Acceptor specificity of amylomaltase from Corynebacterium glutamicum and transglucosylation reaction to synthesize palatinose glucosides

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Acceptor specificity of amylomaltase from Corynebacterium glutamicum and transglucosylation reaction to synthesize palatinose glucosides Wachiraporn Naumthong a , Kazuo Ito b , Piamsook Pongsawasdi a,∗ a b

Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Enzyme Chemistry Laboratory, Graduate School of Science, Osaka City University, Osaka 558 8585, Japan

a r t i c l e

i n f o

Article history: Received 23 April 2015 Received in revised form 15 June 2015 Accepted 1 July 2015 Available online xxx Keywords: Amylomaltase Acceptor specificity Palatinose Palatinose glucosides Transglucosylation

a b s t r a c t Acceptor specificity for intermolecular transglucosylation reaction of amylomaltase from Corynebacterium glutamicum was investigated using starch as glucosyl donor and various saccharide acceptors. Maltooligosaccharides (G1–G4), mannose, palatinose and sucrose were efficient acceptors; the best one was glucose. This amylomaltase preferred hexose sugar containing the same configuration of C2-, C4and C6-hydroxyl groups as glucopyranose. Palatinose was chosen as suitable acceptor for the synthesis of palatinose glucosides (PGs). The optimal condition was to incubate 5 U/ml amylomaltase with 7.5 mM palatinose and 1.0% (w/v) soluble potato starch at 30 ◦ C for 24 h. In addition to PGs, maltooligosaccharides were also produced as by product. The product yield was 67.9%, in which the ratio of PGs to maltooligosaccharides was 1:1. Then PGs were separated by Bio-Gel-P2 column chromatography and analyzed by HPAEC. PG1–PG13 were identified with PG1 and PG2 as major products. NMR analysis showed that the PGs produced are novel products, PG1 and PG2 were a tri- and tetra-saccharide with the structure [O-␣d-glucopyranosyl-(1→4)]n -O-␣-d-glucopyranosyl-(1→6)-d-fructofuranose, where n = 1–2. PG was less sweet than palatinose and sucrose, more hygroscopic with similar prebiotic activity as palatinose. PGs thus have potential to replace sucrose or palatinose in food products for health benefits. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Amylomaltase (EC 2.4.1.25), an intracellular 4-␣-glucanotransferase (4␣GTase), was first found in Escherichia coli as an enzyme involved in maltose metabolism [1,2]. The enzyme was later reported in most thermophilic bacteria and archaea such as Thermus aquaticus [3], Aquifex aeolicus [4] and Pyrobaculum aerophilum IM2 [5]. The corresponding d-enzyme was reported in plants, e.g. potato [6] and cassava [7]. Amylomaltase catalyzes an intermolecular transglucosylation reaction, transferring glucosyl residues from donor to acceptor at free OH group of C4 to produce longer linear oligosaccharides. In addition, this enzyme possesses a unique intramolecular transglucosylation reaction to yield cycloamyloses or large-ring cyclodextrins (LR-CDs) with a degree of polymerization (DP) from 16 onwards. The enzyme has four different activities: disproportionation, cyclization, coupling and hydrolysis, with the first two as main [8,9]. Amylomaltase has many potential applications. Firstly, it is used in the production of

∗ Corresponding author. Tel. +662 218 5423. E-mail address: [email protected] (P. Pongsawasdi).

LR-CDs via cyclization reaction. LR-CD can form inclusion complex with guest molecules by trapping them into hydrophobic cavity resulting in a change of solubility, stability and biological properties of guest molecules [8]. Secondly, amylomaltase is used to modify starch and produce thermo-reversible starch gel with gelatin-like property [5,10] and can be used as fat and cream alternatives in dairy products to improve creaminess in low-fat yogurt [11]. Lastly, the enzyme can be used in the production of linear oligosaccharides and glucoside products through intermolecular transglucosylation reaction. Short-chain isomaltooligosaccharides (IMOs) as DP2–DP6 with prebiotic activity synthesized by combination of amylomaltase and transglucosidase have been reported [12]. (isomaltulose, 6-O-␣-d-glucopyranosyl-dPalatinose fructofuranose) is a disaccharide consisting of glucose and fructose linked by an ␣-1,6 glycosidic bond. Naturally, palatinose is found in honey, sugar cane [13,14] and it can be synthesized by enzymatic method. Palatinose was first produced as a by-product in dextran production process [15]. It was also synthesized by sucrose isomerase from many organisms such as Protaminobacter rubrum [16], Erwinia rhapontici [17], and Pantoea dispersa [18] using sucrose as substrate. Sucrose isomerase catalyzes an isomerization of sucrose, converting ␣-1,2 glycosidic bond to ␣-1,6 glycosidic

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bond [19]. Various immobilized enzymes, such as immobilized ␣-glucosyltransferase from Serratia plymuthica NCIB No. 8285, have also been used in palatinose production [20,21]. Palatinose is hydrolyzed more slowly than sucrose so plasma glucose and insulin levels are gradually increased to reach the maximum level which is lower than that of sucrose [22,23]. For this reason, palatinose is suitable to be used as a sugar substitute [24] and sweetener for diabetics [22,25]. Moreover, palatinose reveals anticariogenic property by inhibition of glycosyltransferase (GTase) activity of Streptococcus mutans. Production of organic acids, plaque and insoluble glucan from sucrose that involved in causing dental caries are inhibited [26–28]. In recent year, the demand for glucoside products in food, drink and cosmetic industries increases, as glucosides usually have better properties, e.g. solubility, stability and bioactivity, than their parent compounds. Most glucoside products have been produced by transferase or hydrolase [29]. For example, 3-O-␣-maltosyll-ascorbate with high stability under oxidative conditions was synthesized from ␣-maltosyl fluoride and l-ascorbic acid catalyzed by mutated cyclodextrin glycosyltransferase (CGTase) [30]. And isomaltooligosaccharides with prebiotic activity were produced by transglucosylation reaction of ␣-glucosidase from Microbacterium sp. using maltose as substrate [31]. However, for palatinose, only one report on the synthesis of palatinose glucosides has been found. The tri- and tetra-saccharide glucoside products of palatinose were synthesized by Thermoanaerobacter brockii kojibiose phosphorylase using ␤-d-glucose 1-phosphate and palatinose as substrates [32]. From our previous study, a novel amylomaltase from Corynebacterium glutamicum ATCC 13032 with low amino acid sequence identity (20–25%) to amylomaltases from Thermus sp. was reported. The enzyme gave cycloamyloses or LR-CD products in the range of DP19-DP50 from its intramolecular transglucosylation activity [33]. Maltooligosylsucrose with anticariogenic property and the prebiotic isomaltooligosaccharides were successfully synthesized by the intermolecular transglucosylation reaction of this amylomaltase [34]. In this study, we here focus on investigation of acceptor specificity for saccharides of C. glutamicum amylomaltase and the synthesis of palatinose glucosides through the intermolecular transglucosylation activity of the enzyme. 2. Materials and Methods 2.1. Materials Maltooligosaccharides: d(+)-glucose, G1 to maltoheptaose, G7; d(+)-mannose, d(+)-cellobiose, d(−)-fructose, l-fucose, lrhamnose and palatinose were purchased from Sigma (USA). d(+)-galactose and sucrose were products from Bio Basic (Canada) while d(−)-arabinose was from BDH Chemical (England). Ribose was obtained from Wako (Japan) while d(+)-allose and d(+)melibiose were products from Tokyo Chemical (Japan). Lactose and raffinose were purchased from Ajax Finechem (Australia). Soluble potato starch was from Scharlau (Spain) and Bio-Gel-P2 beads were from Bio-Rad Laboratories (USA). Ampicillin, glucoamylase from Aspergillus niger, isopropyl ␤-d-1-thiogalactopyranoside (IPTG) and rat intestinal acetone powder were purchased from Sigma (USA). All other chemicals were of analytical grade. 2.2. Preparation and purification of amylomaltase Amylomaltase gene from C. glutamicum ATCC 13032 was expressed in E. coli BL21(DE3) using pET-19b expression vector as previously described [33]. A recombinant clone was cultured in LB medium containing 100 ␮g/ml ampicillin at 37 ◦ C until O.D.600

reached 0.4–0.6, then 0.4 mM IPTG was added to induce the enzyme production. Intracellular crude enzyme was obtained, after cell sonication the enzyme was purified by HisTrap affinity (1 ml, HisTrap FFTM ) column chromatography. Starch transglucosylation activity [33] was measured as described below and protein concentration was determined by Bradford method [35]. 2.3. Determination of acceptor specificity and synthesis of glucoside products Saccharides tested as acceptor substrates were classified into four groups: monosaccharides, which were divided into three subgroups: hexose (glucose, allose, mannose, galactose and fructose), deoxy hexose (fucose and rhamnose) and pentose (arabinose and ribose), disaccharides (maltose, cellobiose, lactose, melibiose, palatinose and sucrose), trisaccharides (maltotriose and raffinose) and oligosaccharides (G4–G7). The acceptor specificity was determined by starch transglucosylation activity of amylomaltase which was assayed as follows. The 600 ␮l reaction mixture containing 0.2%, w/v soluble potato starch, 2.5 mM saccharide and 60 ␮l purified amylomaltase in 50 mM phosphate buffer, pH 6.0 was incubated at 30 ◦ C for 10 min, after that the reaction was stopped by boiling. Then, 100 ␮l of sample was withdrawn to measure the decrease in starch by reacting with 1 ml of iodine solution (0.02% I2 in 0.2% KI, w/v) and A600 was determined. The acceptor with the highest activity was set as 100% of relative activity. The reaction mixtures were analyzed by TLC and HPLC for glucoside products. One unit of starch transglucosylation activity was defined as the amount of enzyme that produces 1% decrease in blue color of starch–iodine complex per minute. 2.3.1. Thin Layer Chromatography (TLC) Sample was spotted on TLC plate (Silica gel 60, Merck) and twice developed in butanol:pyridine:water (5:4:1 by volume) [34]. Glucoside products were detected by spraying the mixture of concentrated sulfuric acid and absolute methanol (1:2 by volume), then heated at 110 ◦ C for 20 min [36]. 2.3.2. High Performance Liquid Chromatography (HPLC) Analysis of glucoside products was performed by HPLC (Shimadzu 10AVP, Japan). The reaction mixture was centrifuged (18,000 × g, 4 ◦ C, 45 min) to remove majority of the remain soluble potato starch before loading onto a Resex RSO-Oligosaccharide Ag+ column (200 × 10.0 mM, Phenomenex, Inc., USA,). The glucoside products were eluted by ultrapure water at 80 ◦ C with a flow rate of 0.2 ml/min and detected by refractive index detector (RID) [34]. The yield of glucoside products was calculated from the equation: Product yield (%) =

Peak area of product × 100 Peak area of acceptor at t0

when peak area of product was determined from the difference between palatinose peak obtained when reaction mixture was treated by glucoamylase and that of untreated. 2.4. Optimization of the synthesis of glucoside products Optimization for the highest glucoside product yield was performed in 50 mM phosphate buffer, pH 6.0 at 30 ◦ C [33] by varying four parameters: palatinose concentration (0–15 mM), soluble potato starch concentration (0–5% w/v), enzyme concentration (0–12 U/ml) and incubation time (0–42 h). Analysis of glucoside products was performed by HPLC.

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2.5. Preliminary characterization of glucoside products The reaction mixture at optimal condition was treated with 40 U/ml of glucoamylase from A. niger and then further incubated at 50 ◦ C for 2 h. After that, the reaction was heated to inactivate glucoamylase and analyzed by HPLC. 2.6. Larger scale preparation, product separation and identification The reaction volume was 100 times enlarged to 60 ml and the synthesis of glucosides was performed under optimized condition. Then, the reaction mixture was concentrated by rotary evaporator to about 5 ml before applied onto a Bio-Gel-P2 column (1.2 × 97 cm) previously equilibrated with ultrapure water. The column was run at room temperature. Palatinose glucosides (PGs) were eluted by ultrapure water at a flow rate of 8 ml/h. Fractions were collected and sugar content was determined by phenol-sulfuric acid method [37] and the PGs were identified by HPAEC. 2.6.1. High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) Selected fractions from Bio-Gel-P2 column were filtrated through a 0.45 ␮m membrane before applied onto a CarboPac-PA1 column (4 × 250 mM, Dionex, USA) and analyzed by HPAEC-PAD (Dionex-300, USA). The elution was by steps of gradient of NaOH and CH3 COONa with a flow rate of 1.0 ml/min at 30 ◦ C. During the first 1–5 min, 150 mM NaOH was used. After that, from 6–43 min, the PGs were eluted by 150 mM NaOH containing 600 mM CH3 COONa. Finally, for the last 2 min, 500 mM NaOH was used as elution buffer [38]. PGs were detected and identified using standard G1–G7 and palatinose as reference compounds. The PGs were separated into two groups: short chain and long chain PGs. Fractions that contained the two groups of PGs were then lyophilized.

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unit was analyzed by 1 H NMR spectra using deuterated water (D2 O) as solvent. 2.7.2. Sweetness test The relative sweetness of 10%, w/v of palatinose, short chain PGs or long chain PGs was determined by measuring the Brix values using Brix Refractometer (Bellingham and Stanley, UK.). Sucrose was used as a reference compound to plot the standard curve between concentration and Degree Brix. ◦ Brix is defined as the amount in g of sucrose in 100 ml of aqueous solution [39]. The Brix value of 10–15% sucrose solution is set as the relative sweetness of 1.0 [40]. 2.7.3. Hygroscopic test 100 mg of palatinose, short chain PGs or long chain PGs was weighed in each preweighed eppendorf tube. Put the tubes in the controlled cabinet at 25 ◦ C and 70% relative humidity with the tube lid open. Then the weight was recorded every 4 h for one day [41]. Hygroscopic value was defined as the increasing in weight of sample, very hygroscopic: increase in mass is equal to or greater than 15%; hygroscopic: increase in mass is equal to or greater than 2% to less than 15%; and slightly hygroscopic: increase in mass is equal to or greater than 0.2% to less than 2% [42]. 2.7.4. Prebiotic activity test The 500 ␮l reaction mixture containing 5 mg of palatinose, short chain PGs or long chain PGs and 100 mg/ml rat intestinal acetone powder in 25 mM acetate buffer, pH 6.0 was incubated at 37 ◦ C for 1 h. The reaction products were analyzed by HPAEC-PAD. 3. Results and Discussion The purified recombinant amylomaltase from C. glutamicum showed a specific activity of 34.7 U/mg protein with a major protein band at 84 kDa, the result of which is the same as that reported previously [33].

2.7. Characterization of properties of glucoside products

3.1. Acceptor specificity

2.7.1. Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) The molecular weight of PGs was analyzed by Liquid Chromatography-Time of Flight Mass Spectrometry (LC-TOF-MS) with positive mode using the mixture of methanol and water (1:9 by volume) as solvent. The linkage between palatinose and glucose

The acceptor specificity of amylomaltase was determined from the starch transglucosylation activity assay using soluble potato starch as a glucosyl donor and various saccharides as acceptors (Fig. 1). Glucose gave the highest activity so it was set as 100% relative activity, followed by allose, maltose and maltotriose. Within the hexose subgroup of monosaccharide, allose, mannose, fructose

Fig. 1. Acceptor specificity of amylomaltase. Soluble potato starch (0.2%, w/v) as glucosyl donor was incubated with 2.5 mM of various saccharides acceptor and enzyme in phosphate buffer, pH 6.0, 30 ◦ C for 10 min, then starch transglucosylation activity was analyzed as described in Section 2.

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and galactose gave 95%, 25%, 18% and 8% of activity as compared to glucose. Hexoses such as fucose and rhamnose, have methyl group instead of CH2 OH at C6 which resulted in decreasing activity to about the same level as galactose. These results suggested that saccharides with aldose functional group are better acceptor when compared to the ketose group. To be a good acceptor, the OH configurations of aldose at position C2, C4 and C6 of glucose were important, with C4 and C6 seems to be more important than C2 while C3 was less important for the enzyme to express its high activity. The result also showed that this amylomaltase preferred hexose to pentose structure since ribose and arabinose gave low activity. The relative activity given by maltooligosaccharide series: maltose > maltotriose > maltotetraose > maltopentaose, suggested that the enzyme preferred the acceptor that consisted of glucose residues connected by ␣-1,4 glycosidic bond and not longer than five glucose units. In the disaccharide and trisaccharide subgroups, maltose gave highest activity followed by maltotriose, palatinose (glucose, ␣-1,6-fructose), sucrose (glucose, ␣-1,2-fructose), cellobiose (glucose, ␤-1,4-glucose), melibiose (galactose, ␣-1,6-glucose), lactose (galactose, ␣-1,4-glucose) and raffinose (galactose, ␣-1,6-glucose, ␣-1,2-fructose). These disaccharides/trisacharides have at least one glucose residue which could act as good acceptor. To our knowledge, only two studies on acceptor specificity of amylomaltase have been reported. In E. coli IFO3806, the efficient acceptors in transglucosylation reaction were glucose, allose, cellobiose, isomaltose, mannose, and xylose, as determined from intensity of product spots on TLC. This E. coli amylomaltase transferred glucosyl residues attached to OH group at C4 of glucose, allose, mannose and xylose producing ␣-1,4 glucoside products which was confirmed by glucoamylase and ␤-amylase treatment and NMR [43]. In another study, 4-␣-glucanotransferase from Pyrococcus kodakaraensis KOD1 showed broad acceptor specificity to various saccharides, G1–G3, isomaltose, cellobiose, sucrose and xylose could act as acceptors especially G1 and xylose were preferred. This archaeal enzyme was specific for saccharides with pyranose structure containing OH configuration at C2, C3 and C4 position as glucose which was different from our enzyme that showed the importance of C2, C4 and C6 in catalysis [44]. When compared with studies in CGTase being classified as GH13 but displays similar catalytic mechanism as amylomaltase which is GH77, the acceptor specificity is different. CGTases from Bacillus circulans A11 [45], Bacillus macerans, and Bacillus megaterium [46] preferred OH groups at C2, C3 and C4 with the same configuration as glucose. From the study of Thermoanaerobacter sp. CGTase, the enzyme activity was also decreased when the length of glucose units increased [47]. Since G1–G4, mannose, palatinose and sucrose were the acceptors giving high to moderate relative activity for C. glutamicum amylomaltase, they were then chosen as substrates for synthesis of glucoside products. At least four to five spots of glucoside products were detected using TLC analysis when G1–G4 or sucrose was used as an acceptor (data not shown). However, these products were not expected to be new products. Maltooligosaccharides and maltooligosylsucrose have previously been synthesized by CGTases from B. macerans [48], and Thermoanaerobacter sp. [49] and by amylomaltase from C. glutamicum [34]. The ␣-1,4 polyglucosylfructosides was synthesized from sucrose by ␣-glucosidase from the digestive juice of Archachatina ventricosa [50] while ␣-1,6 glucosylsucrose was also synthesized from sucrose by mutated ␣glucosidase from Bacillus sp. [51]. Moreover ␣-1,6 maltosylsucrose isomers produced by B. stearothermophilus maltogenic amylase from maltotriose and sucrose substrates showed low sweetness, high hygroscopicity, low Millard reactivity, and high acid and heat stability [52]. In contrast, reports on glucoside products of palatinose and mannose are limited. We found that at least five and seven spots of glucoside products were observed on TLC when

Fig. 2. TLC analysis of the reaction products from amylomaltase incubated with soluble potato starch (0.2%, w/v) and 2.5 mM palatinose/mannose in phosphate buffer, pH 6.0 at 30 ◦ C for 24 h (a) standard G1–G7, (b) soluble potato starch, (c) control reaction without acceptor at 24 h, (d) palatinose, (e)–(f) palatinose as acceptor at 0 and 24 h, (g) mannose, (h)–(i) mannose as acceptor at 0 and 24 h, respectively. PGs = palatinose glucosides, MGs = mannose glucosides.

palatinose and mannose were used as acceptors, respectively (Fig. 2). The products from palatinose and mannose could be predicted as a trisaccharide to heptasaccharide and a disaccharide to octasaccharide, respectively, when compared with standard G1–G7. The quantities of palatinose glucosides (PGs) and mannose glucosides (MGs) were analyzed by HPLC. Eight peaks of PGs at Rt 24, 26, 27, 29, 31, 34, 39 and 44 min were detected with 53.6% yield (Fig. 3B). The smallest PG was at Rt 44 min whereas the largest PG was at Rt 24 min. For mannose acceptor, nine peaks of MGs were observed with only 3.67% yield (data not shown). Judging from product yield, palatinose was chosen as a suitable acceptor for our further study. 3.2. Optimization of the synthesis of glucoside products To optimize the synthesis for the highest yield of glucoside products, the reaction was performed at optimal temperature of 30 ◦ C and pH 6.0 of this amylomaltase [33]. Then four other parameters, concentration of donor, acceptor, enzyme and incubation time, were varied, one at a time. The optimal condition obtained for the synthesis of PGs was to incubate 5 U/ml enzyme with 7.5 mM palatinose and 1.0% (w/v) soluble potato starch in 50 mM phosphate buffer, pH 6.0 at 30 ◦ C for 24 h. Then the yield and linkage of palatinose glucosides was determined by glucoamylase treatment and analyzed by HPLC. Glucoamylase mainly hydrolyzes ␣-1,4 linkage between glucose units in an exo-type from the non-reducing ends of amylose and amylopectin [53] and the glucose residues were released. This enzyme can also hydrolyze ␣-1,6 linkage at the branch points of amylopectin but at a slower rate than the ␣-1,4

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Fig. 3. HPLC chromatogram of palatinose glucosides synthesized by amylomaltase using 1.0% (w/v) soluble potato starch and 7.5 mM palatinose as substrates at (A) 0 and (B) 24 h and (C) palatinose glucosides from (B) was treated with glucoamylase. S = soluble potato starch, P = palatinose, PGs = palatinose glucosides, G1 = glucose. Table 1 The yield of palatinose glucosides and maltooligosaccharides at different ratio of soluble potato starch donor and palatinose acceptor as analyzed by HPLC. Soluble potato starch: palatinose (weight ratio) 1:1 1:2 1:4 1:5 2:1 4:1 5:1

Product yield (%) 55.6 46.7 32.4 27.4 59.3 67.9 62.2

linkage. In Fig. 3C, all of PGs peaks disappeared whereas glucose peak appeared at Rt 62 min suggesting that the transferred glucose units attached to palatinose by an ␣-1,4 linkage, thus confirmed the catalytic action of amylomaltase [54]. However, possibility of PGs having an ␣-1,6 linkage could not be excluded at this stage. When glucosides synthesis was performed at different ratios of starch to palatinose, the yields of glucosides obtained were different (Table 1). At the 1:1 ratio, the total yield of PGs was 55.6%. It was observed that the yield was decreased when the ratio of palatinose in the reaction was increased. On the contrary, the total yield of PGs was increased with the increasing in proportion of soluble potato starch, the highest yield of 67.9% was obtained at the starch to palatinose ratio of 4:1. In previous report, maltooligosylsucrose with 81.7% yield was synthesized by this amylomaltase using raw tapioca starch as a glucosyl donor and sucrose as acceptor [34]. For synthesis of other functional oligosaccharides, IMOs produced by maltogenic amylase in combination with ␣-glucanotransferase gave 68.0% product yield [55] and fructooligosaccharides synthesis catalyzed by fructofuranosidase gave 49.8% yield [56]. Hence, the PGs yield obtained in this study is quite good.

3.3. Larger scale preparation, product separation and identification The reaction mixture for PGs synthesis was up scaled and separated by Bio-Gel-P2 column chromatography, the separation was based on size. Soluble potato starch (peak S), PGs (peak A–E) and palatinose (peak P) were successfully separated (Fig. 4). Five peaks of PGs (A–E) were then identified by HPAEC. From HPAEC analysis, each Bio-Gel-P2 peak consisted of mixtures of products, as exemplified in Fig. 5A and B which were products from fractions at the top of peak E and A, respectively. The HPAEC peak was assigned number of glucose residues attached to palatinose by using palatinose and G1–G7 as reference compounds (Table 2). From Bio-Gel peak E, 13 types of PGs (PG1–PG13) could be identified with long-chain palatinose glucosides (PG7–PG10) as main products (Fig. 5A) whereas peak A consisted of short-chain palatinose glucosides (PG1–PG3) with PG1 (a trisaccharide at Rt 7.8 min) and PG2 (a tetrasaccharide at Rt 8.9 min) as main products (Fig. 5B). It was found that peaks B, C and D consisted of PG1–PG4 (PG2 as major), PG1–PG7 (PG4–PG5 as major) and PG1–PG10 (PG6–PG7 as major), respectively. From the peak area of products from HPLC (Fig. 3) and HPAEC (Fig. 5) profiles, PG1 and PG2 were overall major products of palatinose glucosides synthesis by this amylomaltase. The top fractions from peak A and peak E containing short chain and long chain PGs major products, respectively, were then lyophilized. Palatinose glucosides synthesized by T. brockii Kojibiose phosphorylase using ␤-d-glucose 1-phosphate as glucosyl donor were previously reported. The glucoside products were separated by carbon-celite column chromatography and detected by HPAEC, in which the trisaccharide to tetrasaccharide were main products at the yield ratio of 5:1 [32] while in this study, the trisaccharide to tetrasaccharide major products were obtained at the yield ratio

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Fig. 4. Bio-Gel-P2 chromatogram (column size 1.2 × 97 cm) of reaction products from amylomaltase catalyzed transglucosylation of soluble potato starch and palatinose. Elution was by ultrapure water at room temperature with a flow rate of 8 ml/h, 1 ml fraction was collected. The sugar content was detected by phenol-sulfuric acid method at 490 nm. Peaks A–E were collected as product peaks. Peak S and P were residual starch and palatinose, respectively.

Fig. 5. HPAEC chromatogram of palatinose glucosides (PGs). The PGs were from the top fraction of (A) peak E and (B) peak A separated by Bio-Gel-P2 column in Fig. 4.

of 3:2. In addition, long-chain glucosides were also obtained from amylomaltase catalysis. Further analysis on glycosidic linkage by NMR was performed as described below in Section 3.4.1. In our product identification by HPAEC, it was observed that palatinose glucosides (PG1–PG13) were mixed with maltooligosaccharides (G1–G16) (Fig. 5A) as identified by retention times (Table 2), the glucosyl transfer from starch donor to

palatinose acceptor was competed with such the same transfer to maltooligosaccharides occurring during the intermolecular transglucosylation reaction. The two series of products could be separated by HPAEC (Fig. 5) but not by HPLC Resex RSOOligosaccharide Ag+ column (Fig. 3) of which size separation was achieved. For this reason, the total yield of 67.9% calculated from HPLC peaks of products obtained (as described in Section 3.2) was

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Table 2 HPAEC retention time (Rt) of palatinose glucosides previously separated by Bio-GelP2 column and identified by HPAEC. Peak

Product

HPAEC retention time (min)

Total

Major

A B C

PG1–PG3 PG1–PG4 PG1–PG7

PG1–PG2 PG2 PG4–PG5

D

PG1–PG10

PG6–PG7

E

PG1–PG13

PG7–PG10

7.8, 8.9, 10.4 7.8, 8.9, 10.4, 12.0 7.8, 8.9, 10.4, 12.0, 15.0, 16.9, 19.8 7.8, 8.9, 10.4, 12.0, 15.0, 16.9, 19.8, 20.7, 22.6,24.2 7.8, 8.9, 10.4, 12.0, 15.0, 16.9, 19.8, 20.7, 22.6,24.2, 25.9, 27.4, 28.8

Reference compound Palatinose G1 G2 G3 G4 G5 G6 G7

6.4 3.9 6.8 8.1 9.6 11.5 13.8 16.0

the yield of PGs plus maltooligosaccharides. By HPEAC analysis, the yield ratio of PGs to maltooligosaccharide products was about 1:1 as determined from their peak areas. Thus, the corrected yield of palatinose glucosides in this study should be about 33.9%. 3.4. Characterization and properties of PGs 3.4.1. Mass spectrometry and nuclear magnetic resonance From LC-TOF-MS with positive ions spectrum, PGs at Rt 7.8 and 8.9 min were shown at m/z [M+Na]+ of 527 and 689, respectively. The molecular mass of the compounds was 504 and 666 Da (Fig. 6) corresponding to the size of the trisaccharide (PG1) and tetrasaccharide (PG2) comprising a palatinose linked with one and two glucose residues. The linkage of PG1 was confirmed by 1 H NMR compared to parent palatinose. The peak of anomeric proton of parent palatinose shown as doublet signal at 4.90 ppm

Fig. 6. LC-TOF-MS analysis with positive ions [M+Na]+ for determining the molecular mass of palatinose (A) and palatinose glucosides (B). The palatinose glucosides were from the short chain fraction (peak A) obtained from Bio-Gel-P2 column.

corresponded to the ␣-1,6 linkage [12,32,57,58] so the result confirmed that glucose connected to fructose by ␣-1,6 glycosidic bond. For PG1 spectrum, the peak of ␣-1,6 linkage of palatinose at 4.90 ppm was also found. The peak of anomeric proton appeared as triplet signal at 5.34 ppm corresponded to the ␣-1,4 linkage signal (Fig. 7). Thus, the NMR spectrum of PG1 indicated that the tranferred glucosyl residue linked to palatinose by an ␣-1,4 linkage (Fig. 8) [34,59,60]. Our PGs products were thus different from those synthesized by T. brockii Kojibiose phosphorylase as mentioned in Section 3.3 [32], in which the PGs obtained were of the ␣-1,2 linkage. The structure of PGs could thus be identified as [O-␣-d-glucopyranosyl-(1→4)]n -O-␣d-glucopyranosyl-(1→6)-d-fructofuranose (Fig. 8). This is a new palatinose glucoside product.

Fig. 7. 1 H NMR spectrum of palatinose glucosides using deuterated water as solvent. The palatinose glucosides were from the short chain fraction (peak A) obtained from Bio-Gel-P2 column.

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a series of maltooligosaccharides could be ranked in the increasing order as DP3 > DP4 ≥ DP7 > DP5 > DP6 > DP2 [63]. Neoagarobiose (3-O-(3,6-anhydro-l-galactosyl)-␤-d-galactopyranose) as a disaccharide gave 11 times and 5 times higher in hygroscopic property than sodium hyaluronate and glycerol as humectant in food and cosmetic products, respectively. The PGs in this work might thus be applied as a humectant in food products.

Fig. 8. Structure of palatinose glucosides (PG)n . n = 1–13 glucose residue.

3.4.2. Sweetness test In addition to being referred to the amount of sugar in an aqueous solution, the Brix value can also be used in characterizing the perceived sweetness. A compound which contains a glucophore structure with multiple hydroxyl groups will give sweetness taste [61]. In this study, from the standard curve between sucrose concentration and Brix value (data not shown), the Brix value of 10% of sucrose solution was set as relative sweetness of 1.0 [40]. When compared the Brix values of 10% of palatinose and paltinose glucoside products with that of sucrose, the relative sweetness could be determined. It was found that the relative sweetness of palatinose (0.97) was closed to that of sucrose (set as 1.00). Interestingly, the sweetness of short chain (0.58) and long chain PGs (0.50) was almost twice lower than palatinose and the sweetness of long chain PGs was about 10% less than that of the short chain PGs. The result agrees with previous report that showed the decrease in sweetness of long chain fructooligosaccharides when DP was increased. This low sweetness property is quite useful in various applications of food products where the use of sucrose is restricted by its high sweetness property [62]. The PGs in this study was sweeter than ␣-1,6 maltosylsucrose (0.23) produced by maltogenic amylase [52]. 3.4.3. Hygroscopic test Hygroscopicity was determined by measuring increase in sample weight at constant humidity and temperature. For 24 h testing time, the weight gain was in the order of: long chain PGs > short chain PGs > palatinose (Fig. 9). The result showed that long chain PGs absorbed moisture twice higher than parent palatinose. Reports on hygroscopic property of saccharides and glucosides have been found. For example, hygroscopic property of

3.4.4. Prebiotic activity test Rat intestinal acetone powder consists of ␣-glucosidase, ␣amylase, glucoamylase, isomaltase and sucrase [64], the ␣-1,4 glycosidic bond could be completely hydrolyzed while the ␣-1,6 bond was partially hydrolyzed by this enzyme mixture. In this study, the ␣-1,6 linkage of palatinose could be partially hydrolyzed, the peaks of glucose, fructose and remained palatinose were observed on HPAEC chromatogram (data not shown). For shortchain and long-chain PGs, the ␣-1,4 glycosidic bond connecting glucose units at the non-reducing end of PGs could be completely hydrolyzed and glucose, fructose and palatinose were released whereas the peaks of short-chain and long-chain PGs were disappeared (data not shown). These results suggested that both PGs had similar degree of prebiotic activity to palatinose. This is different from previous report in which the major PG trisaccharide synthesized by T. brockii Kojibiose phosphorylase was hardly hydrolyzed by rat intestinal ␣-glucosidase, thus having a higher prebiotic activity than its parent palatinose [32]. Such difference is due to different linkage of the glucosides obtained from catalysis by amylomaltase and phosphorylase which are of the type ␣-1,4 and ␣-1,2, respectively. 4. Summary Amylomaltase from C. glutamicum was highly specific for saccharide acceptors with hexoaldose moieties that have the same configuration of OH at C2, C4 and C6 as glucose. Oligosaccharides up to 5 glucose units could act as glucosyl acceptor. Palatinose glucosides were successfully synthesized by this enzyme using soluble potato starch as glucosyl donor and palatinose as acceptor. New palatinose glucoside series, PG1–PG13 were produced, with a triand tetra-saccharide, PG1 and PG2, as major products. These glucosides of palatinose have potential as nutraceutical compounds for health benefits. Acknowledgements W. N. received the Chulalongkorn University Graduate Scholarship to Commemorate the 72th Anniversary of His Majesty King

Fig. 9. Relative hygroscopicity of palatinose and palatinose glucosides. The samples were stored in the controlled cabinet at 25 ◦ C and 70% relative humidity. Then the increase in sample weight was monitored for 24 h.

Please cite this article in press as: W. Naumthong, et al., Acceptor specificity of amylomaltase from Corynebacterium glutamicum and transglucosylation reaction to synthesize palatinose glucosides, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.07.003

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Bhumibol Adulyadej from Graduate School. Research funding from IIAC of CU Centenary Academic Development Project and Thailand Research Fund Grant IRG 5780008 are acknowledged.

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