Bente Wischmann

THE DEGREE OF STARCH PHOSPHORYLATION IS RELATED TO THE CHAIN LENGTH DISTRIBUTION OF THE NEUTRAL AND THE PHOSPHORYLATED CHAINS OF AMYLOPECTIN

Abstract The chain length distribution of the amylopectin neutral and phosphorylated α -glucan chains of isoamylase (EC 3.2.1.68)-debranched starch in dependence of starch phosphorylation has been analyzed using high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutral chains of moderately phosphorylated starches showed polymodal distributions with main peaks at dp (degree of polymerisation) 14–15 and 50, respectively. Highly phosphorylated starches specifically showed an increasing proportion of chains with mean dp 19. In the extremely highly phosphorylated curcuma ( Curcuma spp.) starch, this chain population dominated the chain length profile. The sub-population of the corresponding phosphorylated chains from highly phosphorylated starches showed longer retention times than moderately phosphorylated starches on HPAEC. Separation of the phosphorylated chains using anion-exchange chromatography at neutral pH demonstrated a partial separation of chains containing Glc3 P residues and Glc6 P residues, respectively, as well as a separation of chains containing one or several phosphate groups. Major determinants for the observed elution characteristics of phosphorylated chains using HPAEC are suggested to be a combination of chain length (as determined by gel-permeation chromatography), multiple phosphorylation of the chains (as determined from their content of Glc6 P and reducing ends) and finally, the presence of the phosphate group on either the C-3 or the C-6 position on the glucose moieties (as determined by 31 P NMR). The results demonstrate that general rules exist in the relationship between starch phosphorylation and chain length distribution of starches.

Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch.

The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis.

Melting properties and Lintnerisation of potato starch with different degrees of phosphorylation

Lintner dextrins were prepared from size fractionated potato starch granules from two potato varieties (90BKG22 and Lady Rosetta) that contain high or low natural content of esterified phosphate, respectively. The time course of hydrolysis showed the typical two-phase kinetics, with a maximal degree of hydrolysis of between 74% and 81% after 30 days of hydrolysis, except for the fraction of smallest granules of the low phosphorylated variety (low P), which was hydrolysed to 98%. The relative amount of retained glucose-6-P in the Lintner dextrins was 18.6% for the low P variety and 46.6% for the high P variety. However, when calculating the relative distribution of phosphate in the granules, it was shown that approximately 80% (low P) and 35.5% (high P) was located in the amorphous region. Melting characteristics were followed by differential scanning calorimetry (DSC). The DSC endothermic peak became low and broad during the time course of hydrolysis, with rise in enthalpy change, indicating a strong dependency on the amorphous region of the granules. After annealing the same fractions showed the typical raise in gelatinisation temperature and narrowing of gelatinisation peak. The values of the melting temperatures (T o , T m and T c ) are positively correlated to the degree of phosphorylation of the starch dextrin fractions both before and after annealing.

Influence of Phosphate Esters on the Annealing Properties of Starch

The effects of annealing on native potato, waxy maize, and phosphorylated waxy maize starches were compared. Phosphorylated waxy maize starch responded to annealing in a manner between that of the naturally phosphorylated potato starch and that of the native waxy maize starch. The gelatinisation end-point temperature was increased. whereas in the native waxy maize it was decreased. On the other hand. the onset temperature change was much larger in potato starch than in the two waxy maize starches, Steeping also yielded intermediate effects on the phosphorylated waxy maize starch. It was concluded that the phosphate groups have similar effects as they do in the native, naturally phosphorylated potato starch, although the substitution pattern is not entirely the same in the artificially phosphorylated starch.