Adsorption of Wheat alpha-Amylase lsoenzymes to Wheat Starch

By J. G. Sargeant and T. S. Walker, High Wycombe (Great Britain)

Two groups of %-amylase isoenzymes have been identified in germinated wheat, but only one group would adsorb on to undamaged starch granules. This group represented 84,2% of the total a-amylase activity. The non-adsorbing isoenzymes were electrophoretically identical to the a-amylases of immature wheat grain.

1 Introduction

One of the essential processes that occurs when cereal seeds germinate is the de nouo synthesis of hydrolytic enzymes which catalyse the breakdown of carbohydrate and protein storage materials. An important group of these hydrolases are the a-amylases; theenzymes whichdegrade starch. Anumber ofisoenzymes of wheat a-amylase have been reported in the literature [l, 21. At germination, the synthesis of a-amylase is induced by a hormone released from the embryo [3,4, 51. The a-amylases are synthesised within the cells of the aleurone layer, from where they migrate into the starchy endosperm, and hydrolysis of the starch granules begins [6]. Digestion of the starch granule, within the germinating wheat endosperm, is a relatively slow process, because the reaction involves the hydrolysis of an insoluble substrate. It has been postulated that therateofdigestion ofthegranule isprobably dependent upon the amount of enzyme adsorbed to the surface of the granule [7], although the site of adsorption may not necessarily be the site of degradation [8]. In this paper, we wish to present evidence to show that two distinct groups of a-amylase isoenzymes are syntliesised by germinating wheat, and that at physiological concentrations, the adsorption of a-amylase to the starch granule is a prerequisite for the enzymic degradation of the granule.

2 Materials and Methods

2.1 Materials

Winter wheat, variety Champlein (1975 harvest), was obtained from RHM Agriculture Ltd. Soluble starch was supplied by E. Merck Laboratory Chemicals, Darmstadt. Ampholine pH 3.5 - 10 and Ampholine PAG plates pH 3.5 -9.5 were obtained from LKB Instruments Ltd., South Croydon. All other materials used were "Analar" grade chemicals supplied by BDH Ltd. and Sigma Chemical Co.

2.2 Methods

2.2. I Germinationofseeds. Wheat seeds were surfacesterilised in aqueous 1 ?'n ("/,) sodium hypochlorite for 10 min, and then washed thoroughly. Imbibition and germination were carried out on cotton wool pads in light-tight boxes at 18 "C. After the required germination time, the seeds were either stored frozen at - 18 "C or freeze-dried.

Adsorption von Isoenzymen der Weizen-a-Amylase an Weizenstarke. In gekeimtem Weizen wurden zwei Gruppen von %-Amylase- Isoenzymen identifiziert, jedoch wurde nur eine Gruppe von unbeschadigten Starkekornern adsorbiert. Diese Gruppe enthielt 84,2% der gesamten a-Amylaseaktivitat. Die nicht adsorbierenden Isoenzyme waren elektrophoretisch identisch mit den a-Amylasen unreifer Weizenkorner.

2.2.2 a-Amylase assay. a-Amylase was measured by a semi-automated method developed by Smith [9], based on the original Farrand colorimetric method [lo]. In this paper, we define one unit of activity as the amount of enzyme that will produce 1 mg maltose from starch in 3 min at pH 6.9 and

2.2.3 Purification of a-amylases. The method used for the purification of r-amylases from immature wheat and germinated wheat was similar to that described by Kruger [12], but with two modifications. Carboxymethyl cellulose (CMC) chromatography was used instead of acetone precipitation, since this increased the yield of active enzyme. In addition, the technique of glycogen precipitation, as described by Loyter and Schrnmm [ 131, was used, with the substitution of oyster glycogen by rabbit liver glycogen. 2.2.4 Isoelectric focusing. a-Amylase isoenzymes in crude extracts and in purified form were separated by analytical thin-layer polyacrylamide gel electrofocusing. The apparatus (LKB Multiphor) was used in accordance with the makers' instructions. Gels containing carrier ampholytes in the pH range 3.5 to 9.5 were used, and samples were applied using filter paper wicks (0.5 x 1 cm), to which 25 p1 of sample solution had been adsorbed. These sample solutions would normally contain 0.5 - 2 units/ml of a-amylase activity. A constant power of 0.8 watts per cm length of gel was passed through the gel at 4 "C for 14 h. A zymogram was produced by flooding the gel with 1% starch solution (boiled Merck starch) for 5 min at 25°C. The amylolytically-active bands were visualised by immersing the gel in an I,-KI solution? (stocksolution:6.5 g1, + 19.5 KI/l;dilutedto4% "/,before use), for approximately 2 min. Working quantities of the two groups of a-amylase isoenzymes were separated by preparative isoelectric focusing on a bed ofsephadex G-75 (1 1 x 25 x 0.5 cm). This bed was prepared by swelling 5 g of Sephadex in 100 ml of aqueous solution containing 2.5 ml Ampholine (pH 3.5 -9.5) and the enzyme solution from the glycogen precipitation stage (usually 30 ml). The slurry was poured into the bed former and evaporated to a predetermined moisture level at 4';C. After electrofocusing for 16 h at 8 watts, the gel bed was divided into 30 segments using the divider supplied by LKB. The active fractions were desalted and concentrated in an Amicon (Minicon) ultrafiltration cell. The a-amylase so- lutions were stable when stored at 2°C. 2.2.5 Preparation of undamaged starch. Wheat (2 kg Champ- lein) was milled in a Biihler laboratory flour mill, type MLU- 202. The first and second break fractions were combined and

20°C [ll].

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water was added to give a stiff dough from which the starch was washed by hand agitation in water. The starch (350 g approximately) was centrifuged at 2000 xg, and I est jpended in 4 1 ofwater. This washing procedure was repeated t vice and the clean starch was then freeze dried. The starch obtained had a damage value of - 5.7% as determined by the method described by Farrand [ 101. 2.2.6 Determination of a-amylase adsorption. Undamaged starch (100 mg) was slurried in 2 ml distilled water. To this was added a further 0.5 ml of water containing 0.5 units of the enzyme to be tested. After gentle mixing for 2 min, the starch was removed by a centrifugation at 2500 xg and the a-amylase activity remaining in the supernatant was assayed. For each test, a control without starch was also assayed. The difference in a-amylase activity between the two solutions was used to calculate the percentage of enzyme adsorbed by the starch. 2.2.7 Undamaged starch digestion. a-Amylase solutions (0.5 ml containing 1 unit of activity) were added to 00 iiig of undamaged starch, suspended in 4.5 ml distilled water. The mixture was agitated in a rotating water bath (120 rpm) at 25'C for 16 h. The starch remaining undigested was recovered by filtration, dried and weighed. 2.2.8 Determinationofcarbohydrate profile. Samples (1 mg) of the carbohydrate released from starch granules by adsorbed a-amylase, were resolved in a column of Sephadex G-25 (1.6 x 50 cms). A flow rate of7.2 ml/h was used and the eluate was monitored using a total carbohydrate autoanalysis technique based on the method of Dische [14]. A starch hydrolysate of known composition was used as a reference to identify the components of the samples analysed.

3 Results and Discussion

3.1 a-Amylase Activity During Germination

Seeds sampled daily over a 7-day germination period were ground in a mortar and extracted in water, (50 seeds to 50 ml water). After centrifugation (2500 xg), the supernatants were assayed for a-amylase activity on undamaged starch and were also examined by analytical isoelectric focusing, (Figs. 1 and 2). a-Amylase activity appeared at day 2 and the rate of synthesis increased until day 5, whereupon it declined to reach an activity of 5.5 units/seed after 7 days germination. The increase in hydrolytic activity of the extract on undamaged starch during germination closely followed that of a-amylase

6 1 - -Starch Hydrolysis

i n 1 Germination (days )

Figure 1, Comparison of the appearance of a-amylase activity in crudeextracts duringgermination with the activities ofthe extracts on undamaged starch. (Starch hydrolysis : 100 mg undamaged starch digested with 0.25 ml crude extract.)

activity, confirming that this enzyme was directly responsible for raw starch digestion. The zymogram (Fig. 2) shows that the a-amylase of germinated wheat is composed of two groups of isoenzymes with different isoelectric points (PI). Four major a-amylase components were found in one group (group I) and three in the other group (group 11). The PI values of the four major group I isoenzymes and the three group I1 isoenzymes were 6.0 - 6.5 and 4.5 - 4.8 respectively. The former values agree closely with those found by Tkatchuk and Kruger [l] for thea- amylases in germinated Canadian hard red spring wheat and the latter values with those found for the a-amylases of immature wheat by Marchylo et al. [15].

4

5

6

7

P I

l- Group Il isoenzymes

l- Group I isoenzymes

0 1 2 3 4 5 6 7 15821 Germination ldaysl

Figure 2. Zymogram showing the separation of the a-amylase isoenzymes by isoelectric focusing, from the extracts of germinated wheat. (Extracts heated to 70°C for 15 min to inactivate P-amylase.)

3.2 Purification and Separation of the Two Groups of a-Amylases

In order to study the action characteristics of the a-amylases on raw starch, it was necessary to isolate, purify and separate the two groups of isoenzymes. a-Amylase from germinated wheat (day 6) was purified by heat treatment, CMC chromatography and glycogen precipitation, and the two groups of isoenzymes were separated by preparative isoelectric focusing on Sephadex G-75. A zymogram showed the separation of group I and group 11. The a-amylase from immature wheat (Champlein, 20 days post anthesis) was also purified. It was shown that the a- amylase isoenzymes of the immature wheat grain were electrophoretically identical to the Group 11 isoenzymes of germinated wheat. Group I isoenzymes were totally absent in immature wheat and were only synthesised when the seed germinated. From yield data, it was calculated that the group I1 isoenzymes represented 15.8% of the total a-amylase present in the extract of 6-day germinated wheat.

3.3 Adsorption of a-Amylase lsoenzymes onto Undamaged Starch Granules

The adsorption characteristics of the two groups of isoenzymes onto undamaged starch were shown to be different. Group I enzymes were almost totally adsorbed, whereas those of group I1 were virtually unadsorbed. Figure 3 shows the adsorption onto, and subsequent digestion, of undamaged wheat starch in uitro by the purified isoenzymes. The ratio of enzyme to starch and pH of the suspension was the same as that found under physiological conditions after 3 days of germination (i. e. 1.4 units of a-amylase/IOO mg starch; pH 5.6).

Starch/Starke 30 (1978) Nr. 5 , S. 160-163 161

lOOr Group I

80 - E - .

- 2 u c 0 -

- Adsorption

0 - Degradation

Figure 3. starch granules and subsequent starch digestion.

The starch was examined by scanning electron microscopy after 16 h digestion. The granules from the group I digestion showed the characteristic erosions especially around the equatorial groove region of the larger A granules (16). There was no evidence of granule erosion found in the group I1 digestion. Clearly, the adsorption of or-amylase to wheat starch is necessary before degradation of thegranule can occur.

As the group I isoenzymes were absent in the immature (green) seed, the adsorption of both types of a-amylase to immature starch was examined. Starch was extracted from immature wheat at 24,31,41 and 45 days after anthesis, and by a series of aqueous sedimentations, the starch was isolated, washed and then freeze dried. Differences in the adsorption of both groups of isoenzyme were again shown. Figure 4 shows that the adsorption of group I isoenzymes was relatively constant (90 - 100Y0) for each starch. However, the group 11 isoenzymes were adsorbed relatively strongly to starch extracted from grains 26 days after anthesis but as the granules matured, the adsorption decreased to a minimum of 6%. This suggests that these group I1 a-amylase isoenzymes may be active at the surface of the developing starch granules in the immature wheat seed. Possibly, an equilibrium is established, between a-amylase and the starch-synthesising phosphorylase enzymes, which controls the growth of the developing starch granules [17].

Adsorption of a-amylase isoenzymes onto undamaged

- High Mot Wt Material Oligasaccharides

; & O - A 4

\ f E 1 --1 Q , X Group 11

0' i5 i o i5 io d5 @g Days (after anthesis1

Figure 4. Adsorption of the Group I and Group I1 isoenzymes to immature wheat starch.

3.4 Products of Starch Hydrolysis

The products of hydrolysis, released from the surface of undamaged starch granules by the adsorbed group I a- amylases, were removed by continuous elution of the starch with water. The starch (1 g) was packed into a column (1

x 1.5 cm) and 14 units of or-amylase activity were adsorbed. The column was eluted (10 ml/h) for 16 h into liquid N, and the frozen eluant was freeze dried before analysis. Figure 5 shows a Sephadex G-25 fractionation of 1 mg of the carbohydrates released from starch by the adsorbed group I isoenzymes. The carbohydrate consisted of 42.1 O/O high molecular weight material, 47.1 % oligosaccharides and 10.8% maltose, maltotriose and maltotetraose. The high molecular weight material (approximately 5000 mol. wt.) was totally excluded from the gel, and therefore probably represented species of a minimum average chain length of 28 glucose residues. The high molecular weight material was also readily hydrolysed by the non-adsorbing group I1 isoenzymes resulting in the formation of more oligosaccharides and low molecular weight species. Figure 6 shows the hydrolysis of 1 mg of released carbohydrate by 0.1 units of group I1 a- amylase after 4 and 16 h.

I t -

100 90 80 70 60 50 m Elution Volume ( m l l

Figure 6. drate further hydrolysed by 0.1 units of Group I1 isoenzymes.

Whilst there seems to be no doubt that the function of the group I a-amylase isoenzymes is to initiate the degradation of the starch granule, the exact function of the group I1 isoenzymes at germination is uncertain, as they are not adsorbed to, and do not attack, undamaged starch. However, they are active on soluble starch and their function could be to degrade the high molecular weight carbohydrates and oligosaccharides as they are released from the granule by the adsorbed group I isoenzymes. This may be a particularly important function during the early stages of germination, before there is an excess of group I a-amylase available to degrade further the higher oligomers released from the granule, and therefore render them more accessible as an energy source for the germinating seedling.

Sephadex G-25 fractionation of 1 mg released carbohy-

Summary

Isoelectric focusing in polyacrylamide gels has been used to study the appearance of a-amylase isoenzymes during the

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germination of wheat. Two distinct groups of isoenzymes, both synthesised during germination, have been identified. These have been purified and separated from one another, and a fundamental difference in their action on starch has been observed. The four group I isoenzymes (PI 6.0-6.5), representing 84% of the total a-amylase activity, were adsorbed readily onto undamaged starch granules and subsequently degraded them. The three group I1 isoenzymes (PI 4.5 -4.8) were not adsorbed onto mature starch granules and did not hydrolyse them. However, these group I1 isoenzymes were electrophoretically identical to the a- amylases found in immature wheat (24 days post anthesis), and they could be adsorbed onto immature starch granules. However, this affinity decreased rapidly as the starch matured. The function of the non-adsorbed group I1 isoenzymes at germination is uncertain but it is possible that they are present to degrade the high molecular weight (5000 mol. wt.) carbohydrate and oligosaccharides that are released from granules by the adsorbed group I isoenzymes. The experimental evidence suggests that the adsorption of wheat a-amylase to wheat starch is a prerequisite for undamaged starch digestion.

Acknowledgements

The authors wish to thank Dr. J . Taggart and Dr. R. E . Angold for their encouragement and active discussion during the course of the work. We also thank Miss J . E . Foyle for her excellent technical assistance.

References [I] Tkachuk, R., and 1. E. Kruger: Cereal Chem. 51 (1974), 508. [2] Jacobsen, V.,J. G. Scundaliosandd. E. Varnrr: Plant Physiol. 45

(1970), 367. [3] Paleg, L. G . , B. G. Coornbeand M. S. Butrose: Plant Physiol. 37

(1962), 798. [4] Briggs, D. E.: J. Inst. Brew. 69 (1963), 13. [5] Vurner, J . E., G. R. Chandraand M . J . Chrispeels: J. Cell. Comp.

Physiol. 66 (1965), 55. [6] Briggs, D. E. : “Biosynthesis and its Control in Plants”, 219,

Milborrow (Ed.), Academic Press, New York 1973. [7] McLaren, A. D.: Enzymologia 26 (1963), 237. 181 Walker, G. J . , and P. M. H o p e : Biochem. J. 86 (1963). 452. [9] Smith, D. B. : Communication to Technicon Meeting, Novem-

ber 1970. [lo] Farrand, E.: Cereal Chem. 41 (1964), 98. [ l l ] Bernfeld, P. : Methods in Enzymology, I, 149. Ed. by Colobbich

and N . 0. Kaplan, Academic Press, New York 1955. [I21 Kruger, J . E., and R. Tkuchuk: Cereal Chem. 46 (1969), 219. [13] Loyter, A,, and M. Schramm: Biochem. et Biophys. Acta 65

(1962), 200. [34] Dische, Z. , L. B. Shettles and M. Osnos: Arch. Biochem. 22

(1949), 169. [IS] Marchylo, B., J . E. Kruger and G . N . Ircine: Cereal Chem. 53

(1976), 157. [16] Evers, A . D., and E. E. McDermott: Stirke 22 (1970), 23. [17] De Fekete, M. A . R., and G. H . Vieweg: PI. Carbohydrate

Biochem., 127, Pridham(Ed.), Academic Press, NewYork 1974. Address of authors: J . G. Sargeant and T. S. Walker B. Sc., Phd., R H M Research Ltd., (The Lord Rank Centre of Research), Lincoln Road, High Wycombe, Buckinghamshire (Great Britain). (Received: September 27, 1977)

Insoluble Starch Xanthate : Preparation, Stabilization, Scaleup and Use

By R. E. Wing, 6. K. Jasberg and L. L. Navickis, Peoria, Illinois

The addition of magnesium sulfate to xanthation mixtures of Unlosliche Starkexanthate: Darstellung, Stabilisierung, Herstellung crosslinked starch aided isolation, scaleup and room-temperature groDerer Mengen und Verwendung. Der Zusatz von Magnesiumsulfat product stability on storage. Pilot-plant scaleup of laboratory- zu Xanthationengemischen vernetzter Starke fordert die Isolierung, developed procedures also yielded water-insoluble products effective Meng.enherstellung und die Produktstabilitat bei Raumtemperatur. in removing heavy metals from water. Several different processing Die Ubertragung von Laborverfahren in den PilotmaBstab ergab methods are presented to show the ease of preparation and use of the ebenfalls wasserunlosliche Produkte, die geeignet waren. products. Schwermetalle aus Wasser zu entfernen. Verschiedene

Verfahrenswege werden dargelegt, um die einfache Herstellung und die Verwendung der Produkte aufzuzeigen.

1 Introduction

I t has previousIy been shown that water-soluble starch xanthates in combination with cationic polymers form polyelectrolyte complexes that effectively remove heavy metal cations from water [l - 31. Further studies revealed that the cationic polymer could be eliminated by xanthating a highly crosslinked starch to give a water-insoluble product [4 - 81. This product in the sodium form, although effective in heavy metal removal when stored at O’C, was difficult to isolate in a room-temperature stable form. It has been shown [4] that the insoluble-starch xanthate (ISX) increased in room-temperature stability if the product was converted to other salt forms. The order of stability was Mgf2 >> Ca+2

> Na’ > NH;. A detailed description is given on how the addition of magnesium sulfate aids processing of the product and yields a product with improved room-temperature stability. Data are also presented on ways to use ISX for optimum effectiveness in treatment of industrial wastewaters.

2 Experimental

2.1 General Laboratory Xanthation of Crosslinked Starch

A highly crosslinked starch (100 g) is slurried in water (435 ml), and sodium hydroxide (45 g) in water (125 ml) is added. This mixture is stirred 30min. Carbon disulfide

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