J.Roger Stark

The effect of physical damage on the molecular structure of wheat starch

Abstract Physically damaged wheat-starch granules are partly soluble in cold water and, on complete dissolution in methyl sulphoxide, show a limiting viscosity number lower than that of the undamaged granules. Extraction of damaged granules with cold water preferentially leaches out amylopectin of low molecular weight. Aqueous extracts of a commercially milled wheat-flour contained pentosan and α- d -glucan, the latter consisting of 99% of amylopectin and

Partial Purification and Properties of a Bacterial Isoamylase

Isoamylase has been prepared by affinity chromatography of a commercial enzyme-preparation from a strain of Cytophaga (also known as a Flavobacterium or Polyangium). The enzyme was not very stable, but the stability could be improved by calcium ions. The enzyme had a very low but significant activity on pullulan and on alpha-dextrins having maltosyl side-chains. This observation, which is contrary to previous reports, has been related to the specificity of isoamylase and other bacterial debranching-enzymes.

Molecular properties of physically-damaged sorghum starch granules

The effects of physical damage on the molecular properties of starches from varieties of sorghum have been examined by scanning electron microscopy, viscosity measurements, iodine affinity, enzymic debranching and gel-filtration chromatography. The effect of physical damage is most pronounced with waxy sorghum starch, rendering it more than 80% extractable in cold water. After similar mechanical treatment, the other two sorghum starches were only about 50% extractable in cold water. The cold water extracts of these latter sorghum starches contained about 12% more amylopectin than the original starches, indicating that there is a preferential leaching out of amylopectin from the damaged granules. This result is different from previous work on hot aqueous leaching of undamaged starch granules, in which amylose was extracted preferentially.

Molecular modification of barley starch granules by different types of physical treatment

Barley starch granules have been subjected to physical damage under different conditions and the effects examined by iodine staining, viscometry, potentiometric iodine titration, enzymic analysis and gel chromatography. It has been confirmed that, in addition to the physical rupture of the starch granule, the molecular structure of the starch components in the granules is also affected. Amylopectin of low molecular weight was preferentially extracted with cold water from starch granules damaged under various conditions. However, physical damage to starch granules in the presence of water resulted in the extraction of relatively more amylose than in other cases, although the ratio of amylose to amylopectin in the cold water extracts was always less than in the original starch. Extraction of starch granules with propan-1-ol/water (3:1, v/v) made the granules more susceptible to damage, as judged by the quantity of material subsequently extractable with cold water.

The limit dextrinase of the broad bean (Vicia faba L.)

Abstract A limit dextrinase has been isolated from broad-bean flour and purified 800-fold. The enzyme readily hydrolysed branched α-dextrins containing maltosyl or maltotriosyl side-chains, pullulan, and amylopectin β-limit dextrin. Glycogen β-limit dextrins and amylopectin were slowly hydrolysed, but glycogens were barely susceptible to hydrolysis. There was no evidence for the presence of a second debranching enzyme in broad beans.

Some properties of a fungal β-D-glucanase preparation

Abstract A commercial enzyme preparation, of fungal origin, contained a mixture of β- D -glucanases which were fractionated by ion-exchange chromatography to give a mixture of an endo-(1→4)- and an exo-(1→3)-β- D -glucanase. These two enzymes were then separated by molecular-sieve chromatography on Sephadex G-150. The purified exo-(1→3)-β- D -glucanase has a relatively high specificity for (1→3)-β- D -glucosidic linkages, and has no action on lichenin.

Degradation of isolated barley endosperm cell walls by purified endo-(1 → 3)(1 → 4)-β-D-glucanases and malt extracts*†

The structure of isolated barley endosperm cell wall fragments was studied employing malt extracts and two purified endo-(1 → 3)(1 → 4)-β- D -glucanases. The endo-(1 → 3)(1 → 4)-β- D -glucanases were characterised by their activity on various substrates and possessed molecular weights and isoelectric points of 27 kD, 33 kD and pI 8·5 and ⩾ 10·8 respectively. These isoenzymes effected a limited degradation of barley endosperm cell wall polysaccharide, releasing glucose in addition to the expected oligosaccharide products. The release of glucose suggested that β-glucosidases may be associated with the isolated cell wall fragments. The degree of cell wall carbohydrate degradation was increased by the presence of a mixture of basic β-glucanases; whereas crude enzyme extracts from barley malt, containing proteolytic activity, released up to 72% of the total carbohydrate, including arabinoxylan breakdown products. The pattern of the cell wall digestions was, however, not altered by pretreatment of the cell wall fragments with a-amylase and a general proteinase, forwarding the view that proteolysis is not a pre-requisite for endo-β-glucanase attack. Examination of digested endosperm cell wall fragments by electron microscopy indicated that the enzymes of the basic protein fraction from barley malt clearly attacked the inner pitted surface of the walls, although solubilisation of the outer scaly surface, even in the presence of crude enzyme extracts, was less apparent. The observations did not confirm the proposal that the sequential appearance of proteinases and carbohydrases during germination reflect their role in endosperm cell wall breakdown.

The action of isoamylase on the surface of starch granules

There is an apparent conflict regarding the distribution of amylose and amylopectin within cereal-starch granules. When starch was damaged in a laboratory mill, amylopectin was preferentially extracted which cold water’**. The relatively mild damage which occurs during the milling of wheat’ resulted in almost pure amylopcctin being solubilised into cold water. On more severe damage, which results from the use of laboratory milling, starch granules were shown by scanning electron microscopy to crack and craze’ and, although amylopectin was the major component solubilised by cold water, significant amounts of amylose were also present in the cold-water extract2y3. However, experiments on the developing kernels of barley4 showed that, within the large (A-type) granules of barley starch, there was a relatively higher concentration of amylopectin in the core with increasing amounts of amylose towards the periphery. In order to reconcile these apparent differences, it was speculated3s5 that there was a small number of clusters of amylopectin that protruded from the otherwise relatively uniform “hairy-billiard-ball” surface described by Lineback6. The purpose of the present study was to examine the surface of the starch granule, using enzymic techniques. Undamaged starch granules were washed free of any coldwater soluble materials, then incubated with bacterial isoamylase, and the material solubilised was assayed for total carbohydrate and for interaction with iodine. The results (Table I) showed that a small proportion of carbohydrate material was released. On interaction with iodine, the blue value’ and the &,,,, were consistent with data for linear oligosaccharides from debranched amylopectins. The unit chains released by the action of isoamylase on wheat-starch granules were subjected to gel filtration chromatography on Sephadex GSOSF. In Fig. 1, a peak associated with fractions 78-82 did not stain with iodine and corresponded to a mixture of malto-oligosaccharides with an average d.p. of _ 15. The carbohydrate in fractions 48-70 interacted strongly with iodine and corresponded to a d.p. of about 4045. Amylose, if present, would have been excluded from the gel, would have appeared in

The iodine-staining properties and fine structure of some mammalian and invertebrate glycogens

Glycogens from mammalian and invertebrate sources have been compared by measuring the iodine-staining spectra of the debranched polymers and the debranched beta-amylase limit dextrins. From the results, it is concluded that, whereas the interior chains of each group of glycogen are very similar, the exterior chains of the mammalian glycogens generally contain a small number of longer chains which are not found in the invertebrate glycogens.

The molecular structures of some newly formed rat-liver glycogens

Abstract Glycogen has been isolated from the livers of rats which had been fasted and then intubated with d -glucose. The structure of the glycogen, as determined by iodine staining and enzymic methods, was shown to be very similar to that from control animals. There were slight differences in the iodine-staining properties, but not as marked as that previously reported in the literature.