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The size dependence of the average number of branches in amylose
Carbohydrate Polymers 223 (2019) 115134
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
The size dependence of the average number of branches in amylose a
b
c
d
Kai Wang , Francisco Vilaplana , Alex Wu , Jovin Hasjim , Robert G. Gilbert
d,e,f,g,⁎
T
a
College of Food Science, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China Division of Glycoscience, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Centre, SE-106 91, Stockholm, Sweden c Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, 4072, Australia d The University of Queensland, Centre for Nutrition & Food Science, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD, 4072, Australia e Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China f Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China g Jiangsu Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology, College of Agriculture, Yangzhou University, Yangzhou, 225009, China b
A R T I C LE I N FO
A B S T R A C T
Key words: Starch Amylose Molecular structure 2-dimensional Branching
Amylose has a small but significant number of long-chain branches. Two-dimensional size-exclusion chromatography is used to obtain the first measurement of the average number of branches per amylose molecule (from potato tubers) as a function of molecular size. Molecular weight dispersity, average chain length and average amylose molecular weight all increase with increasing size. However, the average number of branches of amylose molecules is weakly, if at all, dependent on size, with 2–4 per molecule except perhaps for the very largest molecules, although for these, the data may suffer from artifacts. Differences in the sizes of amylose molecules is mostly ascribed to variations in chain length. This observation is consistent with the postulate that most branching events occur in the early stage of amylose synthesis, and afterwards the branches are further elongated by granule-bound starch synthases. This gives improved mechanistic understanding of amylose biosynthesis.
1. Introduction Starch consists of two main components: slightly branched amylose and highly branched amylopectin. Although the lesser component of normal starch, amylose plays an important role in determining starch properties (Jane et al., 1999). It is known that a proportion of amylose molecules have branches (Hizukuri, Takeda, Yasuda, & Suzuki, 1981), and attempts have been made to reveal amylose branching structure (Hizukuri & Takagi, 1984; Hizukuri et al., 1981; Jane & Shen, 1993; Takeda, Shirasaka, & Hizukuri, 1984; Takeda, Hizukuri, & Juliano, 1986; Takeda, Maruta, & Hizukuri, 1992; Takeda, Tomooka, & Hizukuri, 1993); however, amylose branching structure, which is species- and variety-dependent, is not yet fully understood. A full description of the branching structure of a complex branched polymer requires an infinite-dimensional distribution function (GrayWeale & Gilbert, 2009); “dimensions” refers here to the mathematical concept of a set of independent variables specifying something (such as the total size and chain length of a molecule, which are independent
quantities giving information about the molecular structure), rather than the physical XYZ spatial dimensions. Various lower-level descriptors can however be measured. One such is the size dependence of the average number of branches per amylose molecule. Understanding amylose branching structure is important because of the strong effect this can have on properties such as mouth-feel of starch-containing foods (Wang, Henry, & Gilbert, 2014), pasting and gelatinization (Tao, Li, Yu, Gilbert, & Li, 2019), and digestion rate (Martinez et al., 2018; Panyoo & Emmambux, 2017; Yu, Tao, & Gilbert, 2018). This is related to the rheology of amylose in solution, a property which is strongly dependent on this branching structure (Li, Prakash, Nicholson, Fitzgerald, & Gilbert, 2016). A two-dimensional (2D) size-exclusion chromatography (SEC) technique (Vilaplana & Gilbert, 2010b, 2011; Vilaplana, Meng, Hasjim, & Gilbert, 2014), termed SEC × SEC, can reveal starch features that are unresolved using other current methods such as conventional SEC. The SEC × SEC method involves two stages. First, preparative SEC is used to collect a number of fractions with different sizes (hydrodynamic radius,
⁎ Corresponding author at: The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD, 4072, Australia. E-mail address: [email protected] (R.G. Gilbert).
https://doi.org/10.1016/j.carbpol.2019.115134 Received 22 June 2019; Received in revised form 21 July 2019; Accepted 25 July 2019 Available online 29 July 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
Carbohydrate Polymers 223 (2019) 115134
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analytical SEC setup after debranching using isoamylase (Wang, Hasjim, Wu, Henry, & Gilbert, 2014; Wu, Li, & Gilbert, 2014). The isoamylase applied was in sufficient excess to ensure complete debranching of amylose samples within 4 h (Ward, Gao, de Bruyn, Gilbert, & Fitzgerald, 2006). Size separation of debranched starch was performed using GRAM precolumn, 100 and 1000 analytical columns (PSS) at a flow rate of 0.6 mL min−1. Pullulan standards with peak molecular weights ranging 342∼2.35 × 106 were used for calibration to convert the SEC elution volume (Vel) to molecular size (R h or V h, where V h = 4/3 π R h3) through the Mark-Houwink equation:
Rh, or the equivalent hydrodynamic volume, Vh, which is the SEC separation parameter) of branched molecules. Because of the inherently lower resolution of preparative SEC, the same sample is also run through an analytical SEC to obtain a more accurate whole-molecule size distribution, although the analytical SEC does not provide sufficient amounts of samples for the following step. In this second step, each size-separated fraction is debranched, and then run through an analytical SEC to obtain the molecular size distribution of the individual chains in each size-separated fraction. In this method, the starch molecules are separated by both size of the whole molecule and the chain length distribution, with the resulting 2D distribution being in terms of both the hydrodynamic radius of branched molecules and the degree of polymerization (DP, X) of the individual chains. The current study employs SEC × SEC to determine the average number of branches of amylose from potato starch as a function of its whole-molecule size. This approach provides a novel way to characterize an important aspect of the branching structure of amylose, and the results revealing the relations of amylose branch number to its molecular size could aid the understanding of the biosynthesis of amylose molecules.
Vh =
2 KM1 + α 5 NA
(1)
where NA is Avogadro’s constant and M the molecular weight (Cave, Seabrook, Gidley, & Gilbert, 2009). The Mark−Houwink parameters K and α of pullulan in DMSO/LiBr solution at 80 °C are 2.424 × 10−4 dL and 0.68, respectively, while K and α for linear starch in DMSO/LiBr at 80 °C are 1.5 × 10−4 dL and 0.743, respectively (PSS, private communication). The resulting distributions are the SEC weight distributions w (logRh) from the refractive index detector, the weight-average molecular weight distributions from the MALLS and RI detectors, and the number average molecular weight distributions from the viscometric detector using the following equation.
2. Materials and methods 2.1. Materials
¯ n (logRh ) = 2 Vh NA M 5 [η]
Commercially available potato-starch amylose and protease from Streptomyces griseus (type XIV) were purchased from Sigma–Aldrich Pty. Ltd. (Castle Hill, NSW, Australia). Isoamylase (from Pseudomonas sp.) was purchased from Megazyme International Ltd. (Bray, Co. Wicklow, Ireland). Pullulan standards with molecular weight range 342 to 1.66·106 Da were purchased from Polymer Standards Service (PSS) GmbH (Mainz, Germany). Dimethyl sulfoxide (DMSO, GR grade for analysis) was purchased from Merck Co. Inc. (Kilsyth, VIC, Australia).
(2)
where NA is Avogadro’s constant and [η] is the intrinsic viscosity from the viscometric detector (Vilaplana & Gilbert, 2010a). To obtain ¯ w (log Rh), a Zimm plot with linear fitting was performed using M WinGPC software (PSS); the differential refractive index (dn/dc) for amylose in DMSO/LiBr solution at 80 °C used was 0.0689 mL g−1, as measured by PSS (Mainz, Germany) (Vilaplana & Gilbert, 2011). For debranched amylose, the DP X was calculated from Rh using the MarkHouwink equation (equation 1), where M = 162 (X – 1) + 180 (see Cave et al. (2009)), and the number distribution of debranched chains (the CLD) Nde(X) was calculated by Nde(X) = wde(log Rh) X–2 (Castro, Dumas, Chiou, Fitzgerald, & Gilbert, 2005).
2.2. Size fractionation Amylose was directly dissolved in DMSO/LiBr solution containing 0.5% (w/w) LiBr (DMSO/LiBr solution). The dissolved amylose samples were fractionated using a preparative SEC setup (AF2000, Postnova Analytics GmbH, Landsberg am Lech, Germany) as reported by Vilaplana and Gilbert (2010b). The DMSO/LiBr solution were used as the mobile phase at a flow rate of 1.5 mL min−1, and separation was carried out using PREP GRAM precolumn, 30, and 3000 columns (PSS). Although these columns are not capable of separating amylopectin molecules with large sizes, they have been proved to have good separation for amylose molecules from various types of starches (Liu et al., 2017; Tao et al., 2019; Wang et al., 2015; Yu et al., 2017). Seven fractions were collected at different elution times, and were recovered by ethanol precipitation followed by centrifugation, for further analysis.
2.4. Average number of branches per amylose molecule The molecular weight dispersity and DP dispersity, denoted ĐM and ¯ w/M ¯ n and ĐX = X¯ de,w/ ĐX, are obtained using the relations ĐM = M ¯ (logVh) is the average molecular weight of the X¯ de,n respectively. M whole amylose molecules in each size fraction, which can be calculated using the following equations in terms of its number-average and weight-average forms, respectively (Gaborieau, Gilbert, Gray-Weale, Hernandez, & Castignolles, 2007): ∞
∫ w (logVh)d logVh ¯ n (logVh ) = M
2.3. Structural characterization of each fraction
0 ∞
∫ w (logVh) M¯ n 1(Vh) d logVh 0
The 2D molecular size distributions of the amylose sample and its size fractions were obtained using a previously described procedure applying 2D SEC and enzymatic debranching (Li, Powell, & Gilbert, 2017; Vilaplana & Gilbert, 2010a, 2010b, 2011). The molecular size distributions of the size separated whole amylose molecules were analyzed using an Agilent 1100 series system (PSS, Mainz, Germany) equipped with a multiple-angle laser light scattering detector (MALLS; BIC-MwA7000, Brookhaven Instrument Corp., USA), a refractive index detector (RI; ShimadzuRID-10A, Shimadzu Corp., Japan) and a viscometric detector (ETA-2010, PSS). Samples were separated using GRAM precolumn, 30 and 3000 analytical columns (PSS) at a flow rate of 0.3 mL min−1 (Vilaplana & Gilbert, 2011). The molecular size distribution of debranched amylose were characterized using the same
∞
∫ M¯ w (Vh) w (logVh)d logVh ¯ w (logVh ) = M
0 ∞
∫ w (logVh) d logVh 0
(3)
X¯ de (Rh ) de(Rh) is the average DP of debranched amylose chains in each size fraction and is given as both number and weight averages ( X¯ de,n and X¯ de,w , respectively) using the following equations: ∞
∫ X Nde (X ) dX X¯ de,n =
0 ∞
∫ Nde (X ) dX 0
2
(4)
Carbohydrate Polymers 223 (2019) 115134
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In addition, the CLDs of all the amylose size fractions (Fig. 2B) exhibited minor peaks/shoulders at DP < 100, suggesting some presence of very short chains, consistent with what had been previously reported in amylose fractions isolated using 1-butanol (Takeda et al., 1984, 1986; Takeda et al., 1992). The current results show that the peak maxima of the two small peaks/shoulders are at around DP 20-30 and 40-50. This is most evident in the largest size amylose molecules (F1). A likely explanation is that these are typical amylopectin peaks observed in starches, as observed in multiple plant sources, for example as in (Wang, Hasjim et al., 2014). It is plausible that purified amylose contains small amylopectin molecules. This means that the small peaks/ shoulders may arise from amylopectin contaminations in the amylose sample. Similar results were observed by Takeda et al. (1984) and Takeda et al. (1986), and it was also proposed that these short chains were from amylopectin impurities.
∫ X 2Nde (X ) dX X¯ de,w
0 ∞
,
∫ XNde (X ) 0
(5)
The average size in each size fraction, R¯ h , was calculated from Eq. (3)as set out by Vilaplana and Gilbert (2010b). The weight- and number-average number of branches per amylose molecule, n¯ w and, n¯ n were calculated for each size fraction as:
¯ w X¯ de,w M0 n¯ w = M
(6)
¯ n X¯ de,n M0 n¯ n = M
(7)
where M0 = 162 = molecular weight of the anhydroglucose monomer and X¯ de,w the weight-average degree of polymerization; there is an obvious equivalent expression for the number average n¯ n . 3. Results and discussion
3.2. Average number of branches per amylose molecule
3.1. Molecular structure of amylose fraction
¯ n and The average molecular size (R¯ h ), average molecular weight (M ¯ w ), average chain-length ( X¯ de ,n and X¯ de, w ), average number of branM ches per molecules (n¯ n and n¯ w ) and dispersity (ĐM and ĐX) of each amylose fraction were calculated using the 2D SEC distributions, with results given in Table 1. The average molecular size (R¯h ) of amylose fractions decreased with increasing fraction order, ranging from 27 to 4.6. F1 had the largest molecules, whereas F7 contained the smallest. ¯ n and M ¯ w values Apparent differences were also observed in both M among different amylose fractions. It was found that amylose with larger molecular sizes tended to have higher molecular weight, as expected. The average chain length ( X¯ de, n and X¯ de ,w ) of each fraction was positively related to amylose molecular size, with X¯ de, n and X¯ de, w values decreasing from 597 and 1350 for F1 to 174 and 211 for F7, respectively. This trend was also observed for amylose from maize and rice (Takeda et al., 1992), suggesting that longer amylose chains would result in larger size of amylose molecules. ĐM and ĐX for F1 (7.29 and 2.26, respectively) were higher than the other size fractions, while differences among other fractions were less apparent (ranging over 1.75∼5.64 and 1.21∼2.26, respectively). To elucidate the relationships of these characteristics to the molecular size of amylose molecules, these parameters are plotted against ¯ n and M ¯w the average size of the fractions, R¯ h , in Fig. 3. It is seen that M increased with R¯ h as expected. The same trends were observed in X¯ de w and X¯ de n indicating the average chain-length of amylose decreased with decreasing molecular size, although all the amyloses had an average DP > 170. The calculated average number of branches per amylose molecule, n¯ n and n¯ w , were significantly higher for the fraction with the largest R¯h
Seven fractions (F1-F7) of different sizes were collected from the potato amylose sample, and the SEC weight distributions of branched (whole molecule) and debranched amylose are shown in Figs. 1 and 2. F1 represents the fraction with the largest amylose molecules, and the molecular size of amylose fraction reduces with increasing fraction number. This is because smaller size molecules require longer time to elute in the SEC columns. All distributions were normalized to same global maximum for presentation. As can be seen in Fig. 1A, the amylose CLDs showed unimodal molecular size distribution, with Rh ranging approximately from 2 to 3000 nm. All size fractions also showed unimodal molecular size distributions (shown in Fig. 1B), and the Rh of all the fractions were within this range. The CLDs of debranched amylose (Fig. 2A) showed a main peak ranging over DP 102∼104, in addition to two small peaks at DP < 100. Similar distributions were observed in the CLDs of debranched amylose size fractions. This indicates that the size-separated amylose samples have mostly long chains, ranging over DP 102∼104, and a small proportion of short chains with length smaller than DP 100. The maximum of the main peak at DP > 100 shifted to smaller DP with increasing fraction number, i.e. F1 showed the longest chain length, while F7 showed the smallest. This means that amylose molecules tend to have shorter chains for fractions collected at later elution times. This trend is similar to that of molecular size distribution, indicating that there is a positive relation between the molecular size and chain length of amylose molecules, which is in accordance with results for amylose from rice, normal maize and high amylose maize reported by Vilaplana and Gilbert (2010b) and Vilaplana et al. (2014).
(F1) than those of other size fractions, which might be due to the presence of residual amylopectin in the purified amylose. Although
Fig. 1. SEC weight distributions, w(log Rh), of branched (whole molecule) (A) potato amylose and (B) each size fraction as a function of molecular size, Rh. F1 – F7 were collected at successive elution times. All distributions normalized to the same maximum height. 3
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Fig. 2. SEC weight distributions, w(log Rh), of (A) debranched potato amylose chains and (B) debranched chains of each size fraction as a function of DP X. All distributions normalized to the same global maximum.
(bar)w = 2∼5. The average branch number of amylose from rice, kuzu, tapioca, and potato were studied by Takeda et al. (1984) and Takeda et al. (1986), and the results showed that amylose from rice has 2∼5 branches (number average), while kuzu, tapioca, and potato amyloses have 5, 8, and 7 branches (number average), respectively. Their results showed that the isolated amylose samples were still contaminated by amylopectin molecules, and therefore the resulting number of branches might be biased. Another study analyzed the number of branches of amylose isolated from maize and rice, and three size fractions were collected respectively, using SEC coupled with low pressure columns. It was reported that the number of branches decreased with reduction in molecular size (9.9, 2.9 and 1.6 for maize amylose size fractions, and 6.8, 2.5 and 1.4 for rice amylose size fractions) (Takeda et al., 1992). However, these results are also very likely to be overestimated, especially for the fraction with the largest molecular size. This is because GPC as used by these workers suffers from band broadening to a greater extent than high pressure SEC, which was used for fractionation in the current study; thus it is likely that a larger amount of small amylopectin molecules eluted together with amylose in these earlier publications. In comparison, less amylopectin impurities would be expected in the amylose fractions in the current study due to the use of high-pressure SEC, and most of these amylopectin contaminations would be in the
Table 1 Weight- and number-average numbers of branches per amylose molecule from commercial amylose from potato* R¯ h (nm)
F1 F2 F3 F4 F5 F6 F7
27.29 18.29 12.95 10.53 8.67 6.88 4.59
Number averages
Weight averages
¯n M
X¯ de, n
n¯ n
¯w M
X¯ de ,w
n¯ w
3.94 × 105 2.82 × 105 1.15 × 105 9.63 × 104 6.83 × 104 7.54 × 104 2.75 × 104
597 528 503 411 295 246 174
4 3 1 1 1 2 1
2.87 × 106 7.35 × 105 6.51 × 105 2.61 × 105 1.85 × 105 1.32 × 105 8.40 × 104
1350 1118 864 647 431 354 211
13 4 5 2 3 2 2
ĐM
ĐX
7.29 2.60 5.64 2.71 2.72 1.75 3.05
2.26 2.12 1.72 1.57 1.46 1.44 1.21
large molecules with Rh > 100 were excluded in the calculation of n¯ n and n¯ w of amylose, a proportion of them may be eluted at elution volumes nominally corresponding to lower Rh values, due to the unavoidable band broadening problem in SEC (Gilbert, 2011). Excluding the largest-size fraction (F1), the n¯ n and n¯ w of other fractions were approximately constant at different molecular sizes (or at least, there is no significant trend with molecular size), with n(bar)n = 1∼3 and n
Fig. 3. Number- and weight-average molecular weight, average chain lengths ( X¯ de ,n X¯ de, w ), number of branches (n¯ n and n¯ w ) per molecule, and dispersities (ĐM and ĐX) of amylose as functions of average molecular size. 4
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termination of branching might also be related to the space limitation during starch granule formation. It is currently accepted that amylose chains are distributed amongst the highly branched amylopectin molecules, which are more preferred by SBEs (Perez & Bertoft, 2010). As a starch granule grows, it becomes increasingly crowded inside starch granules, limiting the accessibility of SBE to amylose chains and further prevent branching. These could explain the observation that amylose molecules have similar branch numbers irrespective of size. If so, differences in the molecular size of various amylose fractions would simply contribute to differences in amylose chain length. For example, amylose in F1 with the largest molecular sizes was found to have longer branches than the other fractions. Sketches of the proposed architectures of branched amylose molecules with various molecular sizes are exhibited in Fig. 4. The elongation and branching of amylose molecules do not impact on each other in amylose synthesis, consistent with the observation that the number of branches is not noticeably dependent on the total molecular size. Elongation of amylose chains may be terminated due to spatial restriction of amylose in starch granules; depletion of substrates might also be involved in amylose synthesis.
Fig. 4. Sketches of the probable relationship between amylose molecular size and branching positions.
fraction with the largest molecular size (F1); hence the results, especially for F2 to F7, are regarded as more reliable. It is noted that with the exclusion of the largest size fraction, the number of branches of amylose from maize and rice were in the range of 1.4˜2.9 (Takeda et al., 1992), which is consistent with results in the present study. In addition, more size fractions (7) were collected in the current study, and fractions with smaller sizes tend to be impacted by band broadening to a lesser extent; thus the relationships between branch number and molecular size in this study are felt to be more reliable. Although it is possible that these results could be impacted by the trace amounts of amylopectin noted above, the actual contributions of the small amylopectin peaks in Fig. 2 to the overall averages in the above equations is negligible.
4. Conclusions A 2D SEC method has been applied to obtain, for the first time, the average number of branches per amylose molecule for potato amylose as a function of its molecular size. The average molecular weight, the average chain-length, and the dispersity of amylose molecules increase with the size of molecules, although the average number of branches per amylose molecule show weak dependence on molecular size, with most molecules having < 5 branches. From this size independence of average number of branches, it seems that amylose molecules with different molecular sizes are likely to differ only in their average chain length, not number of branches. Based on these results, we postulate that in amylose biosynthesis, branching and chain elongation are two separated processes; branching events happen mainly in an early stage of starch synthesis, after which the branches are elongated by GBSS. Amylose molecules with larger sizes would thus have longer chains compared with smaller molecules, but comparable numbers of branches. The present observations can thus provide novel insights into the mechanism of amylose branching and growth in plants.
3.3. Mechanism of amylose biosynthesis 2D SEC revealed here that the average molecular weight and the average chain-length of amylose branches increased with molecular size. However, the average number of branches per molecule was approximately independent of molecular size, with most molecules having < 5 branches based on weight-average data in Table 1. These structural features of amylose molecules have not been reported before using conventional SEC techniques. This observation probably relates to the chain or molecule growthstoppage mechanisms of amylose. Amylose growth is mainly catalyzed by granule-bound starch synthases I (GBSSI), the activity of which is reported to increase in the later stage of grain filling (Dry et al., 1992). This would result in the amylose content increasing over grain filling (Li, Blanco, & Jane, 2007). Starch branching enzymes (SBE) are what catalyze amylose branching, transferring branches with DP > 7 and leaving the remaining branches longer than DP 6 (Wu & Gilbert, 2010; Wu, Morell, & Gilbert, 2013). SBE is reported having multiple isoforms, with differences in their chain length preferences. In addition, SBEI has a higher activity for branching amylose than does SBEII (Guan & Preiss, 1993; Guan, Li, ImparlRadosevich, Preiss, & Keeling, 1997), and tends to transfer longer branches, while SBEII preferentially transfers shorter branches (Takeda, Guan, & Preiss, 1993). From the current results, it is suggested that amylose elongation and branching are somewhat separated processes. Amylose is branched by SBE at an early stage of the biosynthesis, when the branches are at chain lengths suitable for SBE to act. The new branches are then elongated by GBSS. It is previously proposed that GBSSI is the main isoform of GBSS involved in amylose synthesis. It has high affinity to glucan chains, and hence remains associated with the amylose chain until the completion of elongation (Denyer, Waite, Motawia, Moller, & Smith, 1999). In addition, it is probably the case that the formed branches have lengths which prevent the branching activity of SBE. Both mechanisms may be preventing placement of new branches past the initial biosynthesis of amylose. This means that new branches are only formed from a restricted range of chain length, after which more branching cannot occur, while chains can still be elongated. The
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Gilbert, R. G. (2011). Size-separation characterization of starch and glycogen for biosynthesis-structure-property relationships. Analytical and Bioanalytical Chemistry, 399(4), 1425–1438. Gray-Weale, A., & Gilbert, R. G. (2009). General Description of the Structure of Branched Polymers. Journal of Polymer Science Part a-Polymer Chemistry, 47(15), 3914–3930. Guan, H. P., Li, P., ImparlRadosevich, J., Preiss, J., & Keeling, P. (1997). Comparing the properties of Escherichia coli branching enzyme and maize branching enzyme. Archives of Biochemistry and Biophysics, 342(1), 92–98. Guan, H. P., & Preiss, J. (1993). Differentiation of the properties of the branching isozymes from maize (Zea-mays). Plant Physiology, 102(4), 1269–1273. Hizukuri, S., & Takagi, T. (1984). Estimation of the distribution of molecular weight for amylose by the low-angle laser-light-scattering technique combined with high-performance gel chromatography. Carbohydrate Research, 134(1), 1–10. Hizukuri, S., Takeda, Y., Yasuda, M., & Suzuki, A. (1981). Multi-branched nature of amylose and the action of debranching enzymes. Carbohydrate Research, 94(2), 205–213. Jane, J. L., Chen, Y. Y., Lee, L. F., McPherson, A. E., Wong, K. S., Radosavljevic, M., et al. (1999). Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry, 76(5), 629–637. Jane, J. L., & Shen, J. J. (1993). Internal structure of the potato starch granule revealed by chemical gelatinization. Carbohydrate Research, 247, 279–290. Li, C., Powell, P. O., & Gilbert, R. G. (2017). Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm. Amylase, 1, 59–74. Li, H., Prakash, S., Nicholson, T. M., Fitzgerald, M. A., & Gilbert, R. G. (2016). The importance of amylose and amylopectin fine structure for textural properties of cooked rice grains. Food Chemistry, 196, 702–711. Li, L., Blanco, M., & Jane, J.-l. (2007). Physicochemical properties of endosperm and pericarp starches during maize development. Carbohydrate Polymers, 67(4), 630–639. Liu, Y., Chen, J., Luo, S., Li, C., Ye, J., Liu, C., et al. (2017). Physicochemical and structural properties of pregelatinized starch prepared by improved extrusion cooking technology. Carbohydrate Polymers, 175, 265–272. Martinez, M. M., Li, C., Okoniewska, M., Mukherjee, I., Vellucci, D., & Hamaker, B. (2018). Slowly digestible starch in fully gelatinized material is structurally driven by molecular size and A and B1 chain lengths. Carbohydrate Polymers, 197, 531–539. Panyoo, A. E., & Emmambux, M. N. (2017). Amylose-lipid complex production and potential health benefits: A mini-review. Starch-Starke, 69(7–8), 203. Perez, S., & Bertoft, E. (2010). The molecular structures of starch components and their contribution to the architecture of starch granules: A comprehensive review. Starch, 62(8), 389–420. Takeda, Y., Guan, H. P., & Preiss, J. (1993). Branching of amylose by the branching isoenzymes of maize endosperm. Carbohydrate Research, 240, 253–263. Takeda, Y., Hizukuri, S., & Juliano, B. O. (1986). Purification and structure of amylose from rice starch. Carbohydrate Research, 148(2), 299–308. Takeda, Y., Maruta, N., & Hizukuri, S. (1992). Structures of amylose subfractions with different molecular sizes. Carbohydrate Research, 226(2), 279–285.
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
The size dependence of the average number of branches in amylose a
b
c
d
Kai Wang , Francisco Vilaplana , Alex Wu , Jovin Hasjim , Robert G. Gilbert
d,e,f,g,⁎
T
a
College of Food Science, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China Division of Glycoscience, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Centre, SE-106 91, Stockholm, Sweden c Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, 4072, Australia d The University of Queensland, Centre for Nutrition & Food Science, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD, 4072, Australia e Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China f Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China g Jiangsu Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology, College of Agriculture, Yangzhou University, Yangzhou, 225009, China b
A R T I C LE I N FO
A B S T R A C T
Key words: Starch Amylose Molecular structure 2-dimensional Branching
Amylose has a small but significant number of long-chain branches. Two-dimensional size-exclusion chromatography is used to obtain the first measurement of the average number of branches per amylose molecule (from potato tubers) as a function of molecular size. Molecular weight dispersity, average chain length and average amylose molecular weight all increase with increasing size. However, the average number of branches of amylose molecules is weakly, if at all, dependent on size, with 2–4 per molecule except perhaps for the very largest molecules, although for these, the data may suffer from artifacts. Differences in the sizes of amylose molecules is mostly ascribed to variations in chain length. This observation is consistent with the postulate that most branching events occur in the early stage of amylose synthesis, and afterwards the branches are further elongated by granule-bound starch synthases. This gives improved mechanistic understanding of amylose biosynthesis.
1. Introduction Starch consists of two main components: slightly branched amylose and highly branched amylopectin. Although the lesser component of normal starch, amylose plays an important role in determining starch properties (Jane et al., 1999). It is known that a proportion of amylose molecules have branches (Hizukuri, Takeda, Yasuda, & Suzuki, 1981), and attempts have been made to reveal amylose branching structure (Hizukuri & Takagi, 1984; Hizukuri et al., 1981; Jane & Shen, 1993; Takeda, Shirasaka, & Hizukuri, 1984; Takeda, Hizukuri, & Juliano, 1986; Takeda, Maruta, & Hizukuri, 1992; Takeda, Tomooka, & Hizukuri, 1993); however, amylose branching structure, which is species- and variety-dependent, is not yet fully understood. A full description of the branching structure of a complex branched polymer requires an infinite-dimensional distribution function (GrayWeale & Gilbert, 2009); “dimensions” refers here to the mathematical concept of a set of independent variables specifying something (such as the total size and chain length of a molecule, which are independent
quantities giving information about the molecular structure), rather than the physical XYZ spatial dimensions. Various lower-level descriptors can however be measured. One such is the size dependence of the average number of branches per amylose molecule. Understanding amylose branching structure is important because of the strong effect this can have on properties such as mouth-feel of starch-containing foods (Wang, Henry, & Gilbert, 2014), pasting and gelatinization (Tao, Li, Yu, Gilbert, & Li, 2019), and digestion rate (Martinez et al., 2018; Panyoo & Emmambux, 2017; Yu, Tao, & Gilbert, 2018). This is related to the rheology of amylose in solution, a property which is strongly dependent on this branching structure (Li, Prakash, Nicholson, Fitzgerald, & Gilbert, 2016). A two-dimensional (2D) size-exclusion chromatography (SEC) technique (Vilaplana & Gilbert, 2010b, 2011; Vilaplana, Meng, Hasjim, & Gilbert, 2014), termed SEC × SEC, can reveal starch features that are unresolved using other current methods such as conventional SEC. The SEC × SEC method involves two stages. First, preparative SEC is used to collect a number of fractions with different sizes (hydrodynamic radius,
⁎ Corresponding author at: The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD, 4072, Australia. E-mail address: [email protected] (R.G. Gilbert).
https://doi.org/10.1016/j.carbpol.2019.115134 Received 22 June 2019; Received in revised form 21 July 2019; Accepted 25 July 2019 Available online 29 July 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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analytical SEC setup after debranching using isoamylase (Wang, Hasjim, Wu, Henry, & Gilbert, 2014; Wu, Li, & Gilbert, 2014). The isoamylase applied was in sufficient excess to ensure complete debranching of amylose samples within 4 h (Ward, Gao, de Bruyn, Gilbert, & Fitzgerald, 2006). Size separation of debranched starch was performed using GRAM precolumn, 100 and 1000 analytical columns (PSS) at a flow rate of 0.6 mL min−1. Pullulan standards with peak molecular weights ranging 342∼2.35 × 106 were used for calibration to convert the SEC elution volume (Vel) to molecular size (R h or V h, where V h = 4/3 π R h3) through the Mark-Houwink equation:
Rh, or the equivalent hydrodynamic volume, Vh, which is the SEC separation parameter) of branched molecules. Because of the inherently lower resolution of preparative SEC, the same sample is also run through an analytical SEC to obtain a more accurate whole-molecule size distribution, although the analytical SEC does not provide sufficient amounts of samples for the following step. In this second step, each size-separated fraction is debranched, and then run through an analytical SEC to obtain the molecular size distribution of the individual chains in each size-separated fraction. In this method, the starch molecules are separated by both size of the whole molecule and the chain length distribution, with the resulting 2D distribution being in terms of both the hydrodynamic radius of branched molecules and the degree of polymerization (DP, X) of the individual chains. The current study employs SEC × SEC to determine the average number of branches of amylose from potato starch as a function of its whole-molecule size. This approach provides a novel way to characterize an important aspect of the branching structure of amylose, and the results revealing the relations of amylose branch number to its molecular size could aid the understanding of the biosynthesis of amylose molecules.
Vh =
2 KM1 + α 5 NA
(1)
where NA is Avogadro’s constant and M the molecular weight (Cave, Seabrook, Gidley, & Gilbert, 2009). The Mark−Houwink parameters K and α of pullulan in DMSO/LiBr solution at 80 °C are 2.424 × 10−4 dL and 0.68, respectively, while K and α for linear starch in DMSO/LiBr at 80 °C are 1.5 × 10−4 dL and 0.743, respectively (PSS, private communication). The resulting distributions are the SEC weight distributions w (logRh) from the refractive index detector, the weight-average molecular weight distributions from the MALLS and RI detectors, and the number average molecular weight distributions from the viscometric detector using the following equation.
2. Materials and methods 2.1. Materials
¯ n (logRh ) = 2 Vh NA M 5 [η]
Commercially available potato-starch amylose and protease from Streptomyces griseus (type XIV) were purchased from Sigma–Aldrich Pty. Ltd. (Castle Hill, NSW, Australia). Isoamylase (from Pseudomonas sp.) was purchased from Megazyme International Ltd. (Bray, Co. Wicklow, Ireland). Pullulan standards with molecular weight range 342 to 1.66·106 Da were purchased from Polymer Standards Service (PSS) GmbH (Mainz, Germany). Dimethyl sulfoxide (DMSO, GR grade for analysis) was purchased from Merck Co. Inc. (Kilsyth, VIC, Australia).
(2)
where NA is Avogadro’s constant and [η] is the intrinsic viscosity from the viscometric detector (Vilaplana & Gilbert, 2010a). To obtain ¯ w (log Rh), a Zimm plot with linear fitting was performed using M WinGPC software (PSS); the differential refractive index (dn/dc) for amylose in DMSO/LiBr solution at 80 °C used was 0.0689 mL g−1, as measured by PSS (Mainz, Germany) (Vilaplana & Gilbert, 2011). For debranched amylose, the DP X was calculated from Rh using the MarkHouwink equation (equation 1), where M = 162 (X – 1) + 180 (see Cave et al. (2009)), and the number distribution of debranched chains (the CLD) Nde(X) was calculated by Nde(X) = wde(log Rh) X–2 (Castro, Dumas, Chiou, Fitzgerald, & Gilbert, 2005).
2.2. Size fractionation Amylose was directly dissolved in DMSO/LiBr solution containing 0.5% (w/w) LiBr (DMSO/LiBr solution). The dissolved amylose samples were fractionated using a preparative SEC setup (AF2000, Postnova Analytics GmbH, Landsberg am Lech, Germany) as reported by Vilaplana and Gilbert (2010b). The DMSO/LiBr solution were used as the mobile phase at a flow rate of 1.5 mL min−1, and separation was carried out using PREP GRAM precolumn, 30, and 3000 columns (PSS). Although these columns are not capable of separating amylopectin molecules with large sizes, they have been proved to have good separation for amylose molecules from various types of starches (Liu et al., 2017; Tao et al., 2019; Wang et al., 2015; Yu et al., 2017). Seven fractions were collected at different elution times, and were recovered by ethanol precipitation followed by centrifugation, for further analysis.
2.4. Average number of branches per amylose molecule The molecular weight dispersity and DP dispersity, denoted ĐM and ¯ w/M ¯ n and ĐX = X¯ de,w/ ĐX, are obtained using the relations ĐM = M ¯ (logVh) is the average molecular weight of the X¯ de,n respectively. M whole amylose molecules in each size fraction, which can be calculated using the following equations in terms of its number-average and weight-average forms, respectively (Gaborieau, Gilbert, Gray-Weale, Hernandez, & Castignolles, 2007): ∞
∫ w (logVh)d logVh ¯ n (logVh ) = M
2.3. Structural characterization of each fraction
0 ∞
∫ w (logVh) M¯ n 1(Vh) d logVh 0
The 2D molecular size distributions of the amylose sample and its size fractions were obtained using a previously described procedure applying 2D SEC and enzymatic debranching (Li, Powell, & Gilbert, 2017; Vilaplana & Gilbert, 2010a, 2010b, 2011). The molecular size distributions of the size separated whole amylose molecules were analyzed using an Agilent 1100 series system (PSS, Mainz, Germany) equipped with a multiple-angle laser light scattering detector (MALLS; BIC-MwA7000, Brookhaven Instrument Corp., USA), a refractive index detector (RI; ShimadzuRID-10A, Shimadzu Corp., Japan) and a viscometric detector (ETA-2010, PSS). Samples were separated using GRAM precolumn, 30 and 3000 analytical columns (PSS) at a flow rate of 0.3 mL min−1 (Vilaplana & Gilbert, 2011). The molecular size distribution of debranched amylose were characterized using the same
∞
∫ M¯ w (Vh) w (logVh)d logVh ¯ w (logVh ) = M
0 ∞
∫ w (logVh) d logVh 0
(3)
X¯ de (Rh ) de(Rh) is the average DP of debranched amylose chains in each size fraction and is given as both number and weight averages ( X¯ de,n and X¯ de,w , respectively) using the following equations: ∞
∫ X Nde (X ) dX X¯ de,n =
0 ∞
∫ Nde (X ) dX 0
2
(4)
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In addition, the CLDs of all the amylose size fractions (Fig. 2B) exhibited minor peaks/shoulders at DP < 100, suggesting some presence of very short chains, consistent with what had been previously reported in amylose fractions isolated using 1-butanol (Takeda et al., 1984, 1986; Takeda et al., 1992). The current results show that the peak maxima of the two small peaks/shoulders are at around DP 20-30 and 40-50. This is most evident in the largest size amylose molecules (F1). A likely explanation is that these are typical amylopectin peaks observed in starches, as observed in multiple plant sources, for example as in (Wang, Hasjim et al., 2014). It is plausible that purified amylose contains small amylopectin molecules. This means that the small peaks/ shoulders may arise from amylopectin contaminations in the amylose sample. Similar results were observed by Takeda et al. (1984) and Takeda et al. (1986), and it was also proposed that these short chains were from amylopectin impurities.
∫ X 2Nde (X ) dX X¯ de,w
0 ∞
,
∫ XNde (X ) 0
(5)
The average size in each size fraction, R¯ h , was calculated from Eq. (3)as set out by Vilaplana and Gilbert (2010b). The weight- and number-average number of branches per amylose molecule, n¯ w and, n¯ n were calculated for each size fraction as:
¯ w X¯ de,w M0 n¯ w = M
(6)
¯ n X¯ de,n M0 n¯ n = M
(7)
where M0 = 162 = molecular weight of the anhydroglucose monomer and X¯ de,w the weight-average degree of polymerization; there is an obvious equivalent expression for the number average n¯ n . 3. Results and discussion
3.2. Average number of branches per amylose molecule
3.1. Molecular structure of amylose fraction
¯ n and The average molecular size (R¯ h ), average molecular weight (M ¯ w ), average chain-length ( X¯ de ,n and X¯ de, w ), average number of branM ches per molecules (n¯ n and n¯ w ) and dispersity (ĐM and ĐX) of each amylose fraction were calculated using the 2D SEC distributions, with results given in Table 1. The average molecular size (R¯h ) of amylose fractions decreased with increasing fraction order, ranging from 27 to 4.6. F1 had the largest molecules, whereas F7 contained the smallest. ¯ n and M ¯ w values Apparent differences were also observed in both M among different amylose fractions. It was found that amylose with larger molecular sizes tended to have higher molecular weight, as expected. The average chain length ( X¯ de, n and X¯ de ,w ) of each fraction was positively related to amylose molecular size, with X¯ de, n and X¯ de, w values decreasing from 597 and 1350 for F1 to 174 and 211 for F7, respectively. This trend was also observed for amylose from maize and rice (Takeda et al., 1992), suggesting that longer amylose chains would result in larger size of amylose molecules. ĐM and ĐX for F1 (7.29 and 2.26, respectively) were higher than the other size fractions, while differences among other fractions were less apparent (ranging over 1.75∼5.64 and 1.21∼2.26, respectively). To elucidate the relationships of these characteristics to the molecular size of amylose molecules, these parameters are plotted against ¯ n and M ¯w the average size of the fractions, R¯ h , in Fig. 3. It is seen that M increased with R¯ h as expected. The same trends were observed in X¯ de w and X¯ de n indicating the average chain-length of amylose decreased with decreasing molecular size, although all the amyloses had an average DP > 170. The calculated average number of branches per amylose molecule, n¯ n and n¯ w , were significantly higher for the fraction with the largest R¯h
Seven fractions (F1-F7) of different sizes were collected from the potato amylose sample, and the SEC weight distributions of branched (whole molecule) and debranched amylose are shown in Figs. 1 and 2. F1 represents the fraction with the largest amylose molecules, and the molecular size of amylose fraction reduces with increasing fraction number. This is because smaller size molecules require longer time to elute in the SEC columns. All distributions were normalized to same global maximum for presentation. As can be seen in Fig. 1A, the amylose CLDs showed unimodal molecular size distribution, with Rh ranging approximately from 2 to 3000 nm. All size fractions also showed unimodal molecular size distributions (shown in Fig. 1B), and the Rh of all the fractions were within this range. The CLDs of debranched amylose (Fig. 2A) showed a main peak ranging over DP 102∼104, in addition to two small peaks at DP < 100. Similar distributions were observed in the CLDs of debranched amylose size fractions. This indicates that the size-separated amylose samples have mostly long chains, ranging over DP 102∼104, and a small proportion of short chains with length smaller than DP 100. The maximum of the main peak at DP > 100 shifted to smaller DP with increasing fraction number, i.e. F1 showed the longest chain length, while F7 showed the smallest. This means that amylose molecules tend to have shorter chains for fractions collected at later elution times. This trend is similar to that of molecular size distribution, indicating that there is a positive relation between the molecular size and chain length of amylose molecules, which is in accordance with results for amylose from rice, normal maize and high amylose maize reported by Vilaplana and Gilbert (2010b) and Vilaplana et al. (2014).
(F1) than those of other size fractions, which might be due to the presence of residual amylopectin in the purified amylose. Although
Fig. 1. SEC weight distributions, w(log Rh), of branched (whole molecule) (A) potato amylose and (B) each size fraction as a function of molecular size, Rh. F1 – F7 were collected at successive elution times. All distributions normalized to the same maximum height. 3
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Fig. 2. SEC weight distributions, w(log Rh), of (A) debranched potato amylose chains and (B) debranched chains of each size fraction as a function of DP X. All distributions normalized to the same global maximum.
(bar)w = 2∼5. The average branch number of amylose from rice, kuzu, tapioca, and potato were studied by Takeda et al. (1984) and Takeda et al. (1986), and the results showed that amylose from rice has 2∼5 branches (number average), while kuzu, tapioca, and potato amyloses have 5, 8, and 7 branches (number average), respectively. Their results showed that the isolated amylose samples were still contaminated by amylopectin molecules, and therefore the resulting number of branches might be biased. Another study analyzed the number of branches of amylose isolated from maize and rice, and three size fractions were collected respectively, using SEC coupled with low pressure columns. It was reported that the number of branches decreased with reduction in molecular size (9.9, 2.9 and 1.6 for maize amylose size fractions, and 6.8, 2.5 and 1.4 for rice amylose size fractions) (Takeda et al., 1992). However, these results are also very likely to be overestimated, especially for the fraction with the largest molecular size. This is because GPC as used by these workers suffers from band broadening to a greater extent than high pressure SEC, which was used for fractionation in the current study; thus it is likely that a larger amount of small amylopectin molecules eluted together with amylose in these earlier publications. In comparison, less amylopectin impurities would be expected in the amylose fractions in the current study due to the use of high-pressure SEC, and most of these amylopectin contaminations would be in the
Table 1 Weight- and number-average numbers of branches per amylose molecule from commercial amylose from potato* R¯ h (nm)
F1 F2 F3 F4 F5 F6 F7
27.29 18.29 12.95 10.53 8.67 6.88 4.59
Number averages
Weight averages
¯n M
X¯ de, n
n¯ n
¯w M
X¯ de ,w
n¯ w
3.94 × 105 2.82 × 105 1.15 × 105 9.63 × 104 6.83 × 104 7.54 × 104 2.75 × 104
597 528 503 411 295 246 174
4 3 1 1 1 2 1
2.87 × 106 7.35 × 105 6.51 × 105 2.61 × 105 1.85 × 105 1.32 × 105 8.40 × 104
1350 1118 864 647 431 354 211
13 4 5 2 3 2 2
ĐM
ĐX
7.29 2.60 5.64 2.71 2.72 1.75 3.05
2.26 2.12 1.72 1.57 1.46 1.44 1.21
large molecules with Rh > 100 were excluded in the calculation of n¯ n and n¯ w of amylose, a proportion of them may be eluted at elution volumes nominally corresponding to lower Rh values, due to the unavoidable band broadening problem in SEC (Gilbert, 2011). Excluding the largest-size fraction (F1), the n¯ n and n¯ w of other fractions were approximately constant at different molecular sizes (or at least, there is no significant trend with molecular size), with n(bar)n = 1∼3 and n
Fig. 3. Number- and weight-average molecular weight, average chain lengths ( X¯ de ,n X¯ de, w ), number of branches (n¯ n and n¯ w ) per molecule, and dispersities (ĐM and ĐX) of amylose as functions of average molecular size. 4
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termination of branching might also be related to the space limitation during starch granule formation. It is currently accepted that amylose chains are distributed amongst the highly branched amylopectin molecules, which are more preferred by SBEs (Perez & Bertoft, 2010). As a starch granule grows, it becomes increasingly crowded inside starch granules, limiting the accessibility of SBE to amylose chains and further prevent branching. These could explain the observation that amylose molecules have similar branch numbers irrespective of size. If so, differences in the molecular size of various amylose fractions would simply contribute to differences in amylose chain length. For example, amylose in F1 with the largest molecular sizes was found to have longer branches than the other fractions. Sketches of the proposed architectures of branched amylose molecules with various molecular sizes are exhibited in Fig. 4. The elongation and branching of amylose molecules do not impact on each other in amylose synthesis, consistent with the observation that the number of branches is not noticeably dependent on the total molecular size. Elongation of amylose chains may be terminated due to spatial restriction of amylose in starch granules; depletion of substrates might also be involved in amylose synthesis.
Fig. 4. Sketches of the probable relationship between amylose molecular size and branching positions.
fraction with the largest molecular size (F1); hence the results, especially for F2 to F7, are regarded as more reliable. It is noted that with the exclusion of the largest size fraction, the number of branches of amylose from maize and rice were in the range of 1.4˜2.9 (Takeda et al., 1992), which is consistent with results in the present study. In addition, more size fractions (7) were collected in the current study, and fractions with smaller sizes tend to be impacted by band broadening to a lesser extent; thus the relationships between branch number and molecular size in this study are felt to be more reliable. Although it is possible that these results could be impacted by the trace amounts of amylopectin noted above, the actual contributions of the small amylopectin peaks in Fig. 2 to the overall averages in the above equations is negligible.
4. Conclusions A 2D SEC method has been applied to obtain, for the first time, the average number of branches per amylose molecule for potato amylose as a function of its molecular size. The average molecular weight, the average chain-length, and the dispersity of amylose molecules increase with the size of molecules, although the average number of branches per amylose molecule show weak dependence on molecular size, with most molecules having < 5 branches. From this size independence of average number of branches, it seems that amylose molecules with different molecular sizes are likely to differ only in their average chain length, not number of branches. Based on these results, we postulate that in amylose biosynthesis, branching and chain elongation are two separated processes; branching events happen mainly in an early stage of starch synthesis, after which the branches are elongated by GBSS. Amylose molecules with larger sizes would thus have longer chains compared with smaller molecules, but comparable numbers of branches. The present observations can thus provide novel insights into the mechanism of amylose branching and growth in plants.
3.3. Mechanism of amylose biosynthesis 2D SEC revealed here that the average molecular weight and the average chain-length of amylose branches increased with molecular size. However, the average number of branches per molecule was approximately independent of molecular size, with most molecules having < 5 branches based on weight-average data in Table 1. These structural features of amylose molecules have not been reported before using conventional SEC techniques. This observation probably relates to the chain or molecule growthstoppage mechanisms of amylose. Amylose growth is mainly catalyzed by granule-bound starch synthases I (GBSSI), the activity of which is reported to increase in the later stage of grain filling (Dry et al., 1992). This would result in the amylose content increasing over grain filling (Li, Blanco, & Jane, 2007). Starch branching enzymes (SBE) are what catalyze amylose branching, transferring branches with DP > 7 and leaving the remaining branches longer than DP 6 (Wu & Gilbert, 2010; Wu, Morell, & Gilbert, 2013). SBE is reported having multiple isoforms, with differences in their chain length preferences. In addition, SBEI has a higher activity for branching amylose than does SBEII (Guan & Preiss, 1993; Guan, Li, ImparlRadosevich, Preiss, & Keeling, 1997), and tends to transfer longer branches, while SBEII preferentially transfers shorter branches (Takeda, Guan, & Preiss, 1993). From the current results, it is suggested that amylose elongation and branching are somewhat separated processes. Amylose is branched by SBE at an early stage of the biosynthesis, when the branches are at chain lengths suitable for SBE to act. The new branches are then elongated by GBSS. It is previously proposed that GBSSI is the main isoform of GBSS involved in amylose synthesis. It has high affinity to glucan chains, and hence remains associated with the amylose chain until the completion of elongation (Denyer, Waite, Motawia, Moller, & Smith, 1999). In addition, it is probably the case that the formed branches have lengths which prevent the branching activity of SBE. Both mechanisms may be preventing placement of new branches past the initial biosynthesis of amylose. This means that new branches are only formed from a restricted range of chain length, after which more branching cannot occur, while chains can still be elongated. The
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