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Transfructosylation reaction in cured tobacco leaf (Nicotiana tabacum)
Journal of Bioscience and Bioengineering VOL. 116 No. 6, 666e671, 2013 www.elsevier.com/locate/jbiosc
Transfructosylation reaction in cured tobacco leaf (Nicotiana tabacum) Atsushi Nagai,1, 3, * Toshiki Mine,2, x Takeshi Yamamoto,2, x and Hiroyuki Wariishi3 Tobacco Science Research Center, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan,1 Plant Innovation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan,2 and Department of Innovative Science and Bioenvironmental Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan3 Received 25 February 2013; accepted 27 May 2013 Available online 8 July 2013
Tobacco plant was known to be a non-fructan-storing plant. However, we demonstrated that fructo-oligosaccharides (FOSs) were formed in cured tobacco leaf on adding sucrose to the leaf in our previous report (Nagai et al., J. Agric. Food Chem., 60, 6606e6612, 2012). Also, it was expected from the results obtained in previous study that FOSs were generated by enzymatic reaction in cured tobacco leaf. The purpose of this study is to confirm and understand the mechanisms of above-mentioned FOSs formation. Thus, we tried to purify the enzymes related to the production of FOSs. The enzymes were extracted from pulverized cured tobacco leaf (burley type leaf), and were purified by charcoal treatment, ultrafiltration, and several chromatography techniques. As a result, one of the enzymes was purified up to 414-fold. It was revealed that this enzyme was acid invertase exhibiting maximum transfructosylation activity at pH 6.0, 60 C. In addition, general properties of this enzyme were also investigated. The enzyme purified in this study enhanced the ratio of FOSs formation under the condition of high concentrated sucrose. From these results, it was suggested that this enzyme participated in the formation of FOSs in tobacco leaf after curing. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cured tobacco; Invertase; Remaining activity; Heat stability; Transfructosylation]
Tobacco leaves obtained from commercial tobacco plant undergo many processes after harvest (1). Curing is the first process, and its purpose is to remove the moisture from tobacco leaves. This process also plays a fundamental role in giving tobacco its flavor and aroma. The dried leaves are called cured tobacco leaves. Sugars are the main components in cured tobacco leaves, and are also key compounds directly influencing the aroma/taste of cigarette smoke (2e5). Therefore, investigating the sugar composition is an important issue in managing tobacco product quality. Recently, we reported on the formation of fructo-oligosaccharides (FOSs) in cured tobacco leaf (6). It was strongly expected that FOSs were formed through enzymatic reaction using sucrose as the substrate in cured leaf with low moisture content. The structures of FOSs identified in cured tobacco leaves are shown in Fig. 1. FOSs are condensates of fructose in which fructose residues are bound by b-linkage with or without a single glucose unit in each molecule. Polymerized FOSs, which is called fructan, is known as one of the reserve carbohydrates and has been found in plants, algae, and bacteria (7,8). While fructan is found in approximately
* Corresponding author at: Tobacco Science Research Center, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan. Tel.: þ81 45 345 5165; fax: þ81 45 973 6781. E-mail addresses: [email protected], [email protected] (A. Nagai). x Current address: Product Science Division, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan.
15% of flowering plants, tobacco is classified as a non-fructanstoring plant due to its lack of ability to accumulate fructan (9). In fact, up to present, there is no report on fructosyltransferase purified from tobacco plant, and some literature has actually reported on the absence of FOSs in fresh tobacco leaf without transformation (10,11). For this reason, FOSs formation in cured tobacco leaf has been conjectured to be the cause of a side reaction by invertase (b-fructofuranosidase, EC 3.2.1.26) rather than by fructosyltransferase as typified by sucrose: sucrose 1-fructosyltransferase (1-SST: EC 2.4.1.99). Invertases functionally catalyze the hydrolytic cleave of the terminal non-reducing b-D-fructofuranoside residue in molecules. On the other hand, an additional property, which catalyzes transfructosylation reactions resulting in the formation of kestose-type trisaccharides, has also been reported (12e15). Invertases are widely distributed in various organisms including bacteria, fungi and plants, and are ubiquitously present in higher plants (16e18). Concerning tobacco, acid invertase from cultured tobacco cells has been reported (19). Although many studies of plant invertases have been reported, little attention has been given to the effect of its remaining activity in plant materials that have lost their biological activity (20). Commercially tobacco crop was generally subjected to various processes such as curing, threshing and re-drying after harvest. Nevertheless, remaining enzyme activity relating to the formation of FOSs was clearly observed in the cured tobacco leaf, and the activity exerted influence toward sugar compositions in cured tobacco leaf (6). In this paper, we will focus on enzyme relating to the formation of FOSs in cured tobacco leaf.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.033
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columns were connected, GE Healthcare UK Ltd.) equilibrated with buffer D. After loading, the bound protein was washed with 50 mL of buffer D and then eluted with 100 mL of buffer E: 20 mM Bis-Tris, pH 6.0, containing 500 mM NaCl, 1 M a-D-methylglucopyranoside. The flow rate for column loading, washing and elution was 1 mL/min. For further purification or enzyme assay, the eluted solution was carefully replaced several times with another buffer by ultrafiltration. Gel filtration chromatography Before performing gel filtration chromatography, samples were concentrated to volumes less than 500 mL by use of Amicon Ultra centrifugal tubes (30,000-Da cutoff filter, Millipore Co.). The concentrate was loaded to a Superdex 200 HR16/60 gel filtration column (GE Healthcare UK Ltd.), which had been equilibrated with buffer E: 20 mM Bis-Tris, pH 6.0, containing 100 mM NaCl. The void volume was 42 mL. The flow rate for sample loading and for elution was 0.5 mL/min. The fraction size was 2e5 mL. This procedure was performed using an ÄCTA design FPLC equipped with Frac-950 fraction collector (GE Healthcare UK Ltd.).
FIG. 1. Structures of fructooligosaccharides identified in cured tobacco leaves.
MATERIALS AND METHODS Materials Cured tobacco leaf (growing district: Brazil; leaf type: air-cured leaf) was stored in a warehouse of Japan Tobacco Inc. Cured tobacco leaf was pulverized into 0.5e1.0 mm mesh powder using a mill (Nara Machinery Co., Ltd., Tokyo, Japan). The tobacco material contained approximately 10% moisture (wet basis). Moisture measurement was performed by the following procedure. Approximately 1 g of pulverized tobacco powder was weighed, and then dried using a rotary oven (Tsukasa Co., Ltd., Tokyo, Japan) at 80 C for 3 h. After drying, the sample was cooled to room temperature in a desiccator for 1 h and then weighed again. The reduction in weight was taken to be the moisture content. Small-scale preparation for crude enzyme solution from cured tobacco leaf Pulverized tobacco powder (2 g) were placed in a vial, extracted with 100 mL of iced buffer: 4.8 mM citric acide10.2 mM disodium phosphate buffer [15 mM McIlvaine buffer], pH 5.4. The sample was homogenized and extracted by ultrasonication using a Bransonic cleaner (Branson Ultrasonic Co., Danbury, CT, USA), maintaining the temperature below 4 C for 30 min. The extract was filtered using a paper filter (Whatman no. 60) and a cellulose acetate membrane filter with pore size of 0.20 mm (Whatman, GE Healthcare UK Ltd., Buckinghamshire, UK). Sixty milliliters of filtrate was taken and subjected to ultrafiltration by use of Amicon Ultra centrifugal tubes (30,000-Da cutoff filter, Millipore Co., MA, USA). The concentrate was washed 3 times, collected and finally adjusted to 12 mL with extract buffer. This solution is defined as crude enzyme solution (CES). Preparation for crude extract from cured tobacco leaf One kilogram of pulverized tobacco powder was taken in 5 L of ice-cold buffer A: 15 mM McIlvaine buffer, pH 5.4, containing 0.1% polyoxyethylene (10) octylphenyl ether (Triton X-100), and was stirred while maintaining the temperature below 4 C for 1 h. The heterogeneous extract was filtrated with two layers of Miracloth (Merck Ltd., Darmstadt, Germany) and centrifuged at 4800 g for 30 min. The brown supernatant was collected and defined as crude extract. Unless otherwise noted, all steps including the following further purification were performed at 0e4 C. Activated charcoal treatment Activated charcoal (12 w/v%, acid washed with phosphoric and sulfuric acids grade, SigmaeAldrich Co., MO, USA) was added to the crude extract and vigorously stirred for 5 min. Treated black solution was filtrated with diatomaceous earth (non-washed, SiO2 approximately 90% grade, SigmaeAldrich Co.). The diatomaceous earth was washed several times with buffer A before use. The light brown filtrate was passed through a Nalgene polyethersulfone membrane with pore size of 0.45 mm (Nalge Nunc International Co., NY, USA). The homogeneous enzyme solution was then ultrafiltrated using 30,000-Da cutoff filters (Millipore Co.). At the end of ultrafiltration, the buffer solvent was replaced with buffer B: 20 mM Bis-Tris, pH 6.0, containing 0.1% Triton X-100. Finally, dark brown enzyme solution was collected and adjusted to 50 mL. Q sepharose anion-exchange chromatography 50 mL of the dark brown enzyme solution was passed through a Whatman cellulose acetate membrane filter with pore size of 0.20 mm (GE Healthcare UK Ltd.). The filtrate was loaded on a column (resins: Q sepharose fast flow, GE Healthcare) equilibrated with binding buffer (buffer B). The prepared column volume was 100 mL. The bound protein was washed with 300 mL of binding buffer and then eluted in 200 mL of buffer C: 20 mM Bis-Tris, pH 6.0, containing 0.1% Triton X-100, 300 mM NaCl. The flow rate for column loading, washing, and elution was 5 mL/min. For the next step of purification, the eluted buffer solution was concentrated using 30,000-Da cutoff filters (Millipore Co.), and its solvent was replaced with buffer D: 20 mM Bis-Tris, pH 6.0, containing 500 mM NaCl, 1 mM MnCl2, and 1 mM CaCl2. Finally, light brown enzyme solution was collected and adjusted to 50 mL. Concanavalin A sepharose affinity chromatography The light brown enzyme solution, which had been fractionated by anion-exchange chromatography, was applied to Concanavalin A Sepharose 4B Fast Flow (5 mL of the two
Gel filtration chromatography of crude enzyme from fresh leaf tobacco Fresh leaf tobacco (Nicotiana tabacum) was cultivated and harvested from a field managed by Japan Tobacco Inc. Veins of leaves were removed. After this, all procedures were carried out at 0e4 C. 350 g of fresh tissue was homogenized in 700 mL iced buffer A. The homogenate was filtered through two layers of Miracloth (Merck Ltd., Germany). The filtrate was slowly poured into 3 L of iced acetone at 30 C. The mixed solution was centrifuged at 6000 g for 15 min. The precipitate was collected and dried under reduced pressure. 19 g of white powder was obtained. 4 g of the powder was placed in a vial, and extracted with 80 mL of iced buffer E. The extract was centrifuged at 4800 g for 30 min. 40 mL of the supernatant was taken and subjected to ultrafiltration by use of 30,000 Da cutoff filters. The concentrate was adjusted to 2.0 mL with buffer E. The insoluble matter was filtered with a cellulose acetate membrane filter (pore size of 0.20 mm, GE Healthcare UK Ltd.). 1.0 mL of the solution was loaded on GFC. The conditions of GFC were same as it described previously (see Gel filtration chromatography). Assay of enzyme activity The reaction mixture containing 50 mM McIlvaine buffer (pH 5.0), 100 mM sucrose and appropriately diluted-enzyme solution was incubated for 15 min at 50 C. The reaction was stopped by addition of 1 M Na2CO3 (quenched solution: approximately pH 10). In the case of measurement of transfructosylation activity, quenching was performed by rapid cooling and ultrafiltration using Amicon Ultra microcentrifugal tubes (30,000-Da cutoff filter, Millipore Co.) at 4 C. Invertase activity was basically determined by measuring the amount of glucose liberated. In the test of substrate specificity using other substrates such as raffinose and stachyose, hydrolytic activity was determined by measuring the amount of free fructose. The concentrations of glucose and fructose were determined using F-kit Glucose (Hexokinase method) or F-kit Glucose/Fructose (enzyme-coupling method) according to the manufacturer’s instructions (Roche Applied Science, Basel, Switzerland). The transfructosylation activity was determined by measuring the amount of 1kestose formed. Quantification of 1-kestose was performed by the use of an Agilent 1200 HPLC system equipped with a 6410 Triple Quadrupole MSD (LC/MS/MS, Agilent Technology, CA, USA). One nkatal is defined as the amount of enzyme that can catalyzes the formation of 1 nmol of products per second. With the characterization of purified enzyme, corresponding parameters were changed (i.e., pH, constituents of buffer, temperature, concentration of substrate and so on). In the case of studying the effect of metal ions on invertases, assays were carried out using acetate buffer instead of McIlvaine buffer to avoid a chelating effect by citrate as a buffer constituent. LC/MS/MS conditions LC/MS/MS system was used for the analyses of FOSs formed in the test of enzyme assays. A simplified method of our previous work was used (6). Briefly, chromatographic separations were carried out using a Develosil RP Aqueous column (150 1.5 mm I.D., Nomura Chemical Co., Ltd., Japan) under the following conditions: isocratic elution (5 mM ammonium acetate); flow rate 0.1 mL/min; run time 30 min; column temperature 30 C; injection volume 2 mL. Ionization using ESI/MS was performed under the following conditions: negative ion mode; capillary voltage 4000 V; drying gas temperature 350 C; gas flow 11 L/min; nebulizer pressure 35 psi. Product ion chromatography (PIC) for profiling the analyte, and multiple reaction monitoring (MRM) for quantification of 1-kestose, were achieved under the following conditions: fragmentor voltage 140 V; collision energy 10e20 V; precursor ions m/z 503 (RT 0e10 min), m/z 665 (RT 10e25 min); scan range on PIC m/z 100e700; scan time on PIC 500 ms; product ions on MRM m/z 221, 323, 485; dwell time on MRM 200 ms. Determination of protein concentration Protein concentration was determined by Bradford’s method (21). BSA was used as a standard protein. Poly-acrylamide gel electrophoresis and staining Electrophoresis for purified invertase was carried out in 7.5% gel by the method of Blue-native polyacrylamide gel electrophoresis (BN-PAGE) (22). Protein on BN-PAGE was visualized with Coomassie Brilliant Blue (CBB) or by Periodic Acid-Schiff (PAS) method (23).
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TABLE 1. Purification of invertases from cured tobacco leaf. Purification step
Crude extract Activated charcoal treatmentc Q sepharose Con A sepharose IV1 Superdex 200d Superdex 200 IV2 Superdex 200 IV3 a b c d
Activitya (nkatb)
11175 3858 2267 1204 57 319 172
Protein (mg)
3258 195 71.3 10.3 0.04 1.48 1.82
Specific activity (nkat/mg protein) 3.43 19.8 31.8 117 1420 215 94.4
Yield (%)
35 20 11 0.5 2.9 1.5
Purification (fold)
5.8 9.3 34 414 63 28
Defined as catalyzing the formation of glucose. The amount of enzyme catalyzing the formation of 1 nmol glucose per second. Includes ultrafiltration. IV1 was purified by re-loading twice.
FIG. 2. Gel filtration chromatography (GFC) of enzyme solution prepared from tobacco leaves. (A) The first chromatogram of enzyme solution from cured tobacco leaf after other purification steps. (B) The second chromatogram by re-loading sample purified by the first GFC. (C) GFC of enzyme solution which were prepared from fresh leaf tobacco.
Confirmation of enzyme activity in CES We first confirmed the enzymatic reactive properties of CES using sucrose as substrate. The result confirmed that CES generated both hydrolysate (glucose) and condensate (1-kestose) products. Moreover, the rate of product formation showed pH dependence and temperature dependence. Hydrolysate was most efficiently produced at pH 5.0, 55 C, while condensate was most efficiently produced at pH 5.7, 55 C. These results at least indicated that tobacco extract contained one or more active enzymes relating to the formation of glucose and 1kestose. Here, on the hypothesis that invertases were related to the formation of FOSs in cured tobacco leaves, we attempted purification of the invertases. Purification of invertases Cured tobacco leaf commonly contains huge amounts of soluble components. Brown pigment is a typical contaminant whose physiochemical property is close to that of protein. Melanoidin is known to be one of the brown pigments in processed food, and has high molecular mass and electrical charge on its polymer surface (24). In some cases, proteins and peptides may be constituents of melanoidin (25). As we had expected, common initial purification methods such as ammonium sulfate precipitation and organic solvent precipitation were not effective in the removal of brown pigments. However, activated charcoal treatment effectively separated a large part of brown pigment from invertase activity in crude extract. Active proteins in treated solution were concentrated by ultrafiltration using a 30,000-Da cutoff filter to also serve in the removal of low molecular contaminants. Concentrates were then partially purified through Q sepharose anion-exchange chromatography and Concanavalin A Sepharose affinity chromatography. These steps efficiently separated the active invertases from brown pigments and raised specific activity (Table 1). After affinity chromatography, the solution gave a faint yellow color. Partially purified soluble acid invertases were further separated by gel filtration chromatography (GFC). Fig. 2A shows the result of the first loading on GFC. After finishing the first GFC, the forward active fraction shown as IV1 þ IV2 was collected, and concentrated by ultrafiltration. The concentrate was re-loaded on GFC (Fig. 2B). As a result, GFC showed three peaks of invertase activities. The first active fraction in Fig. 2B was defined as IV1. The purification level of IV1 was 414-fold toward the crude extract. The other two active fractions were also evaluated. The
FIG. 3. BN-PAGE of purified IV1. The staining methods and the amounts of protein loaded in each lane are: (A) CBB, 3 mg; (B) CBB, 15 mg; (C) PAS, 3 mg.
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TABLE 2. Characterization of IV1 from cured tobacco leaf. Parameters Optimum pH Optimum temp. ( C) Inhibition rate (%) 200 mM MnCl2 10 mM CuCl2 10 mM ZnCl2 10 mM HgCl2 10 mM pyridoxal
Glucose formation 5.0 60 30.0 60.6 29.4 73.7 18.3
1-Kestose formation 6.0 60
1.0 0.4 1.2 3.1 1.0
61.7 53.8 34.7 40.8 31.3
1.8 1.0 0.2 0.3 0.7
second and the third peaks, which were respectively defined as IV2 and IV3, were partially purified up to the levels of 63 and 28-fold. According to the elution volume of IV1 on GFC, IV1 had a huge molecular mass of about relative molecular mass (Mr) 590 k. IV2 and IV3 showed Mr 300 k and 70 k, respectively (Fig. S1). Plant invertases generally have molecular masses from 50 k to 70 k (26). Concerning tobacco, acid invertase purified from cultured tobacco cells (N. tabacum BY-2 cells) has molecular mass of about 70 k (19). The molecular mass of a cell-wall invertase coded in a tobacco cDNA has been estimated 61 k (27). As compared with these reports, IV1 and IV2 were huge. In addition, GFC for crude enzymes prepared from fresh leaf tobacco indicated the absence of IV1 and IV2 (shown in Fig. 2C). SDS-PAGE in reducing condition of purified IV1 resulted in smeared shape. On the other hand, Protein band were observed on BN-PAGE (shown in Fig. 3). From these facts, we expected that IV1 and IV2 were in a kind of condensed state, with invertase, other proteins, polysaccharides and melanoidin and so on. General property of IV1 The general properties of purified invertases, mainly IV1, were investigated. The results of characterization of IV1 are shown in Table 2. IV1 clearly comprised acid invertases, having optimum pH 5.0, approximately 60% relative activity at pH 3.5, and less than 30% at pH 8.0 (Fig. S2). Concerning transfructosylation, optimum pH was different from the case of hydrolysis. Both hydrolysis and transfructosylation reactions were most accelerated at 60 C. It is reported that acid invertase activity is often inhibited by heavy metal ions typified by Hg2þ, suggesting the participation of sulfhydryl group at the catalytic site (28). Pyridoxal is also well-known as a typical inhibitor against invertase activity (14,29,30). In this study, IV1 was similarly inhibited by Cu2þ, Zn2þ, Hg2þ, and pyridoxal. Mn2þ showed a slight inhibitory effect. Co2þ, Ni2þ, and alkaline-earth metals such as Mg2þ and Ca2þ, did not inhibit IV1 activity. While the inhibition ratio was often different between hydrolysis and transfructosylation reactions, both reaction rates decreased simultaneously in all cases. Substrate specificity is summarized in Table 3. IV1 cleaved raffinose, stachyose, and 1-kestose with high efficiencies of over 70% when compared with the case of sucrose.
FIG. 4. Heat-stability of IV1. Buffer solution containing IV1 was heated at each temperature in a heat block for 300e1800 s, before enzyme activity assays. The assays were carried out by the method described in Materials and methods.
IV1 hardly worked on melezitose, maltose or trehalose. IV1 recognizes substrates that include b-fructosyl residues. A series of results suggested that IV1 was certainly acid invertase. Heat stability of IV1 was investigated as part of verification of remaining activity in cured tobacco leaves. Buffer solution containing IV1 was heated at several temperatures in heat blocks for 300e1800 s. Subsequently, invertase assays were carried out. Fig. 4 shows heat stability of IV1, that is, changes of activity with time. As the results, IV1 retained over 70% of its activity during heat-treatment for 15 min at 55 C, and was deactivated at an accelerating rate over 60 C. Transfructosylation activity Concerning purified invertase (IV1), we investigated its transfructosylation activity to explore the possibility that the invertase could be responsible for the formation of fructooligosaccharides in our previous study. Fig. 5 shows product ion chromatograms of reacted solution with 100 mM sucrose using IV1. It was clarified that the peak intensity of 1-kestose increased with time.
TABLE 3. Substrate specificities of IV1. Substratea
Sucrose Raffinose Stachyose 1-Kesotse Melezitose Maltose Trehalose a b c d e
Activity [nkat/mL] (relative activity, %b) [Fru]c
[Glc]c
1.21 (90) 0.98 (73) 1.03 (76) 0.97 (72) N.D.d e e
1.35 (100) N.D. N.D. Tracee N.D. N.D. Trace
100 mM substrates were used in each test. The rate of glucose formation using sucrose as a substrate is defined as 100%. Fructose and glucose were quantified using F-kit (see Materials and methods). N.D. means less than 0.03 nkat/mL. Trace means less than 0.07 nkat/mL.
FIG. 5. Product ion chromatography (PIC) and multiple reaction monitoring (MRM) performed by LC/MS/MS in negative-ion mode. The precursor ions on PICs were m/z 503 and 665. (A) PIC of standard chemicals, (B) PIC of enzymatic reaction solution, (C, D) MRM chromatograms of product ions of m/z 323 (C) and 221 (D) from those precursor ions of m/z 503.
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J. BIOSCI. BIOENG., structures are interesting. These results have suggested that the remaining enzyme in dried leaves may act unlike that in the living plant. Highly processed food material should be managed the remained enzyme activities even if the processed material has little moisture about 10%. This type of knowledge is indispensable from the view-point of enzymology not only for regulation techniques of tobacco components, but also for the advancement of food science. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.05.033. ACKNOWLEDGMENTS
FIG. 6. Reaction schemes of sucrose.
In this time, other minor peaks that were probably considered isomers of 1-kestose were simultaneously increased. However, nistose, whose degree of polymerization was four, was not detected. Although the other active fractions on GFC (IV2 and IV3) similarly showed transfructosylation activity, they did not produce nistose, either. An enzyme that contributes to the formation of nistose in cured tobacco leaves may be discriminated from purified invertases (IV1, IV2, and IV3). Subsequently, with regard to IV1, 1-kestose production rate was compared with glucose production rate. Glucose is thought to be inevitably produced with any reaction that includes hydrolysis or transfructosylation as shown in Fig. 6 (31). Fig. 7 shows the relationship between sucrose concentration and the production rate of glucose and 1-kestose. The rate of glucose production was saturated at lower concentration of sucrose than 1kestose production rate. The ratio of 1-kestose production toward all reactions increased over 13% at 300 mM sucrose. Sucrose could be concentrated in cured tobacco leaves containing little moisture (approximately 10%DB). Although there are still unclear portions such as delocalization of constituents associated with cell destruction during the curing process, the difference in sucrose concentration is possibly one reason for specific metabolism in cured tobacco leaves. That is, FOSs formation observed in previous work would be explainable by remaining invertase activity and low moisture environment. In this paper, we demonstrated purification of active invertase from cured tobacco leaf, and reported on its character focusing on transfructosylation activity and general properties. At least, three active fractions on GFC were confirmed, and all of them have had transfructosylation activity. It was also clarified that IV1 showed transfructosylation activity more notably with high-sucrose concentration. Furthermore, the origin of IV1 and its detailed
FIG. 7. The change in sucrose concentrations and the ratio of transfructosylation activity by IV1. Open circles indicate rates of 1-kestose formation. Closed circles indicate rates of glucose formation.
Special appreciation is extended to all at Plant Innovation Center. Especially the authors would like to thank Ichiro Oka and Keiji Suyama for various support including tobacco cultivation. We also thank Mari Kawase for providing technical assistance. References 1. Tso, T. C.: Seed to smoke, pp. 1e31, in: Davis, D. L. and Nielsen, M. T. (Eds.), Tobacco: production chemistry and technology. Blackwell Science, Oxford, UK (1999). 2. Leffingwell, J. C.: Leaf chemistry: basic chemical constituents of tobacco leaf and differences among tobacco types, pp. 265e284, in: Davis, D. L. and Nielsen, M. T. (Eds.), Tobacco: production chemistry and technology. Blackwell Science, Oxford, UK (1999). 3. Weeks, W. W.: Leaf chemistry: relationship between leaf chemistry and organoleptic properties of tobacco smoke, pp. 304e312, in: Davis, D. L. and Nielsen, M. T. (Eds.), Tobacco: production, chemistry and technology. Blackwell Science, Oxford, UK (1999). 4. Baker, R. R., Pereira da Silva, J. R., and Smith, G.: The effect of tobacco ingredients on smoke chemistry. Part I: flavourings and additives, Food Chem. Toxicol., 42S, S3eS37 (2004). 5. Baker, R. R., Pereira da Silva, J. R., and Smith, G.: The effect of tobacco ingredients on smoke chemistry. Part II: casing ingredients, Food Chem. Toxicol., 42S, S39eS52 (2004). 6. Nagai, A., Yamamoto, T., and Wariishi, H.: Identification of fructo- and maltooligosaccharides in cured tobacco leaves (Nicotiana tabacum), J. Agric. Food Chem., 60, 6606e6612 (2012). 7. Biggs, D. R. and Hancock, K. R.: Fructan 2000, Trends Plant Sci., 6, 8e9 (2001). 8. Banguela, A. and Hernández, L.: Fructans: from natural sources to transgenic plants, Biotechnol. Appl., 23, 202e210 (2006). 9. Hendry, G. A. F.: Evolutionary origins and natural functions of fructans e a climatological, biogeographic and mechanistic appraisal, New Phytol., 123, 3e14 (1993). 10. Pilon-Smits, E. A. H., Ebskamp, M. J. M., Paul, M. J., Jeuken, M. J. W., Weisbeek, P. J., and Smeekens, S. C. M.: Improved performance of transgenic fructan-accumulating tobacco under drought stress, Plant Physiol., 107, 125e130 (1995). 11. Sprenger, N., Schellenbaum, L., Dun, K. V., Boller, T., and Wiemken, A.: Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose: fructan 6-fructosyltransferase, FEBS Lett., 400, 355e358 (1997). 12. Cairns, A. J. and Ashton, J. E.: The interpretation of in vitro measurements of fructosyl transferase activity: an analysis of patterns of fructosyl transfer by fungal invertase, New Phytol., 118, 23e34 (1991). 13. Ende, W. V. D. and Laere, A. V.: Purification and properties of an invertase with sucrose: sucrose fructosyltransferase (SST) activity from the roots of Cichorium intybus L, New Phytol., 12, 31e37 (1993). 14. Obenland, D. M., Simmen, U., Boller, T., and Wiemken, A.: Purification and characterization of three invertases from barley (Hordeum vulgare L.) leaves, Plant Physiol., 101, 1331e1339 (1993). 15. Agopian, R. G. D., Purgatto, E., Cordenunsi, B. R., and Lajolo, F. M.: Synthesis of fructooligosaccharides in banana ‘Prata’ ana its relation to invertase activity and sucrose accumulation, J. Agric. Food Chem., 57, 10765e10771 (2009). 16. Ishimoto, M. and Nakamura, A.: Purification and properties of b-fructofuranosidase from Clostridium perfringens, Biosci. Biotechnol. Biochem., 61, 599e603 (1997). 17. Belcarz, A., Ginalska, G., Lobarzewski, J., and Panel, C.: The novel non-glycosylated invertase from Candida utilis (the properties and the conditions of production and purification), Biochim. Biophys. Acta, 1594, 40e53 (2002). 18. Lee, H. S. and Sturm, A.: Purification and characterization of neutral and alkaline invertase from carrot, Plant Physiol., 112, 1513e1533 (1996). 19. Nakamura, M., Hagimori, M., and Matsumoto, T.: Purification and characterization of acid invertase from cultured tobacco cells, Agric. Biol. Chem., 52, 3157e3158 (1988). 20. Nakanishi, K. and Yokotsuka, K.: Characterization of thermostable invertase from wine grapes, J. Ferment. Bioeng., 69, 16e22 (1990).
VOL. 116, 2013 21. Bradford, M. M.: A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 22. Schägger, H. and Jagow, G. V.: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem., 199, 223e231 (1991). 23. Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J.: Glycoprotein staining following electrophoresis on acrylamide gels, Anal. Biochem., 30, 148e152 (1969). 24. Homma, S., Terasawa, N., Kubo, T., Yoneyama-Ishii, N., Aida, K., and Fujimaki, M.: Changes in chemical properties of melanoidin by oxidation and reduction, Biosci. Biotechnol. Biochem., 61, 533e535 (1997). 25. Smaniotto, A., Bertazzo, B., Comai, S., and Traldi, P.: The role of peptides and proteins in melanoidin formation, J. Mass. Spectrom., 44, 410e418 (2009).
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26. Fotopoulos, V.: Plant invertases: structure, function and regulation of a diverse enzyme family, J. Biol. Res., 4, 127e137 (2005). 27. Greiner, S., Weil, M., Krausgrill, S., and Rausch, T.: A tobacco cDNA coding for cell-wall invertase, Plant Physiol., 108, 825e826 (1995). 28. Sturm, A.: Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning, Plant Physiol., 121, 1e7 (1999). 29. Pressey, R.: Inhibition of invertases by pyridoxal and its analogues, Biochim. Biophys. Acta, 159, 414e416 (1968). 30. Lopez, M. E., Vattuone, M. A., and Sampietro, A. R.: Partial purification and properties of invertase carica papaya fruits, Phytochemistry, 27, 3077e3081 (1988). 31. Edelman, J. and Jefford, T. G.: The mechanism of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus, New Phytol., 67, 517e531 (1968).
Transfructosylation reaction in cured tobacco leaf (Nicotiana tabacum) Atsushi Nagai,1, 3, * Toshiki Mine,2, x Takeshi Yamamoto,2, x and Hiroyuki Wariishi3 Tobacco Science Research Center, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan,1 Plant Innovation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan,2 and Department of Innovative Science and Bioenvironmental Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan3 Received 25 February 2013; accepted 27 May 2013 Available online 8 July 2013
Tobacco plant was known to be a non-fructan-storing plant. However, we demonstrated that fructo-oligosaccharides (FOSs) were formed in cured tobacco leaf on adding sucrose to the leaf in our previous report (Nagai et al., J. Agric. Food Chem., 60, 6606e6612, 2012). Also, it was expected from the results obtained in previous study that FOSs were generated by enzymatic reaction in cured tobacco leaf. The purpose of this study is to confirm and understand the mechanisms of above-mentioned FOSs formation. Thus, we tried to purify the enzymes related to the production of FOSs. The enzymes were extracted from pulverized cured tobacco leaf (burley type leaf), and were purified by charcoal treatment, ultrafiltration, and several chromatography techniques. As a result, one of the enzymes was purified up to 414-fold. It was revealed that this enzyme was acid invertase exhibiting maximum transfructosylation activity at pH 6.0, 60 C. In addition, general properties of this enzyme were also investigated. The enzyme purified in this study enhanced the ratio of FOSs formation under the condition of high concentrated sucrose. From these results, it was suggested that this enzyme participated in the formation of FOSs in tobacco leaf after curing. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cured tobacco; Invertase; Remaining activity; Heat stability; Transfructosylation]
Tobacco leaves obtained from commercial tobacco plant undergo many processes after harvest (1). Curing is the first process, and its purpose is to remove the moisture from tobacco leaves. This process also plays a fundamental role in giving tobacco its flavor and aroma. The dried leaves are called cured tobacco leaves. Sugars are the main components in cured tobacco leaves, and are also key compounds directly influencing the aroma/taste of cigarette smoke (2e5). Therefore, investigating the sugar composition is an important issue in managing tobacco product quality. Recently, we reported on the formation of fructo-oligosaccharides (FOSs) in cured tobacco leaf (6). It was strongly expected that FOSs were formed through enzymatic reaction using sucrose as the substrate in cured leaf with low moisture content. The structures of FOSs identified in cured tobacco leaves are shown in Fig. 1. FOSs are condensates of fructose in which fructose residues are bound by b-linkage with or without a single glucose unit in each molecule. Polymerized FOSs, which is called fructan, is known as one of the reserve carbohydrates and has been found in plants, algae, and bacteria (7,8). While fructan is found in approximately
* Corresponding author at: Tobacco Science Research Center, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan. Tel.: þ81 45 345 5165; fax: þ81 45 973 6781. E-mail addresses: [email protected], [email protected] (A. Nagai). x Current address: Product Science Division, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan.
15% of flowering plants, tobacco is classified as a non-fructanstoring plant due to its lack of ability to accumulate fructan (9). In fact, up to present, there is no report on fructosyltransferase purified from tobacco plant, and some literature has actually reported on the absence of FOSs in fresh tobacco leaf without transformation (10,11). For this reason, FOSs formation in cured tobacco leaf has been conjectured to be the cause of a side reaction by invertase (b-fructofuranosidase, EC 3.2.1.26) rather than by fructosyltransferase as typified by sucrose: sucrose 1-fructosyltransferase (1-SST: EC 2.4.1.99). Invertases functionally catalyze the hydrolytic cleave of the terminal non-reducing b-D-fructofuranoside residue in molecules. On the other hand, an additional property, which catalyzes transfructosylation reactions resulting in the formation of kestose-type trisaccharides, has also been reported (12e15). Invertases are widely distributed in various organisms including bacteria, fungi and plants, and are ubiquitously present in higher plants (16e18). Concerning tobacco, acid invertase from cultured tobacco cells has been reported (19). Although many studies of plant invertases have been reported, little attention has been given to the effect of its remaining activity in plant materials that have lost their biological activity (20). Commercially tobacco crop was generally subjected to various processes such as curing, threshing and re-drying after harvest. Nevertheless, remaining enzyme activity relating to the formation of FOSs was clearly observed in the cured tobacco leaf, and the activity exerted influence toward sugar compositions in cured tobacco leaf (6). In this paper, we will focus on enzyme relating to the formation of FOSs in cured tobacco leaf.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.033
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columns were connected, GE Healthcare UK Ltd.) equilibrated with buffer D. After loading, the bound protein was washed with 50 mL of buffer D and then eluted with 100 mL of buffer E: 20 mM Bis-Tris, pH 6.0, containing 500 mM NaCl, 1 M a-D-methylglucopyranoside. The flow rate for column loading, washing and elution was 1 mL/min. For further purification or enzyme assay, the eluted solution was carefully replaced several times with another buffer by ultrafiltration. Gel filtration chromatography Before performing gel filtration chromatography, samples were concentrated to volumes less than 500 mL by use of Amicon Ultra centrifugal tubes (30,000-Da cutoff filter, Millipore Co.). The concentrate was loaded to a Superdex 200 HR16/60 gel filtration column (GE Healthcare UK Ltd.), which had been equilibrated with buffer E: 20 mM Bis-Tris, pH 6.0, containing 100 mM NaCl. The void volume was 42 mL. The flow rate for sample loading and for elution was 0.5 mL/min. The fraction size was 2e5 mL. This procedure was performed using an ÄCTA design FPLC equipped with Frac-950 fraction collector (GE Healthcare UK Ltd.).
FIG. 1. Structures of fructooligosaccharides identified in cured tobacco leaves.
MATERIALS AND METHODS Materials Cured tobacco leaf (growing district: Brazil; leaf type: air-cured leaf) was stored in a warehouse of Japan Tobacco Inc. Cured tobacco leaf was pulverized into 0.5e1.0 mm mesh powder using a mill (Nara Machinery Co., Ltd., Tokyo, Japan). The tobacco material contained approximately 10% moisture (wet basis). Moisture measurement was performed by the following procedure. Approximately 1 g of pulverized tobacco powder was weighed, and then dried using a rotary oven (Tsukasa Co., Ltd., Tokyo, Japan) at 80 C for 3 h. After drying, the sample was cooled to room temperature in a desiccator for 1 h and then weighed again. The reduction in weight was taken to be the moisture content. Small-scale preparation for crude enzyme solution from cured tobacco leaf Pulverized tobacco powder (2 g) were placed in a vial, extracted with 100 mL of iced buffer: 4.8 mM citric acide10.2 mM disodium phosphate buffer [15 mM McIlvaine buffer], pH 5.4. The sample was homogenized and extracted by ultrasonication using a Bransonic cleaner (Branson Ultrasonic Co., Danbury, CT, USA), maintaining the temperature below 4 C for 30 min. The extract was filtered using a paper filter (Whatman no. 60) and a cellulose acetate membrane filter with pore size of 0.20 mm (Whatman, GE Healthcare UK Ltd., Buckinghamshire, UK). Sixty milliliters of filtrate was taken and subjected to ultrafiltration by use of Amicon Ultra centrifugal tubes (30,000-Da cutoff filter, Millipore Co., MA, USA). The concentrate was washed 3 times, collected and finally adjusted to 12 mL with extract buffer. This solution is defined as crude enzyme solution (CES). Preparation for crude extract from cured tobacco leaf One kilogram of pulverized tobacco powder was taken in 5 L of ice-cold buffer A: 15 mM McIlvaine buffer, pH 5.4, containing 0.1% polyoxyethylene (10) octylphenyl ether (Triton X-100), and was stirred while maintaining the temperature below 4 C for 1 h. The heterogeneous extract was filtrated with two layers of Miracloth (Merck Ltd., Darmstadt, Germany) and centrifuged at 4800 g for 30 min. The brown supernatant was collected and defined as crude extract. Unless otherwise noted, all steps including the following further purification were performed at 0e4 C. Activated charcoal treatment Activated charcoal (12 w/v%, acid washed with phosphoric and sulfuric acids grade, SigmaeAldrich Co., MO, USA) was added to the crude extract and vigorously stirred for 5 min. Treated black solution was filtrated with diatomaceous earth (non-washed, SiO2 approximately 90% grade, SigmaeAldrich Co.). The diatomaceous earth was washed several times with buffer A before use. The light brown filtrate was passed through a Nalgene polyethersulfone membrane with pore size of 0.45 mm (Nalge Nunc International Co., NY, USA). The homogeneous enzyme solution was then ultrafiltrated using 30,000-Da cutoff filters (Millipore Co.). At the end of ultrafiltration, the buffer solvent was replaced with buffer B: 20 mM Bis-Tris, pH 6.0, containing 0.1% Triton X-100. Finally, dark brown enzyme solution was collected and adjusted to 50 mL. Q sepharose anion-exchange chromatography 50 mL of the dark brown enzyme solution was passed through a Whatman cellulose acetate membrane filter with pore size of 0.20 mm (GE Healthcare UK Ltd.). The filtrate was loaded on a column (resins: Q sepharose fast flow, GE Healthcare) equilibrated with binding buffer (buffer B). The prepared column volume was 100 mL. The bound protein was washed with 300 mL of binding buffer and then eluted in 200 mL of buffer C: 20 mM Bis-Tris, pH 6.0, containing 0.1% Triton X-100, 300 mM NaCl. The flow rate for column loading, washing, and elution was 5 mL/min. For the next step of purification, the eluted buffer solution was concentrated using 30,000-Da cutoff filters (Millipore Co.), and its solvent was replaced with buffer D: 20 mM Bis-Tris, pH 6.0, containing 500 mM NaCl, 1 mM MnCl2, and 1 mM CaCl2. Finally, light brown enzyme solution was collected and adjusted to 50 mL. Concanavalin A sepharose affinity chromatography The light brown enzyme solution, which had been fractionated by anion-exchange chromatography, was applied to Concanavalin A Sepharose 4B Fast Flow (5 mL of the two
Gel filtration chromatography of crude enzyme from fresh leaf tobacco Fresh leaf tobacco (Nicotiana tabacum) was cultivated and harvested from a field managed by Japan Tobacco Inc. Veins of leaves were removed. After this, all procedures were carried out at 0e4 C. 350 g of fresh tissue was homogenized in 700 mL iced buffer A. The homogenate was filtered through two layers of Miracloth (Merck Ltd., Germany). The filtrate was slowly poured into 3 L of iced acetone at 30 C. The mixed solution was centrifuged at 6000 g for 15 min. The precipitate was collected and dried under reduced pressure. 19 g of white powder was obtained. 4 g of the powder was placed in a vial, and extracted with 80 mL of iced buffer E. The extract was centrifuged at 4800 g for 30 min. 40 mL of the supernatant was taken and subjected to ultrafiltration by use of 30,000 Da cutoff filters. The concentrate was adjusted to 2.0 mL with buffer E. The insoluble matter was filtered with a cellulose acetate membrane filter (pore size of 0.20 mm, GE Healthcare UK Ltd.). 1.0 mL of the solution was loaded on GFC. The conditions of GFC were same as it described previously (see Gel filtration chromatography). Assay of enzyme activity The reaction mixture containing 50 mM McIlvaine buffer (pH 5.0), 100 mM sucrose and appropriately diluted-enzyme solution was incubated for 15 min at 50 C. The reaction was stopped by addition of 1 M Na2CO3 (quenched solution: approximately pH 10). In the case of measurement of transfructosylation activity, quenching was performed by rapid cooling and ultrafiltration using Amicon Ultra microcentrifugal tubes (30,000-Da cutoff filter, Millipore Co.) at 4 C. Invertase activity was basically determined by measuring the amount of glucose liberated. In the test of substrate specificity using other substrates such as raffinose and stachyose, hydrolytic activity was determined by measuring the amount of free fructose. The concentrations of glucose and fructose were determined using F-kit Glucose (Hexokinase method) or F-kit Glucose/Fructose (enzyme-coupling method) according to the manufacturer’s instructions (Roche Applied Science, Basel, Switzerland). The transfructosylation activity was determined by measuring the amount of 1kestose formed. Quantification of 1-kestose was performed by the use of an Agilent 1200 HPLC system equipped with a 6410 Triple Quadrupole MSD (LC/MS/MS, Agilent Technology, CA, USA). One nkatal is defined as the amount of enzyme that can catalyzes the formation of 1 nmol of products per second. With the characterization of purified enzyme, corresponding parameters were changed (i.e., pH, constituents of buffer, temperature, concentration of substrate and so on). In the case of studying the effect of metal ions on invertases, assays were carried out using acetate buffer instead of McIlvaine buffer to avoid a chelating effect by citrate as a buffer constituent. LC/MS/MS conditions LC/MS/MS system was used for the analyses of FOSs formed in the test of enzyme assays. A simplified method of our previous work was used (6). Briefly, chromatographic separations were carried out using a Develosil RP Aqueous column (150 1.5 mm I.D., Nomura Chemical Co., Ltd., Japan) under the following conditions: isocratic elution (5 mM ammonium acetate); flow rate 0.1 mL/min; run time 30 min; column temperature 30 C; injection volume 2 mL. Ionization using ESI/MS was performed under the following conditions: negative ion mode; capillary voltage 4000 V; drying gas temperature 350 C; gas flow 11 L/min; nebulizer pressure 35 psi. Product ion chromatography (PIC) for profiling the analyte, and multiple reaction monitoring (MRM) for quantification of 1-kestose, were achieved under the following conditions: fragmentor voltage 140 V; collision energy 10e20 V; precursor ions m/z 503 (RT 0e10 min), m/z 665 (RT 10e25 min); scan range on PIC m/z 100e700; scan time on PIC 500 ms; product ions on MRM m/z 221, 323, 485; dwell time on MRM 200 ms. Determination of protein concentration Protein concentration was determined by Bradford’s method (21). BSA was used as a standard protein. Poly-acrylamide gel electrophoresis and staining Electrophoresis for purified invertase was carried out in 7.5% gel by the method of Blue-native polyacrylamide gel electrophoresis (BN-PAGE) (22). Protein on BN-PAGE was visualized with Coomassie Brilliant Blue (CBB) or by Periodic Acid-Schiff (PAS) method (23).
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TABLE 1. Purification of invertases from cured tobacco leaf. Purification step
Crude extract Activated charcoal treatmentc Q sepharose Con A sepharose IV1 Superdex 200d Superdex 200 IV2 Superdex 200 IV3 a b c d
Activitya (nkatb)
11175 3858 2267 1204 57 319 172
Protein (mg)
3258 195 71.3 10.3 0.04 1.48 1.82
Specific activity (nkat/mg protein) 3.43 19.8 31.8 117 1420 215 94.4
Yield (%)
35 20 11 0.5 2.9 1.5
Purification (fold)
5.8 9.3 34 414 63 28
Defined as catalyzing the formation of glucose. The amount of enzyme catalyzing the formation of 1 nmol glucose per second. Includes ultrafiltration. IV1 was purified by re-loading twice.
FIG. 2. Gel filtration chromatography (GFC) of enzyme solution prepared from tobacco leaves. (A) The first chromatogram of enzyme solution from cured tobacco leaf after other purification steps. (B) The second chromatogram by re-loading sample purified by the first GFC. (C) GFC of enzyme solution which were prepared from fresh leaf tobacco.
Confirmation of enzyme activity in CES We first confirmed the enzymatic reactive properties of CES using sucrose as substrate. The result confirmed that CES generated both hydrolysate (glucose) and condensate (1-kestose) products. Moreover, the rate of product formation showed pH dependence and temperature dependence. Hydrolysate was most efficiently produced at pH 5.0, 55 C, while condensate was most efficiently produced at pH 5.7, 55 C. These results at least indicated that tobacco extract contained one or more active enzymes relating to the formation of glucose and 1kestose. Here, on the hypothesis that invertases were related to the formation of FOSs in cured tobacco leaves, we attempted purification of the invertases. Purification of invertases Cured tobacco leaf commonly contains huge amounts of soluble components. Brown pigment is a typical contaminant whose physiochemical property is close to that of protein. Melanoidin is known to be one of the brown pigments in processed food, and has high molecular mass and electrical charge on its polymer surface (24). In some cases, proteins and peptides may be constituents of melanoidin (25). As we had expected, common initial purification methods such as ammonium sulfate precipitation and organic solvent precipitation were not effective in the removal of brown pigments. However, activated charcoal treatment effectively separated a large part of brown pigment from invertase activity in crude extract. Active proteins in treated solution were concentrated by ultrafiltration using a 30,000-Da cutoff filter to also serve in the removal of low molecular contaminants. Concentrates were then partially purified through Q sepharose anion-exchange chromatography and Concanavalin A Sepharose affinity chromatography. These steps efficiently separated the active invertases from brown pigments and raised specific activity (Table 1). After affinity chromatography, the solution gave a faint yellow color. Partially purified soluble acid invertases were further separated by gel filtration chromatography (GFC). Fig. 2A shows the result of the first loading on GFC. After finishing the first GFC, the forward active fraction shown as IV1 þ IV2 was collected, and concentrated by ultrafiltration. The concentrate was re-loaded on GFC (Fig. 2B). As a result, GFC showed three peaks of invertase activities. The first active fraction in Fig. 2B was defined as IV1. The purification level of IV1 was 414-fold toward the crude extract. The other two active fractions were also evaluated. The
FIG. 3. BN-PAGE of purified IV1. The staining methods and the amounts of protein loaded in each lane are: (A) CBB, 3 mg; (B) CBB, 15 mg; (C) PAS, 3 mg.
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TABLE 2. Characterization of IV1 from cured tobacco leaf. Parameters Optimum pH Optimum temp. ( C) Inhibition rate (%) 200 mM MnCl2 10 mM CuCl2 10 mM ZnCl2 10 mM HgCl2 10 mM pyridoxal
Glucose formation 5.0 60 30.0 60.6 29.4 73.7 18.3
1-Kestose formation 6.0 60
1.0 0.4 1.2 3.1 1.0
61.7 53.8 34.7 40.8 31.3
1.8 1.0 0.2 0.3 0.7
second and the third peaks, which were respectively defined as IV2 and IV3, were partially purified up to the levels of 63 and 28-fold. According to the elution volume of IV1 on GFC, IV1 had a huge molecular mass of about relative molecular mass (Mr) 590 k. IV2 and IV3 showed Mr 300 k and 70 k, respectively (Fig. S1). Plant invertases generally have molecular masses from 50 k to 70 k (26). Concerning tobacco, acid invertase purified from cultured tobacco cells (N. tabacum BY-2 cells) has molecular mass of about 70 k (19). The molecular mass of a cell-wall invertase coded in a tobacco cDNA has been estimated 61 k (27). As compared with these reports, IV1 and IV2 were huge. In addition, GFC for crude enzymes prepared from fresh leaf tobacco indicated the absence of IV1 and IV2 (shown in Fig. 2C). SDS-PAGE in reducing condition of purified IV1 resulted in smeared shape. On the other hand, Protein band were observed on BN-PAGE (shown in Fig. 3). From these facts, we expected that IV1 and IV2 were in a kind of condensed state, with invertase, other proteins, polysaccharides and melanoidin and so on. General property of IV1 The general properties of purified invertases, mainly IV1, were investigated. The results of characterization of IV1 are shown in Table 2. IV1 clearly comprised acid invertases, having optimum pH 5.0, approximately 60% relative activity at pH 3.5, and less than 30% at pH 8.0 (Fig. S2). Concerning transfructosylation, optimum pH was different from the case of hydrolysis. Both hydrolysis and transfructosylation reactions were most accelerated at 60 C. It is reported that acid invertase activity is often inhibited by heavy metal ions typified by Hg2þ, suggesting the participation of sulfhydryl group at the catalytic site (28). Pyridoxal is also well-known as a typical inhibitor against invertase activity (14,29,30). In this study, IV1 was similarly inhibited by Cu2þ, Zn2þ, Hg2þ, and pyridoxal. Mn2þ showed a slight inhibitory effect. Co2þ, Ni2þ, and alkaline-earth metals such as Mg2þ and Ca2þ, did not inhibit IV1 activity. While the inhibition ratio was often different between hydrolysis and transfructosylation reactions, both reaction rates decreased simultaneously in all cases. Substrate specificity is summarized in Table 3. IV1 cleaved raffinose, stachyose, and 1-kestose with high efficiencies of over 70% when compared with the case of sucrose.
FIG. 4. Heat-stability of IV1. Buffer solution containing IV1 was heated at each temperature in a heat block for 300e1800 s, before enzyme activity assays. The assays were carried out by the method described in Materials and methods.
IV1 hardly worked on melezitose, maltose or trehalose. IV1 recognizes substrates that include b-fructosyl residues. A series of results suggested that IV1 was certainly acid invertase. Heat stability of IV1 was investigated as part of verification of remaining activity in cured tobacco leaves. Buffer solution containing IV1 was heated at several temperatures in heat blocks for 300e1800 s. Subsequently, invertase assays were carried out. Fig. 4 shows heat stability of IV1, that is, changes of activity with time. As the results, IV1 retained over 70% of its activity during heat-treatment for 15 min at 55 C, and was deactivated at an accelerating rate over 60 C. Transfructosylation activity Concerning purified invertase (IV1), we investigated its transfructosylation activity to explore the possibility that the invertase could be responsible for the formation of fructooligosaccharides in our previous study. Fig. 5 shows product ion chromatograms of reacted solution with 100 mM sucrose using IV1. It was clarified that the peak intensity of 1-kestose increased with time.
TABLE 3. Substrate specificities of IV1. Substratea
Sucrose Raffinose Stachyose 1-Kesotse Melezitose Maltose Trehalose a b c d e
Activity [nkat/mL] (relative activity, %b) [Fru]c
[Glc]c
1.21 (90) 0.98 (73) 1.03 (76) 0.97 (72) N.D.d e e
1.35 (100) N.D. N.D. Tracee N.D. N.D. Trace
100 mM substrates were used in each test. The rate of glucose formation using sucrose as a substrate is defined as 100%. Fructose and glucose were quantified using F-kit (see Materials and methods). N.D. means less than 0.03 nkat/mL. Trace means less than 0.07 nkat/mL.
FIG. 5. Product ion chromatography (PIC) and multiple reaction monitoring (MRM) performed by LC/MS/MS in negative-ion mode. The precursor ions on PICs were m/z 503 and 665. (A) PIC of standard chemicals, (B) PIC of enzymatic reaction solution, (C, D) MRM chromatograms of product ions of m/z 323 (C) and 221 (D) from those precursor ions of m/z 503.
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J. BIOSCI. BIOENG., structures are interesting. These results have suggested that the remaining enzyme in dried leaves may act unlike that in the living plant. Highly processed food material should be managed the remained enzyme activities even if the processed material has little moisture about 10%. This type of knowledge is indispensable from the view-point of enzymology not only for regulation techniques of tobacco components, but also for the advancement of food science. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.05.033. ACKNOWLEDGMENTS
FIG. 6. Reaction schemes of sucrose.
In this time, other minor peaks that were probably considered isomers of 1-kestose were simultaneously increased. However, nistose, whose degree of polymerization was four, was not detected. Although the other active fractions on GFC (IV2 and IV3) similarly showed transfructosylation activity, they did not produce nistose, either. An enzyme that contributes to the formation of nistose in cured tobacco leaves may be discriminated from purified invertases (IV1, IV2, and IV3). Subsequently, with regard to IV1, 1-kestose production rate was compared with glucose production rate. Glucose is thought to be inevitably produced with any reaction that includes hydrolysis or transfructosylation as shown in Fig. 6 (31). Fig. 7 shows the relationship between sucrose concentration and the production rate of glucose and 1-kestose. The rate of glucose production was saturated at lower concentration of sucrose than 1kestose production rate. The ratio of 1-kestose production toward all reactions increased over 13% at 300 mM sucrose. Sucrose could be concentrated in cured tobacco leaves containing little moisture (approximately 10%DB). Although there are still unclear portions such as delocalization of constituents associated with cell destruction during the curing process, the difference in sucrose concentration is possibly one reason for specific metabolism in cured tobacco leaves. That is, FOSs formation observed in previous work would be explainable by remaining invertase activity and low moisture environment. In this paper, we demonstrated purification of active invertase from cured tobacco leaf, and reported on its character focusing on transfructosylation activity and general properties. At least, three active fractions on GFC were confirmed, and all of them have had transfructosylation activity. It was also clarified that IV1 showed transfructosylation activity more notably with high-sucrose concentration. Furthermore, the origin of IV1 and its detailed
FIG. 7. The change in sucrose concentrations and the ratio of transfructosylation activity by IV1. Open circles indicate rates of 1-kestose formation. Closed circles indicate rates of glucose formation.
Special appreciation is extended to all at Plant Innovation Center. Especially the authors would like to thank Ichiro Oka and Keiji Suyama for various support including tobacco cultivation. We also thank Mari Kawase for providing technical assistance. References 1. Tso, T. C.: Seed to smoke, pp. 1e31, in: Davis, D. L. and Nielsen, M. T. (Eds.), Tobacco: production chemistry and technology. Blackwell Science, Oxford, UK (1999). 2. Leffingwell, J. C.: Leaf chemistry: basic chemical constituents of tobacco leaf and differences among tobacco types, pp. 265e284, in: Davis, D. L. and Nielsen, M. T. (Eds.), Tobacco: production chemistry and technology. Blackwell Science, Oxford, UK (1999). 3. Weeks, W. W.: Leaf chemistry: relationship between leaf chemistry and organoleptic properties of tobacco smoke, pp. 304e312, in: Davis, D. L. and Nielsen, M. T. (Eds.), Tobacco: production, chemistry and technology. Blackwell Science, Oxford, UK (1999). 4. Baker, R. R., Pereira da Silva, J. R., and Smith, G.: The effect of tobacco ingredients on smoke chemistry. Part I: flavourings and additives, Food Chem. Toxicol., 42S, S3eS37 (2004). 5. Baker, R. R., Pereira da Silva, J. R., and Smith, G.: The effect of tobacco ingredients on smoke chemistry. Part II: casing ingredients, Food Chem. Toxicol., 42S, S39eS52 (2004). 6. Nagai, A., Yamamoto, T., and Wariishi, H.: Identification of fructo- and maltooligosaccharides in cured tobacco leaves (Nicotiana tabacum), J. Agric. Food Chem., 60, 6606e6612 (2012). 7. Biggs, D. R. and Hancock, K. R.: Fructan 2000, Trends Plant Sci., 6, 8e9 (2001). 8. Banguela, A. and Hernández, L.: Fructans: from natural sources to transgenic plants, Biotechnol. Appl., 23, 202e210 (2006). 9. Hendry, G. A. F.: Evolutionary origins and natural functions of fructans e a climatological, biogeographic and mechanistic appraisal, New Phytol., 123, 3e14 (1993). 10. Pilon-Smits, E. A. H., Ebskamp, M. J. M., Paul, M. J., Jeuken, M. J. W., Weisbeek, P. J., and Smeekens, S. C. M.: Improved performance of transgenic fructan-accumulating tobacco under drought stress, Plant Physiol., 107, 125e130 (1995). 11. Sprenger, N., Schellenbaum, L., Dun, K. V., Boller, T., and Wiemken, A.: Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose: fructan 6-fructosyltransferase, FEBS Lett., 400, 355e358 (1997). 12. Cairns, A. J. and Ashton, J. E.: The interpretation of in vitro measurements of fructosyl transferase activity: an analysis of patterns of fructosyl transfer by fungal invertase, New Phytol., 118, 23e34 (1991). 13. Ende, W. V. D. and Laere, A. V.: Purification and properties of an invertase with sucrose: sucrose fructosyltransferase (SST) activity from the roots of Cichorium intybus L, New Phytol., 12, 31e37 (1993). 14. Obenland, D. M., Simmen, U., Boller, T., and Wiemken, A.: Purification and characterization of three invertases from barley (Hordeum vulgare L.) leaves, Plant Physiol., 101, 1331e1339 (1993). 15. Agopian, R. G. D., Purgatto, E., Cordenunsi, B. R., and Lajolo, F. M.: Synthesis of fructooligosaccharides in banana ‘Prata’ ana its relation to invertase activity and sucrose accumulation, J. Agric. Food Chem., 57, 10765e10771 (2009). 16. Ishimoto, M. and Nakamura, A.: Purification and properties of b-fructofuranosidase from Clostridium perfringens, Biosci. Biotechnol. Biochem., 61, 599e603 (1997). 17. Belcarz, A., Ginalska, G., Lobarzewski, J., and Panel, C.: The novel non-glycosylated invertase from Candida utilis (the properties and the conditions of production and purification), Biochim. Biophys. Acta, 1594, 40e53 (2002). 18. Lee, H. S. and Sturm, A.: Purification and characterization of neutral and alkaline invertase from carrot, Plant Physiol., 112, 1513e1533 (1996). 19. Nakamura, M., Hagimori, M., and Matsumoto, T.: Purification and characterization of acid invertase from cultured tobacco cells, Agric. Biol. Chem., 52, 3157e3158 (1988). 20. Nakanishi, K. and Yokotsuka, K.: Characterization of thermostable invertase from wine grapes, J. Ferment. Bioeng., 69, 16e22 (1990).
VOL. 116, 2013 21. Bradford, M. M.: A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 22. Schägger, H. and Jagow, G. V.: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem., 199, 223e231 (1991). 23. Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J.: Glycoprotein staining following electrophoresis on acrylamide gels, Anal. Biochem., 30, 148e152 (1969). 24. Homma, S., Terasawa, N., Kubo, T., Yoneyama-Ishii, N., Aida, K., and Fujimaki, M.: Changes in chemical properties of melanoidin by oxidation and reduction, Biosci. Biotechnol. Biochem., 61, 533e535 (1997). 25. Smaniotto, A., Bertazzo, B., Comai, S., and Traldi, P.: The role of peptides and proteins in melanoidin formation, J. Mass. Spectrom., 44, 410e418 (2009).
TRANSFRUCTOSYLATION REACTION IN CURED TOBACCO LEAF
671
26. Fotopoulos, V.: Plant invertases: structure, function and regulation of a diverse enzyme family, J. Biol. Res., 4, 127e137 (2005). 27. Greiner, S., Weil, M., Krausgrill, S., and Rausch, T.: A tobacco cDNA coding for cell-wall invertase, Plant Physiol., 108, 825e826 (1995). 28. Sturm, A.: Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning, Plant Physiol., 121, 1e7 (1999). 29. Pressey, R.: Inhibition of invertases by pyridoxal and its analogues, Biochim. Biophys. Acta, 159, 414e416 (1968). 30. Lopez, M. E., Vattuone, M. A., and Sampietro, A. R.: Partial purification and properties of invertase carica papaya fruits, Phytochemistry, 27, 3077e3081 (1988). 31. Edelman, J. and Jefford, T. G.: The mechanism of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus, New Phytol., 67, 517e531 (1968).