Preparation and Identification of 6²−α-Maltotriosyl-Maltotriose Using a Commercial Pullulanase

Abstract

In this article, 6²−α-maltotriosyl-maltotriose was consecutively prepared by hydrolysis and reverse synthesis action of Bacillus Acidopullulyticus pullulanase using pullulan as a substrate. Maltotriose syrup was obtained by hydrolyzing of pullulan with pullulanase. The optimal reverse synthesis conditions were then investigated and the optimum conditions were as follows: maltotriose syrup concentration, 70–75%; time, 40 h; pH, 4.0; temperature, 65°C; amount of pullulanase, 120 U. Under these conditions, the relative conversion rate of reverse synthesis reached up to 26%. The products were further isolated by preparative high performance liquid chromatography on Lichrospher NH₂ column. 6²−α-Maltotriosyl-maltotriose was purified and maltotriose syrup was collected and recycled as the substrate for reverse synthesis. Structure of 6²−α-maltotriosyl-maltotriose was elucidated by electrospray ionization mass spectrometry, fourier transform infrared spectroscopy, ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, and heteronuclear multiple bond correlation. These results suggested that this was a promising way of preparation of 6²−α-maltotriosyl-maltotriose.

Keywords:

• Pullulanase
• Pullulan
• Reverse synthesis
• Preparation
• 6²−α-Maltotriosyl-maltotriose

INTRODUCTION

With the development of carbohydrate chemistry, oligosaccharides demand more and more attention due to their important biological functionality and molecular diversity.^[1−3] However, chemical synthesis of oligosaccharides is more laborious than that of other biological macromolecules. Multifarious protections and de-protection strategies are essential for the achievement of the desired oligosaccharides.^[45] These complicated processes cause limitations to the application and research of oligosaccharides. Enzymatic synthesis of oligosaccharides is a promising alternative to the chemical method. The panose and isopanose were prepared in good yield from hydrolyzates of pullulan.^[6] Li et al.^[7] reported a simple method of preparing diverse neoagaro-oligosaccharides with β-agarase. 6ʹ-α-Maltosyl-maltotriose and 6″-α-glucosyl-maltotriose were prepared from Novamyl degradation of branched cylodextrins.^[8]

Pullulan is an exocellular polysaccharide containing a succession of maltotriose linked by α-(1, 6) glycosidic bonds and 7% maltotetraose.^[910] The distinctive physical traits of pullulan are attributable to the regular linkages pattern.^[11] Pullulanase (EC 3.2.1.41) is a member of the α-amylase family and specifically cleaves α-(1, 6) glycosidic bonds in starch, amylopectin, and pullulan. Maltotriose syrup has been prepared by hydrolyzing of pullulan with pullulanase.^[1213] Reversible action of pullulanase was verified through condensation reactions, and the smallest known substrate for the reverse action of pullulanase was maltose.^[14] The reversibility of hydrolysis for pullulanase has been employed to produce branched cyclodextrins, such as maltosyl-β-cyclodextrin.^[15] The specific synthesis of 6²−α-maltotriosyl-maltotriose by chemical process is complicated and difficult. Therefore, it is of interest to investigate the way for producing 6²−α-maltotriosyl-maltotriose by hydrolysis and reverse synthesis action of pullulanase consecutively.

MATERIALS AND METHOD

Materials

Pullulan (purity >98%) was purchased from Threewin Trading Co., Ltd (Shanghai). Pullulanase from Bacillus acidopullulyticus was purchased from Genencor Inc. (Wuxi, China). All other chemicals and reagents were of reagent grade.

Assay of Pullulanase Activity

Pullulanase activity was determined by measuring the amount of enzyme to release reducing sugars during the incubation with pullulan. In this assay, the reaction mixture containing 0.5 mL of 1% (w/v) pullulan in 20 mM acetate buffer (pH 5.0) and 0.5 mL of enzyme sample was incubated at 50°C for 30 min. The reducing sugar released was determined by dinitrosalisyilic acid method (DNS).^[16] One unit of pullulan activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of reducing sugars per minute. A V-1800 spectrophotometer (Mapada, Shanghai, China) was used for all assays.

General Procedures for the Preparation of 6²−α-Maltotriosyl-Maltotriose

Preparation of maltotriose syrup was performed through a modification of the method of Wu et al.^[12] and Singh et al.^[13] Pullulan was dissolved in 50 mM acetate buffer to make a solution of a concentration of 6% (w/v). Then, 100 ml of pullulan solution was incubated with pullulanase (8 U/ml) under agitation of 100 rpm at 45°C for 8 h. To investigate the optimal reverse synthesis condition, the resulting maltotriose syrup was concentrated in vacuo. Pullulanase was added into the maltotriose syrup and the reaction mixture was stirred with a speed of 100 rpm at 60°C for 48 h. After the reaction, the mixture was heated at 100°C for 15 min to inactivate the enzyme and centrifuged to remove the precipitate. The relative conversion rate (RC) was used for evaluating the degree of reverse synthesis:

The peak area of 6²−α-maltotriosyl-maltotriose and the peak area of maltotriose were detected and calculated by high performance liquid chromatography (HPLC).

Purification of 6²−α-Maltotriosyl-Maltotriose

6²−α-Maltotriosyl-maltotriose was isolated by preparative HPLC from the reaction supernatant. The conditions of this process are as follows: column, Lichrospher NH₂ (5 μm 10 × 300 mm); solvent system, acetonitrile-water (63:37, v/v); column temperature, 35°C; flow rate, 3 ml/min; detector, refractive index detector. The unreacted maltotriose syrup were collected and recycled as substrate for reverse systhesis.

HPLC, Mass Spectrometry (MS), and Nuclear Magnetic Resonance (NMR) Analysis

The product was analyzed by HPLC under the following conditions: column, Lichrospher NH₂ (5 μm 4.6 × 250 mm); solvent system, acetonitrile-water (70:30, v/v); column temperature, 35°C; flow rate, 1 ml/min; detector, refractive index detector. Electrospray Ionization mass spectrometry (ESIMS) analysis of 6²−α-maltotriosyl-maltotriose was performed using a Waters Platform ZMD 4000 mass spectrometer with electro spray ionization. ¹H nuclear magnetic resonance (¹H NMR), ¹³C nuclear magnetic resonance (¹³C NMR), and heteronuclear multiple bond correlation (HMBC) spectra were carried out on a Bruker AV400 MHz (Bruker, Rneinstetten, Germany). Samples were solved in D₂O with tetramethylsilane (TMS) as an internal standard.

Statistical Analysis

The data were expressed as means of triplicate. Statistical significance was assessed with one-way analysis of variance (ANOVA) using ORIGIN 7.5 (OriginLab Inc., USA). A value of p < 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

Effect of Maltotriose Syrup Concentration on the Preparation of 6²−α-Maltotriosyl-Maltotriose

Effects of maltotriose syrup concentration on the reverse synthesis of pullulanase were shown in Fig. 1 (T1). RC was found to increase with increasing concentration of maltotriose syrup. The optimum concentration for preparation of 6²−α-maltotriosyl-maltotriose was 65% (w/w). This indicated that the reverse synthesis activity of pullulanase depended on the concentration and tended to be enhanced with the increase of maltotriose syrup. The result was confirmed with previous reports. Little tetrasaccharide were synthesized at a maltose concentration of under 5%.^[14] Maltosyl cyclodextrins were prepared through the reverse reaction of pullulanase incubated with concentration of 70–75%.^[15]

FIGURE 1 T1: Effect of maltotriose syrup concentration on the preparation of 6²−α-maltotriosyl-maltotriose; T2: Effect of time on the preparation of 6²−α-maltotriosyl-maltotriose; T3: Effect of pH on the preparation of 6²−α-maltotriosyl-maltotriose: T4: Effect of temperature on the preparation of 6²−α-maltotriosyl-maltotriose; and T5: Effect of pullulanase amount on the preparation of 6²−α-maltotriosyl-maltotriose.

Effect of Time on the Preparation of 6²−α-Maltotriosyl-Maltotriose

The time course experiment for reverse synthesis of 6²−α-maltotriosyl-maltotriose was presented in Fig. 1 (T2). The results showed that RC increased with the proceeding of reaction and had an obvious increase within 40 h. Thereafter, RC increased slowly with the proceeding of reaction. This indicated that the consumption of maltotriose syrup decreased the reverse synthesis activity of pullulanase. This was in accordance with the preceding results that the concentration was crucial for reverse synthesis activity of pullulanase.

Effect of pH on the Preparation of 6²−α-Maltotriosyl-Maltotriose

Figure 1 (T3) presents the influence of reaction pH on RC. A wide pH range from 3.5 to 5.5 was applicable to the reverse synthesis reaction. The maximum RC reached 26% at a pH of 4.0. This characteristic was in agreement with the operational pH (4.0–4.5) for formation of maltosyl cyclodextrins.^[15] Compared with the optimum pH 5.0 for hydrolysis,^[12] it shifted to a more acidic value.

Effect of Temperature on the Preparation of 6²−α-Maltotriosyl-Maltotriose

The influence of the reaction temperature on RC was evaluated as shown in Fig. 1 (T4). It was evident that RC was optimal at 65°C. In contrast, optimal temperatures for hydrolysis of pullulanase were obtained at 45°C^[13] and 50°C.^[12] The result showed that higher temperature promoted the reverse synthesis activity of pullulanase and more activation energy was required. Moreover, higher temperature accelerated the diffusion between substrate and pullulanase. These results agree well with previous reports claiming the crucial role of temperature in improving reaction rate.^[1718]

Effect of Pullulanase Amount on the Preparation of 6²−α-Maltotriosyl-Maltotriose

As shown in Fig. 1 (T5), the increase of pullulanase loading from 30 to 120 U resulted in linear increases of RC. The conversion of maltotriose maintained 26% at high levels of pullulanase (120 U). Compared with hydrolysis, more pullulanase was required in the reverse synthesis reaction. This characteristic was in good agreement with the preparation of branched cyclodextrins.^[15]

Identification of 6²−α-Maltotriosyl-Maltotriose

The FTIR spectrum of 6²−α-maltotriosyl-maltotriose is shown in Fig. 2. The product exhibited an absorption peak at 3400 cm⁻¹, which was the characteristic of the –OH group. There was an apparent absorption at 2927 cm⁻¹ corresponding to the –CH– group. The FTIR absorption of the product at 1150 cm⁻¹ was the absorption of the –CO– group. The characteristic bands at 847 cm⁻¹ were attributed to α-glycosidic linkage and there was no characteristic absorption of β-glycosidic linkage in FTIR spectrum. From the FTIR spectrum of the product it was shown that the structure of the product was similar to that of maltotriose. The ESI-MS spectrum of 6²−α-maltotriosyl-maltotriose showed [M-H]⁺ at m/z 989.3 and [M-H]⁻ at m/z 991.3 (Fig. 3). The molecular weight was corresponding to six glucose moleculars.

FIGURE 2 FTIR spectrum of 6²−α-maltotriosyl-maltotriose.

FIGURE 3 ESIMS spectra of 6²−α-maltotriosyl-maltotriose in the negative and positive mode.

The ¹H NMR spectrum of 6²−α-maltotriosyl-maltotriose is shown in Fig. 4a. Figure 4 These signals were assigned on the basis of comparison with other spectra of branched oligosaccharides.^[819−21] The spectrum comprised four kinds of anomeric proton assigned to H-1 of internal anomeric (δ 5.34–5.20), H-1 of branched point (δ 4.87), and H-1 of the reducing end included α and β forms (δ 5.15 and δ 4.56). The structure of 6²−α-maltotriosyl-maltotriose was elucidated by these data and guidelines.^[22] It was deduced that there were four internal α1,4-linkages, one α1,6-branch, and one reducing end (α + β form). The ¹³C NMR spectrum of 6²−α-maltotriosyl-maltotriose is shown in Fig. 4b. Compared with other ¹³C NMR spectra of branched oligosaccharides,^[19−22] the resonance peaks in the ¹³C NMR spectrum were assigned to glucose residue. Four kinds of C-1 signals were observed: C-1s of internal anomeric (δ 101.85–101.21), C-1 of branched point (δ 100.31), and C-1 of the reducing end included α and β forms (δ 93.61 and δ 97.51). The C-6 signals of glucopyranose residues normally appeared at the highest field. The peaks at δ 62.67–62.29 could be attributed to C-6 of internal anomeric. The C-6 resonance of the branched point was shifted to the lower field at δ 68.38. The large downfield shifts indicated that branched maltotriose residues were linked to the respective residues. Two kinds of C-4 signals appeared at δ 71.24 and δ 79.88–78.99. The peak at δ 71.24 was due to the C-4 of the reducing end and the shift of C-4 resonance was caused by the glycosylation of hydroxyl group. This downfield shift of C resonance was confirmed by previous research.^[420] In addition, the HMBC spectra allowed for determination of the order of the branched point. As shown in Fig. 4c, C-4 and H-1 of the product had an α1,4-linkage at the inner residues (δ 79.23, 5.26); C-1 and H-4 of the product was found to couple to signals (δ 101.58, 3.24); C-6 and H-1 of the product had an α1,4-linkage at branched points (δ 68.38, 4.85). These results suggest that the product was 6²−α-maltotriosyl-maltotriose.

FIGURE 4 (A) ¹H NMR spectrum of 6²−α-maltotriosyl-maltotriose; (B) ¹³C NMR spectrum of 6²−α-maltotriosyl-maltotriose; and (C) HMBC spectrum of 6²−α-maltotriosyl-maltotrios.

CONCLUSION

Preparation of maltotriose syrup was performed through the hydrolysis of pullulan with a commercial pullulanase. 6²−α-Maltotriosyl-maltotriose can be prepared through the reverse synthesis of the same enzyme. The optimum reverse synthesis conditions were as follows: maltotriose syrup concentration, 70–75%; time, 40 h; pH, 4.0; temperature, 65°C; amount of pullulanase, 120 U. The product could be isolated directly through Lichrospher NH₂ column by preparative HPLC. The structural identities of the product were determined by the analysis of ESIMS, FTIR, ¹H NMR, ¹³C NMR, and HMBC. The result showed that the oligosaccharide was a 6²−α-maltotriosyl-maltotriose.

FUNDING

This study was also supported by the National Natural Science Foundation of China (Nos. 31230057 and 31071490), the National ‘Twelfth Five-Year’ Plan for Science & Technology Support of China (2012BAD37B02), and The Youth Foundation of Hubei Province Education (Q20132602).