofuranosidase. One of these enzymes (bFFaseI)
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showed transfructosylation activity in highly concentrated sucrose solution, whereas the other enzyme (bFFaseII) catalyzed only sucrose hydrolysis.5 We extracted these b-fructofuranosidases from strain NBRC100959 mycelia that were cultivated in liquid medium containing 2% sucrose and separated the enzymes using ion exchange column chromatography. Each purified enzyme was added to 4 mL of buffer containing 1.2 g of SucNAc [SucNAc concentration, 30% (w/v)]. SucNAc is almost saturated in this reaction solution. Although bFFaseII did not act on SucNAc (Fig. 1A), bFFaseI generated oligosaccharides that were predicted to be transfructosylation products (products 1 and 2 in Fig. 1B). Generation of fructose was not observed in the reaction solution containing bFFaseI, indicating that SucNAc was scarcely hydrolyzed by the enzyme in this reaction condition (Fig. 1B). These results indicated that the bFFaseI-catalyzed transfructosylation reaction proceeded efficiently in the solution containing 30% SucNAc. It was confirmed by HPLC analysis that the oligosaccharides except product 1 and 2 were not generated during the reaction for 8 h (data not shown). Products 1 and 2 were purified by charcoal column chromatography and medium-pressure liquid chromatography using an UltraPack NH-40C column. No other carbohydrate peaks were detected in HPLC analyses of the purified oligosaccharide samples. The structures of the purified products were then determined using MS and NMR spectrometry. The FAB-MS spectra of products 1 and 2 corresponded to [M+H]+ species at m/z 546 and m/z 709 (Supplementary Fig. S1), respectively, suggesting that product 1 is a trisaccharide consisting of two fructose residues and one GlcNAc residue and that product 2 is a tetrasaccharide consisting of three fructose residues and one GlcNAc residue. The assignments of these compounds from 1H and 13C NMR signals (Supplementary Figs. S2–S4) are shown in Figure 2A and B, respectively. The anomeric hydrogen of GlcNAc (G-H-1) in each product was found to have a coupling constant (J1,2) of 3.6 or 3.7 Hz, indicating that GlcNAc exists in product 1 and 2 with a-anomeric form. In all fructose units of each product, strong NOE cross peaks were observed between H-1 and H3, indicating that their configurations are of b-anomeric form. The positions of the glycosidic linkages were determined using heteronuclear multiple bond correlation HMBC-NMR experiments. The anomeric hydrogen of GlcNAc (G-H-1) in product 1 exhibited a cross peak with C-2 of fructose 1 (F1-C-2), and H-1 of fructose 1 (F1-H-1) exhibited cross peaks with C-2 of fructose 2 (F2-C-2). In product 2, the anomeric hydrogen of GlcNAc (G-H-1) exhibited a cross peak with C-2 of fructose 1 (F1-C-2), whereas H-1 of fructose 1 (F1-H-1) exhibited cross peaks with C-2 of fructose 2 (F2-C-2), and H-1 of fructose 2 (F2-H-1) exhibited cross peaks with C-2 of fructose 3 (F3-C-2). These results enabled us to identify products
Figure 1. TLC analysis of the compounds produced by the action of bFFaseII (A) and bFFaseI (B). Products in the reaction mixture were identified based on the following standards: SucNAc (a), GlcNAc (b), and fructose (c). Products 1 (Rf value; 0.51) and 2 (Rf value; 0.20) are indicated by arrows.
1 and 2 as b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2M1)-2acetamido-2-deoxy-a-D-glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl(2?1)-b- D -fructofuranosyl-(2M1)-2-acetamido-2-deoxy- a D -glucopyranoside (N-acetylnystosamine, NysNAc), respectively. These compounds are novel oligosaccharides. Amounts of 1-KesNAc and NysNAc obtained from the reaction mixtures after 8 h of shaking were 374 mg and 133 mg, respectively. From the elemental analysis data shown below, it was confirmed that samples of 1-KesNAc and NysNAc contained bound water of two molecules and five molecules per one molecule, respectively. 1-KesNAc2H2O: Calcd for C20H39NO18: C, 41.31; H, 6.76; N, 2.41. Found: C, 41.56; H, 6.90; N, 2.42. NysNAc5H2O: Calcd for C26H55NO26: C, 39.15; H, 6.85; N, 1.76. Found: C, 39.49; H, 6.95; N, 1.89. The specific rotations ð½a20 D Þ of these oligosaccharides were measured in consideration of bound water, and following values were obtained; 1-KesNAc, 36.2° (c = 0.413, H2O) and NysNAc, 9.85° (c = 0.408, H2O). 3.2. Preparation of A. oryzae mycelia for the production of 1KesNAc and NysNAc In the previous studies, we successfully produced SucNAc from sucrose and GlcNAc using dried A. oryzae mycelia containing bFFaseI and bFFaseII.5 The whole-cell catalysis method presents several advantages for the enzymatic production of oligosaccharides: (1) extraction of the enzyme from the culture is unnecessary, and (2) the enzyme can be easily removed from the reaction mixture. We therefore attempted to produce 1-KesNAc and NysNAc from SucNAc using A. oryzae mycelia containing b-fructofuranosidase. Before conducting this experiment, we investigated the hydrolytic properties of bFFaseII against 1-KesNAc and NysNAc in both low- (20 mM) and high-concentration (200 mM) solutions of each oligosaccharide. The results confirmed that bFFaseII hydrolyzes 1KesNAc and NysNAc under both conditions, and SucNAc and fructose were obtained as end products (Supplementary Fig. S5). bFFaseII did not participate in the synthesis of 1-KesNAc and NysNAc from SucNAc (Fig. 1A). These data indicate that the presence of bFFaseII is disadvantageous for the production of these oligosaccharides using bFFaseI. Therefore, in order to synthesize 1-KesNAc and NysNAc using the whole-cell catalysis method, we prepared dried A. oryzae mycelia containing bFFaseI as the major b-fructofuranosidase. Two types of b-fructofuranosidases were previously found in A. oryzae KB, and it was confirmed that one of the enzymes hydrolyzed sucrose to glucose and fructose and the other catalyzed a transfructosylation reaction to produce fructooligosaccharides from sucrose.15,16 These reports indicated that the amount of each enzyme produced by strain KB was influenced by the concentration of sucrose in the liquid culture medium. We therefore cultivated A. oryzae NBRC100959 in liquid culture media containing various concentrations of sucrose [1%, 2%, 3%, 6%, and 12% (w/v)] and prepared dried granules consisting of Celite 535 and mycelia. bFFaseI and bFFaseII were extracted from the dried mycelia and separated by ion exchange column chromatography (Supplementary Fig. S6), after which the total activity of each enzyme was determined (Table 1). Only very small levels of bFFaseII were produced in mycelia proliferated in media containing 3%, 6%, or 12% sucrose. However, bFFaseI was produced in all of the dried mycelia samples, and its level was highest in mycelia cultivated in media containing 6% or 12% sucrose. The presence of b-fructofuranosidase in the supernatant of A. oryzae culture broth was examined using ion exchange column chromatography. The quantity of bFFaseII in the supernatant increased with increasing sucrose concentration, whereas bFFaseI was not detected in any of the culture supernatants (Supplementary Table S1). These data indicate that
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Figure 2. Identification of compounds produced by the action of bFFaseI. 1H and 13C NMR shifts and the structures of product 1 (A) and product 2 (B). 1H NMR data (chemical shift in ppm; multiplicity; coupling constant in Hz) and 13C NMR chemical shifts (ppm) are designated with arrows and dashed line arrows, respectively. Bent double-headed arrows indicate HMBC cross peaks.
Table 1 Total enzyme activities of bFFaseI and bFFaseII extracted from A. oryzae mycelia cultivated in media containing various concentrations of sucrose Sucrose concentration in culture medium (%)
1 2 3 6 12
findings, we decided to use dried samples of A. oryzae mycelia, which proliferated in medium containing 6% sucrose, for wholecell catalysis production of 1-KesNAc and NysNAc from SucNAc.
Total enzyme activity (U) bFFaseI
bFFaseII
8.35 8.99 6.80 16.4 16.5
23.9 14.4 0.358 0.756 0.789
bFFaseI and bFFaseII were extracted from the soft granules containing approximately 0.43 g of dried mycelia of A. oryzae. After (NH4)2SO4 precipitation, these two enzymes were separated by ion exchange column chromatography (column size; / 1.5 8.5 cm) using TOYOPEARL SuperQ-650M resin. The fractions containing each enzyme were combined, and then total sucrose hydrolysis activity (U) of each enzyme was determined.
though bFFaseII is produced in A. oryzae mycelia cultivated under the presence of high concentrations of sucrose, this enzyme is secreted into the culture fluid under these conditions. Based on these
3.3. Production of 1-KesNAc and NysNAc from SucNAc using A. oryzae mycelia 1-KesNAc and NysNAc were produced by adding soft granules composed of dried A. oryzae mycelia and Celite 535 to 2 mL of buffer solution containing 0.6 g of SucNAc [SucNAc concentration, 30% (w/v)]. The time course of oligosaccharide generation is shown in Figure 3A. As the concentration of SucNAc decreased, 1-KesNAc and NysNAc were generated as major products (Fig. 3B). After 8 h of shaking, 190 mg of 1-KesNAc and 60 mg of NysNAc were isolated using two types of column chromatography. During the early stage of the reaction (0–8 h), the level of 1-KesNAc rapidly increased in proportion to the rapid decrease in the level of SucNAc. Most of the 1-KesNAc was produced after 4 h (33.5% of the total carbohydrate composition). The following phenomena were observed during the latter stage of the reaction (8–24 h): (1) the level
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Figure 3. Oligosaccharide production by A. oryzae mycelia containing bFFaseI as the major b-fructofuranosidase. (A) TLC analysis of the oligosaccharides formed in the enzymatic reaction. Products in the reaction mixture were identified based on the following standards: SucNAc (a), GlcNAc (b), fructose (c), 1-KesNAc (d), and NysNAc (e). (B) Carbohydrate composition in the reaction solution as determined by HPLC analysis. Retention time (min) of the carbohydrates in HPLC: GlcNAc, 8.56; fructose, 9.89; SucNAc, 13.8; 1-KesNAc, 21.0; and NysNAc, 31.0. Symbols: s, SucNAc; h, GlcNAc; 4 , fructose; d , 1-KesNAc; j , NysNAc; N , other oligosaccharides.
tion-catalyzing b-fructofuranosidase (bFFaseI) from A. oryzae NBRC100959. Moreover, we successfully synthesized both 1-KesNAc and NysNAc using a whole-cell catalysis method involving dried A. oryzae NBRC100959 mycelia containing bFFaseI as the major b-fructofuranosidase. This whole-cell catalysis method facilitates efficient synthesis of 1-KesNAc and NysNAc because extraction and purification of bFFaseI from the mycelia are unnecessary. This method enables large-scale production of 1-KesNAc and NysNAc because the mycelia can be used as immobilized enzymes for use in a bioreactor. We plan to develop 1-KesNAc and NysNAc as functional oligosaccharides. Further attempts will be made in the large-scale production of 1-KesNAc and NysNAc using an A. oryzae mycelia packed-bed column reactor and in the investigations of the possible physiological uses of these oligosaccharides with samples produced using the bioreactor. Acknowledgment We thank Dr. Hitoshi Sato of the Japan Sugar Refiners’ Association for his support. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2013.10. 006. References 1. 2. 3. 4. 5.
Figure 4. Schematic representation of oligosaccharide synthesis by the transfructosylation action of bFFaseI.
of 1-KesNAc decreased, (2) there was almost no decrease in the level of SucNAc, and (3) the level of NysNAc increased. From these data, we concluded that three separate transfructosylation reactions (A, B, and C in Fig. 4) occur during whole-cell catalysis, of which A and B are the major reactions occurring during the early stage, and C is the major reaction occurring during the latter stage.
6. 7. 8. 9. 10. 11. 12. 13. 14.
4. Conclusion 15.
Two novel oligosaccharides, 1-KesNAc and NysNAc, were synthesized from SucNAc using a partially purified transfructosyla-
16.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine using b-fructofuranosidase from Aspergillus oryzae Toshiyuki Nishio a,⇑, Mai Juami a, Toru Wada a, Yuta Sugimoto a, Hiroki Senou a, Wataru Komori a, Chiseko Sakuma b, Takako Hirano a, Wataru Hakamata a, Mitsuru Tashiro c a b c
Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan Department of Chemistry, College of Science and Technology, Meisei University, 2-1-1 Hodokubo, Hino, Tokyo 191-8506, Japan
a r t i c l e
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Article history: Received 22 July 2013 Received in revised form 3 October 2013 Accepted 7 October 2013 Available online 12 October 2013 Keywords: N-Acetyl-1-kestosamine N-Acetylnystosamine N-Acetylsucrosamine b-Fructofuranosidase Transfructosylation
a b s t r a c t Mycelia of Aspergillus oryzae NBRC100959 contain 2 types of b-fructofuranosidases, transfructosylationcatalyzing enzyme (bFFaseI), and hydrolysis-catalyzing enzyme (bFFaseII). Using bFFaseI extracted from the mycelia of strain NBRC100959, two novel oligosaccharides consisting of GlcNAc and fructose, b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2?1)b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside (N-acetylnystosamine, NysNAc), were synthesized from b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside (N-acetylsucrosamine, SucNAc). We next planned to synthesize 1-KesNAc and NysNAc using A. oryzae mycelia. However, it was thought that the presence of bFFaseII is disadvantageous for the production of these oligosaccharides by bFFaseI, because bFFaseII hydrolyzed 1-KesNAc and NysNAc. We succeeded to produce A. oryzae mycelia containing bFFaseI as the major b-fructofuranosidase, by increasing sucrose concentration in the culture medium. Then, using a dried sample of these A. oryzae mycelia, reaction for the oligosaccharide production was performed. As the results, 190 mg of 1-KesNAc and 60 mg of NysNAc were obtained from 0.6 g of SucNAc. This whole-cell catalysis method facilitates the synthesis of 1-KesNAc and NysNAc because extraction and purification of bFFaseI from mycelia are unnecessary. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Various oligosaccharides that exhibit physiologically useful activities have been developed as functional oligosaccharides, some of which are used as dietary supplements. These oligosaccharides are produced by enzymatic reaction from abundant carbohydrates such as starch, sucrose, and lactose.1–4 Most functional oligosaccharides therefore consist of combinations of only three kinds of monosaccharides: D-glucose, D-fructose, and D-galactose. In order to expand the uses of functional oligosaccharides, we think it is important that other monosaccharides be incorporated. Toward this end, we attempted the enzymatic production of oligosaccharides that incorporate the amino sugar derivative 2-acetamide-2-deoxy-D-glucose (GlcNAc). This monosaccharide can be obtained from the biomass polysaccharide chitin, a b-(1,4) polymer of GlcNAc. Using dried Aspergillus oryzae mycelia containing the transfructosylation-catalyzing enzyme b-fructofuranosidase as a whole-cell catalyst, we recently synthesized b-D-fructofuranosyl-(2M1)-2-acetamido-2-deoxy-a-D-glucopyranoside ⇑ Corresponding author. Tel./fax: +81 466 84 3951. E-mail address: [email protected] (T. Nishio). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.10.006
(N-acetylsucrosamine, SucNAc) from sucrose and GlcNAc.5 Moreover, we established the methodology for large-scale production of this disaccharide using a packed-bed column reactor with A. oryzae mycelia.6 SucNAc is a disaccharide in which the glucose residue in sucrose is replaced with GlcNAc. We are now investigating the physiological function of this disaccharide. Fructooligosaccharides such as 1-kestose [b-D-fructofuranosyl(2?1)-b- D -fructofuranosyl-(2M1)- a - D -glucopyranoside] and nystose [b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2?1)-bD-fructofuranosyl-(2M1)-a-D-glucopyranoside] are representative functional oligosaccharides, and it has been shown that they enhance bowel health by promoting an increase in the number of enterobacteria, such as the bifidobacteria.7–9 Based upon these studies, we attempted the synthesis of oligosaccharides with structures in which the glucose residues of fructooligosaccharides were replaced with GlcNAc. Several reports have described the production of fructooligosaccharides from sucrose by exploiting the transfructosylation action of b-fructofuranosidases from various strains of Aspergillus.10–15 We therefore attempted enzymatic synthesis of target oligosaccharides from SucNAc using both partially purified A. oryzae transfructosylation-catalyzing b-fructofuranosidase and A. oryzae mycelia containing this
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enzyme. In the present report, the results of these studies are described. 2. Experimental 2.1. Materials GlcNAc and D-fructose were purchased from Funakoshi Corp. (Tokyo, Japan) and Wako Pure Chemical Industries, Ltd (Osaka, Japan), respectively. SucNAc was prepared according to procedures we previously reported.6 All other chemicals were of analytical grade. Charcoal (particle size, 63–300 lm) for column chromatography and diatomaceous earth (Celite 535) were purchased from Wako Pure Chemical Industries, Ltd. The mold cell wall dissolution enzyme Yatalase was obtained from Takara Bio Inc. (Shiga, Japan). Aspergillus oryzae NBRC100959 was obtained from the Biotechnology Section of the National Institute of Technology and Evaluation (Chiba, Japan). 2.2. Analysis of carbohydrates Qualitative and quantitative analyses of carbohydrates were carried out using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. TLC plates (Silica gel 60, 0.25 mm, Merck KGaA, Darmstadt, Germany) were developed twice using a solvent of 1-butanol/pyridine/H2O 8:3:1 (v/v/v) as the mobile phase, and resolved compounds were visualized by spraying the plates with a solution containing phosphomolybdic acid followed by heating.5 HPLC analyses were performed using an LC-10AS pump (Shimadzu Corp., Kyoto, Japan) equipped with a Shodex RI-71 differential refractometer (Showa Denko K.K., Tokyo, Japan) and a COSMOSIL Sugar-D column (u 4.6 250 mm, Nacalai Tesque, Kyoto, Japan). The system was operated under isocratic conditions using a solvent of acetonitrile/H2O 77:23 (v/v) as the mobile phase (flow rate, 0.8 mL min1). The amounts of various oligosaccharides in the reaction mixture were estimated by comparing the area of each oligosaccharide peak with the peak areas of standard samples. SucNAc reaction products were characterized using 1H and 13C NMR spectrometry as well as mass spectrometry (MS). The 1H and 13C NMR spectra were recorded in D2O at 25 °C for N-acetyl-1-kestosamine and 10 °C for N-acetylnystosamine using a Varian NMR system 600 spectrometer (Varian, Palo Alto, CA, USA). The 2D 1H–1H DQF-COSY, 1H–1H TOCSY, 1H–13C HSQC, 1H–13C HSQC-TOCSY, and 1 H–13C HMBC spectra were acquired to make 1H and 13C assignments. The 1H and 13C chemical shifts were relative to sodium 2,2-dimethyl-2-silapentane-5-sulfonate-d6 as an external standard. Mass spectra were recorded using a JMX SX-102A instrument (JEOL Ltd, Tokyo, Japan) under positive-ion fast atom bombardment (FAB) ionization conditions. Optical rotation was measured in H2O at 20 °C using a P-1030 polarimeter (JASCO Corp., Tokyo, Japan). 2.3. Partial purification of b-fructofuranosidase from A. oryzae mycelia Soft granules consisting of Celite 535 and dried A. oryzae NBRC100959 mycelia [mycelia content of granules, 24.5%, (w/w)] containing two types of b-fructofuranosidases were prepared according to procedures we previously reported.5 Although strain NBRC100959 dies out during the process of preparing the dried mycelia, the intracellular b-fructofuranosidases remain in their active form. The mycelial content of the dried granules was calculated by subtracting the weight of Celite 535 used from the weight of the granules. Soft granules containing approximately
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5 g of dried A. oryzae mycelia were suspended in 400 mL of 10 mM sodium phosphate buffer (pH 6.0) containing 0.6 M (NH4)2SO4 and 400 mg of Yatalase, and the suspension was incubated with gentle shaking at 30 °C for 5 h, after which insoluble material was removed from the suspension by suction filtration using no. 5A filter paper (Kiriyama Glass Co., Tokyo, Japan). Proteins in the resulting filtrate were precipitated with (NH4)2SO4 between 30% and 90% saturation and were collected by centrifugation (9100g, 4 °C, 15 min). The b-fructofuranosidase-containing precipitate was dissolved in 10 mL of 10 mM sodium citrate buffer (pH 6.0) and then dialyzed sufficiently against the same buffer to obtain a crude enzyme solution, which was loaded onto a TOYOPEARL SuperQ-650M anion exchange resin (TOSOH Corp., Tokyo, Japan) column (u 2 20 cm) pre-equilibrated with 10 mM sodium citrate buffer (pH 6.0). The b-fructofuranosidases were separated and eluted from the column with a linear gradient from 0 to 0.25 M NaCl in the same buffer. Sucrose hydrolysis activity in the eluate fractions was measured, and fractions containing the different b-fructofuranosidases were collected separately and concentrated using an Amicon Ultra-15 10 K centrifugal filter device (EMD Millipore Corp., Billerica, MA, USA) to obtain solutions of partially purified enzyme. b-Fructofuranosidase hydrolytic activity was assayed using sucrose [0.5% (w/v) in buffer] according to previously reported procedures.5 One unit (U) of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 lmol of sucrose per min under the assay conditions used. 2.4. Production and purification of oligosaccharides In experiment 1 (oligosaccharide production using partially purified enzyme), 4 mL of 20 mM sodium citrate buffer (pH 5.5) containing 4.0 U of partially purified b-fructofuranosidase and 1.2 g of SucNAc was incubated at 30 °C with shaking. In experiment 2 (oligosaccharide production using dried A. oryzae NBRC100959 mycelia), soft granules containing 14 mg of dried mycelia were added to 2 mL of 20 mM sodium citrate buffer (pH 5.5) containing 0.6 g of SucNAc, and the reaction mixture was incubated at 30 °C with shaking. Mycelia were removed from the mixture by suction filtration using no. 5A filter paper to obtain the reaction solution. At specific intervals during the enzymatic reaction in each experiment, the progress of oligosaccharide production was monitored using TLC and HPLC. After 8 h of incubation, the reaction solution was loaded onto a charcoal column (u 3.0 30 cm; solvent, H2O), and the monosaccharides included in the reaction mixture were eluted with water. Oligosaccharides that adhered to the charcoal were eluted with a solvent of 2-PrOH/H2O 3:7 (v/v). Fractions containing the products were collected and concentrated by evaporation, and the target oligosaccharides were further purified by chromatography using an UltraPack NH-40C column [Yamazen Science Inc., Osaka, Japan; u 2.6 30 cm; solvent, 2-PrOH/H2O 7:3 (v/v); flow rate, 8 mL min1]. Fractions containing the oligosaccharide were collected and concentrated by evaporation, and the resulting sample was lyophilized, resulting in a white powder as the final product. 3. Results and discussion 3.1. Oligosaccharide production from SucNAc using bfructofuranosidases extracted from A. oryzae mycelia Previously, we found that the mycelia of A. oryzae NBRC100959 cultivated in liquid medium containing 2% (w/v) sucrose expressed two types of b-fructofuranosidase. One of these enzymes (bFFaseI)
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showed transfructosylation activity in highly concentrated sucrose solution, whereas the other enzyme (bFFaseII) catalyzed only sucrose hydrolysis.5 We extracted these b-fructofuranosidases from strain NBRC100959 mycelia that were cultivated in liquid medium containing 2% sucrose and separated the enzymes using ion exchange column chromatography. Each purified enzyme was added to 4 mL of buffer containing 1.2 g of SucNAc [SucNAc concentration, 30% (w/v)]. SucNAc is almost saturated in this reaction solution. Although bFFaseII did not act on SucNAc (Fig. 1A), bFFaseI generated oligosaccharides that were predicted to be transfructosylation products (products 1 and 2 in Fig. 1B). Generation of fructose was not observed in the reaction solution containing bFFaseI, indicating that SucNAc was scarcely hydrolyzed by the enzyme in this reaction condition (Fig. 1B). These results indicated that the bFFaseI-catalyzed transfructosylation reaction proceeded efficiently in the solution containing 30% SucNAc. It was confirmed by HPLC analysis that the oligosaccharides except product 1 and 2 were not generated during the reaction for 8 h (data not shown). Products 1 and 2 were purified by charcoal column chromatography and medium-pressure liquid chromatography using an UltraPack NH-40C column. No other carbohydrate peaks were detected in HPLC analyses of the purified oligosaccharide samples. The structures of the purified products were then determined using MS and NMR spectrometry. The FAB-MS spectra of products 1 and 2 corresponded to [M+H]+ species at m/z 546 and m/z 709 (Supplementary Fig. S1), respectively, suggesting that product 1 is a trisaccharide consisting of two fructose residues and one GlcNAc residue and that product 2 is a tetrasaccharide consisting of three fructose residues and one GlcNAc residue. The assignments of these compounds from 1H and 13C NMR signals (Supplementary Figs. S2–S4) are shown in Figure 2A and B, respectively. The anomeric hydrogen of GlcNAc (G-H-1) in each product was found to have a coupling constant (J1,2) of 3.6 or 3.7 Hz, indicating that GlcNAc exists in product 1 and 2 with a-anomeric form. In all fructose units of each product, strong NOE cross peaks were observed between H-1 and H3, indicating that their configurations are of b-anomeric form. The positions of the glycosidic linkages were determined using heteronuclear multiple bond correlation HMBC-NMR experiments. The anomeric hydrogen of GlcNAc (G-H-1) in product 1 exhibited a cross peak with C-2 of fructose 1 (F1-C-2), and H-1 of fructose 1 (F1-H-1) exhibited cross peaks with C-2 of fructose 2 (F2-C-2). In product 2, the anomeric hydrogen of GlcNAc (G-H-1) exhibited a cross peak with C-2 of fructose 1 (F1-C-2), whereas H-1 of fructose 1 (F1-H-1) exhibited cross peaks with C-2 of fructose 2 (F2-C-2), and H-1 of fructose 2 (F2-H-1) exhibited cross peaks with C-2 of fructose 3 (F3-C-2). These results enabled us to identify products
Figure 1. TLC analysis of the compounds produced by the action of bFFaseII (A) and bFFaseI (B). Products in the reaction mixture were identified based on the following standards: SucNAc (a), GlcNAc (b), and fructose (c). Products 1 (Rf value; 0.51) and 2 (Rf value; 0.20) are indicated by arrows.
1 and 2 as b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl-(2M1)-2acetamido-2-deoxy-a-D-glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl(2?1)-b- D -fructofuranosyl-(2M1)-2-acetamido-2-deoxy- a D -glucopyranoside (N-acetylnystosamine, NysNAc), respectively. These compounds are novel oligosaccharides. Amounts of 1-KesNAc and NysNAc obtained from the reaction mixtures after 8 h of shaking were 374 mg and 133 mg, respectively. From the elemental analysis data shown below, it was confirmed that samples of 1-KesNAc and NysNAc contained bound water of two molecules and five molecules per one molecule, respectively. 1-KesNAc2H2O: Calcd for C20H39NO18: C, 41.31; H, 6.76; N, 2.41. Found: C, 41.56; H, 6.90; N, 2.42. NysNAc5H2O: Calcd for C26H55NO26: C, 39.15; H, 6.85; N, 1.76. Found: C, 39.49; H, 6.95; N, 1.89. The specific rotations ð½a20 D Þ of these oligosaccharides were measured in consideration of bound water, and following values were obtained; 1-KesNAc, 36.2° (c = 0.413, H2O) and NysNAc, 9.85° (c = 0.408, H2O). 3.2. Preparation of A. oryzae mycelia for the production of 1KesNAc and NysNAc In the previous studies, we successfully produced SucNAc from sucrose and GlcNAc using dried A. oryzae mycelia containing bFFaseI and bFFaseII.5 The whole-cell catalysis method presents several advantages for the enzymatic production of oligosaccharides: (1) extraction of the enzyme from the culture is unnecessary, and (2) the enzyme can be easily removed from the reaction mixture. We therefore attempted to produce 1-KesNAc and NysNAc from SucNAc using A. oryzae mycelia containing b-fructofuranosidase. Before conducting this experiment, we investigated the hydrolytic properties of bFFaseII against 1-KesNAc and NysNAc in both low- (20 mM) and high-concentration (200 mM) solutions of each oligosaccharide. The results confirmed that bFFaseII hydrolyzes 1KesNAc and NysNAc under both conditions, and SucNAc and fructose were obtained as end products (Supplementary Fig. S5). bFFaseII did not participate in the synthesis of 1-KesNAc and NysNAc from SucNAc (Fig. 1A). These data indicate that the presence of bFFaseII is disadvantageous for the production of these oligosaccharides using bFFaseI. Therefore, in order to synthesize 1-KesNAc and NysNAc using the whole-cell catalysis method, we prepared dried A. oryzae mycelia containing bFFaseI as the major b-fructofuranosidase. Two types of b-fructofuranosidases were previously found in A. oryzae KB, and it was confirmed that one of the enzymes hydrolyzed sucrose to glucose and fructose and the other catalyzed a transfructosylation reaction to produce fructooligosaccharides from sucrose.15,16 These reports indicated that the amount of each enzyme produced by strain KB was influenced by the concentration of sucrose in the liquid culture medium. We therefore cultivated A. oryzae NBRC100959 in liquid culture media containing various concentrations of sucrose [1%, 2%, 3%, 6%, and 12% (w/v)] and prepared dried granules consisting of Celite 535 and mycelia. bFFaseI and bFFaseII were extracted from the dried mycelia and separated by ion exchange column chromatography (Supplementary Fig. S6), after which the total activity of each enzyme was determined (Table 1). Only very small levels of bFFaseII were produced in mycelia proliferated in media containing 3%, 6%, or 12% sucrose. However, bFFaseI was produced in all of the dried mycelia samples, and its level was highest in mycelia cultivated in media containing 6% or 12% sucrose. The presence of b-fructofuranosidase in the supernatant of A. oryzae culture broth was examined using ion exchange column chromatography. The quantity of bFFaseII in the supernatant increased with increasing sucrose concentration, whereas bFFaseI was not detected in any of the culture supernatants (Supplementary Table S1). These data indicate that
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Figure 2. Identification of compounds produced by the action of bFFaseI. 1H and 13C NMR shifts and the structures of product 1 (A) and product 2 (B). 1H NMR data (chemical shift in ppm; multiplicity; coupling constant in Hz) and 13C NMR chemical shifts (ppm) are designated with arrows and dashed line arrows, respectively. Bent double-headed arrows indicate HMBC cross peaks.
Table 1 Total enzyme activities of bFFaseI and bFFaseII extracted from A. oryzae mycelia cultivated in media containing various concentrations of sucrose Sucrose concentration in culture medium (%)
1 2 3 6 12
findings, we decided to use dried samples of A. oryzae mycelia, which proliferated in medium containing 6% sucrose, for wholecell catalysis production of 1-KesNAc and NysNAc from SucNAc.
Total enzyme activity (U) bFFaseI
bFFaseII
8.35 8.99 6.80 16.4 16.5
23.9 14.4 0.358 0.756 0.789
bFFaseI and bFFaseII were extracted from the soft granules containing approximately 0.43 g of dried mycelia of A. oryzae. After (NH4)2SO4 precipitation, these two enzymes were separated by ion exchange column chromatography (column size; / 1.5 8.5 cm) using TOYOPEARL SuperQ-650M resin. The fractions containing each enzyme were combined, and then total sucrose hydrolysis activity (U) of each enzyme was determined.
though bFFaseII is produced in A. oryzae mycelia cultivated under the presence of high concentrations of sucrose, this enzyme is secreted into the culture fluid under these conditions. Based on these
3.3. Production of 1-KesNAc and NysNAc from SucNAc using A. oryzae mycelia 1-KesNAc and NysNAc were produced by adding soft granules composed of dried A. oryzae mycelia and Celite 535 to 2 mL of buffer solution containing 0.6 g of SucNAc [SucNAc concentration, 30% (w/v)]. The time course of oligosaccharide generation is shown in Figure 3A. As the concentration of SucNAc decreased, 1-KesNAc and NysNAc were generated as major products (Fig. 3B). After 8 h of shaking, 190 mg of 1-KesNAc and 60 mg of NysNAc were isolated using two types of column chromatography. During the early stage of the reaction (0–8 h), the level of 1-KesNAc rapidly increased in proportion to the rapid decrease in the level of SucNAc. Most of the 1-KesNAc was produced after 4 h (33.5% of the total carbohydrate composition). The following phenomena were observed during the latter stage of the reaction (8–24 h): (1) the level
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Figure 3. Oligosaccharide production by A. oryzae mycelia containing bFFaseI as the major b-fructofuranosidase. (A) TLC analysis of the oligosaccharides formed in the enzymatic reaction. Products in the reaction mixture were identified based on the following standards: SucNAc (a), GlcNAc (b), fructose (c), 1-KesNAc (d), and NysNAc (e). (B) Carbohydrate composition in the reaction solution as determined by HPLC analysis. Retention time (min) of the carbohydrates in HPLC: GlcNAc, 8.56; fructose, 9.89; SucNAc, 13.8; 1-KesNAc, 21.0; and NysNAc, 31.0. Symbols: s, SucNAc; h, GlcNAc; 4 , fructose; d , 1-KesNAc; j , NysNAc; N , other oligosaccharides.
tion-catalyzing b-fructofuranosidase (bFFaseI) from A. oryzae NBRC100959. Moreover, we successfully synthesized both 1-KesNAc and NysNAc using a whole-cell catalysis method involving dried A. oryzae NBRC100959 mycelia containing bFFaseI as the major b-fructofuranosidase. This whole-cell catalysis method facilitates efficient synthesis of 1-KesNAc and NysNAc because extraction and purification of bFFaseI from the mycelia are unnecessary. This method enables large-scale production of 1-KesNAc and NysNAc because the mycelia can be used as immobilized enzymes for use in a bioreactor. We plan to develop 1-KesNAc and NysNAc as functional oligosaccharides. Further attempts will be made in the large-scale production of 1-KesNAc and NysNAc using an A. oryzae mycelia packed-bed column reactor and in the investigations of the possible physiological uses of these oligosaccharides with samples produced using the bioreactor. Acknowledgment We thank Dr. Hitoshi Sato of the Japan Sugar Refiners’ Association for his support. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2013.10. 006. References 1. 2. 3. 4. 5.
Figure 4. Schematic representation of oligosaccharide synthesis by the transfructosylation action of bFFaseI.
of 1-KesNAc decreased, (2) there was almost no decrease in the level of SucNAc, and (3) the level of NysNAc increased. From these data, we concluded that three separate transfructosylation reactions (A, B, and C in Fig. 4) occur during whole-cell catalysis, of which A and B are the major reactions occurring during the early stage, and C is the major reaction occurring during the latter stage.
6. 7. 8. 9. 10. 11. 12. 13. 14.
4. Conclusion 15.
Two novel oligosaccharides, 1-KesNAc and NysNAc, were synthesized from SucNAc using a partially purified transfructosyla-
16.
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