An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches

Biochimica et Biophysica Acta 1525 (2001) 29^36

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An investigation of the action of porcine pancreatic K-amylase on native and gelatinised starches Suzanne L. Slaughter, Peter R. Ellis, Peter J. Butterworth * Biopolymers Group, Division of Life Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford St., London SE1 8WA UK Received 25 July 2000; received in revised form 19 October 2000; accepted 19 October 2000

Abstract The action of pancreatic K-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine K-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0 ] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat /Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100³C, kcat /Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat /Km is not a simple one. The value of kcat= Km rises to reach a maximum at a pre-treatment temperature of 75³C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although K-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : K-Amylase ; Starch; Catalytic e¤ciency; Retrogradation

1. Introduction Starch is an important carbohydrate in the human diet and contributes signi¢cantly to the exogenous supply of glucose and total food energy intake. In a typical British diet, starch contributes about 60^70% of the `available' or `glycaemic' carbohydrate [1,2], which is de¢ned as the carbohydrate fraction digested by K-amylase in the upper gastrointestinal tract and absorbed into the portal blood (mainly as glucose). The traditional view of starch digestion, as seen in many standard textbooks of biochemistry

* Corresponding author. Fax: +44-20-7848 4500 ; E-mail : [email protected]

and physiology, is that starch, whatever the source, is digested rapidly at more or less the same rate and to the same extent. It has been known for many years, however, that in response to di¡erent starch-rich foods containing isoglucidic amounts of available carbohydrate, large di¡erences can be found in the postprandial rise in blood glucose and insulin levels [3]. This awareness led to starchy foodstu¡s being ranked according to their postprandial glycaemic and insulinaemic indices [4,5]. Numerous digestibility studies, in vitro and in vivo, have demonstrated that starchy foods are digested at markedly di¡erent rates [1,6,7]. Also, it is known that a variable fraction of ingested starch can escape digestion in the small intestine altogether, and is later fermented by bacterial enzymes in the colon, the main products of which are short chain fatty acids, e.g. butyric acid [1,8^10].

0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 1 6 2 - 8

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A full explanation for the observed di¡erences in the glycaemic index of foods remains to be established but several factors are of undoubted importance. These include (a) the macrostructure and properties of starchy foods (e.g. plant tissues containing intracellular starch granules, starch-gluten matrix of wheat bread), (b) physico-chemical structure and properties of the starch per se at the granule and molecular levels (e.g. granule size and amylose:amylopectin ratio), which vary enormously depending on botanical source, and (c) presence of other dietary components (e.g. dietary ¢bre and lipid) and anti-nutrients (e.g. proteinaceous K-amylase inhibitors). [7,11^16]. Many of these factors will be in£uenced by food processing conditions (e.g. hydrothermal treatment gelatinises the starch) [14]. The presence of dietary ¢bre (mainly non-starch polysaccharides) in particular, can strongly inhibit the rate and extent of digestion of available carbohydrate in a number of ways, depending critically on the type of polysaccharides consumed [7,17^ 19]. Pancreatic K-amylase is present in high activity in the lumen of the small intestine [20,21] and it has been widely assumed that the enzyme does not contribute signi¢cantly to the rate-limiting process governing carbohydrate digestion and absorption. Because of di¡erences in the assay systems and in the de¢nition of a unit of activity, calculations of amylase concentration based on modern knowledge of the turnover numbers of homogeneous enzyme preparations cannot be made precisely. If it is assumed however, that human and porcine K-amylases have similar turnover numbers, activity values reported in the literature [20,21] suggest a range of concentrations from 5 to 15 nM approximately. This translates into substantial amounts of catalytic activity but to be e¡ective, the enzyme must interact directly with its substrate, a process that will be greatly in£uenced by the structure of the starch granule. The enzyme's known speci¢city for K-1,4 glycosidic linkages and the knowledge that many botanical starches have a high amylopectin content also imposes a limit on the catalytic action of amylase to 3 or 4 glucose residues distant from an K-1,6 branch point. The properties of salivary and pancreatic amylases have been studied for many years culminating in elucidation of 3-D structures for human and porcine pancreatic amylases by X-ray crystallography [22,23]. The structures are an excellent resource for

interpreting observed catalytic properties, but in spite of the long history of study of amylases there is a paucity of reliable experimental data apart from one or two notable exceptions [24,25]. Standard enzyme kinetic work is based on a model in which interaction occurs between enzyme and substrate molecules in solution. Although the starch may be gelatinised, it is not in true solution and as stated above, its structure will be greatly in£uenced by the botanical source and previous processing history (e.g. heat treatment, milling). We now report a kinetic study of K-amylase acting on starches of several sources both in granular and gelatinised forms and consider the implications of a soluble enzyme acting upon a solid substrate. The varieties were chosen on the basis of their known di¡erences in physico-chemical properties, amylose/amylopectin content and the fact that these starches make a signi¢cant contribution to human diet. 2. Materials and methods 2.1. Starch granules and reagents Puri¢ed starch granules, with their moisture contents in brackets, of wheat (9.1%), potato (18.5%), normal rice (11.2%) and waxy rice (10.5%) were gifts from respectively Rank Hovis MacDougal Research Ltd. (High Wycombe, Bucks, UK), the National Starch and Chemical Corporation (Manchester, UK) and Cairn Chemical Ltd. (Chesham, Bucks UK). Isolated amylopectin was a gift from Unilever Research (Sharnbrook, Beds, UK). Porcine pancreatic K-amylase (type 1) was obtained from Sigma Chemical Co. (Poole, Dorset, UK). This is a twice crystallised product that is free of other proteins. General chemicals were purchased from BDH Laboratory Supplies (Poole, Dorset, UK). Some characteristics of the various starches are shown in Table 1. 2.2. Preparation of starch suspensions Starch granules were suspended in PBS [26], pH 7.4 and agitated gently by swirling the mixture for 20 min in a conical £ask. This was carried out either at room temperature and so designated `native' or in a heated water bath

Table 1 Characteristics of native and fully gelatinised starch samples Starch source and type

Damaged starch (%)

Total amylose content (%)

Solubility of gelatinised starch (%)

Proportion of amylose in solubles (%)

Swelling power (g/g)

Wheat Potato Normal rice Waxy rice

4 3 1 61

27.5 þ 1.1 23.2 þ 1.8 16.1 þ 0.2 1.4 þ 0.2

28.2 þ 3.0 20.5 þ 1.3 21.5 þ 0.3 23.8 þ 0.9

99.0 þ 0.3 99.9 þ 4.5 13.9 þ 0.2 5.1 þ 0.5

22.2 þ 0.6 35.1 þ 1.4 24.2 þ 0.3 35.4 þ 0.4

The results for damaged starch are the mean values of two determinations but all other entries in the table refer to the mean and S.E.M. of four determinations.

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for experiments where the e¡ects of heat-treatment were to be investigated. The £asks were sealed with glass marbles to restrict water loss by evaporation during heating and the £ask and contents were weighed both prior to and after the 20 min period so that any losses could be made good. The concentration of the starch suspensions was 1% for most `routine' assays but suspensions ranging in concentration of 0.05^1% were used in assays in which kinetic parameters were being determined. The suspensions were not stored but prepared fresh and used immediately for each experiment. 2.3. Starch damage The estimate of the percentage of granules that had been damaged during the puri¢cation of the native samples was estimated from the fraction, examined by light microscopy, of the granules that become stained by 0.2% aqueous Congo Red solution. Only damaged granules take up the dyestu¡. Duplicate counts were performed and 100 granules were counted in each case. 2.4. Starch solubility and swelling Swelling was determined by the method of Leach et al. [27]. Known quantities of heat treated starch samples were centrifuged at 2500 rpm for 20 min. The supernatant was removed and the sediment weighed. Aliquots of the supernatant were dried in an oven at 120³C to constant weight and the residue weighed. The swelling power (g/g on a dry weight basis) was calculated from the formula : (mass of sedimented gel)/(mass of total starch3mass of soluble starch). 2.5. Estimation of amylose Portions of 0.1 ml of solubilised starch preparations or of supernatants from heated starch suspensions, were added, with mixing, to 5 ml of 0.5% trichloroacetic acid followed by 0.05 ml of I2 -KI solution (1.27 g I2 +3 g KI per litre). After immediate mixing, colour was allowed to develop for 30 min. before reading the absorbance at 620 nm. Amylose concentrations per litre of solution were approximated by multiplying the absorbance values by a factor of 45.8 [28] after allowance for a dilution factor of 51.5. 2.6. Di¡erential scanning calorimetry (DSC) Measurements of gelatinisation: onset (To ), peak (Tp ) and conclusion (Tc ) temperatures and enthalpy of gelatinisation were made on a Perkin-Elmer DSC-7 calorimeter. Small samples (15^35 mg) of starch contained in a slurry with PBS, pH 7.4, were hermetically sealed in 40 Wl aluminium pans, placed in the DSC machine and heated from 20 to 90³C at a rate of 5³C/min. Values of To , Tp and vH

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were calculated from data output obtained from 3 or 4 runs for each starch sample. The ¢nal starch concentrations were in the range of 17^25% (w/w). 2.7. Digestion of starch with porcine pancreatic K-amylase Aliquots (3 ml) of freshly prepared starch suspension were transferred to 6 ml plastic Falcon tubes which were placed on a rotating table (33.3 rpm) at a ¢xed angle to give constant end-over-end mixing. The table was located in an incubator maintained at 37³C. After 30 min, 10 Wl of a suitably diluted solution of amylase in PBS containing 0.1 mg/ml BSA was added to the tubes. At 30 s, and then at further timed intervals of a few min up to a total of 120, aliquots of 200 Wl were removed from each reaction tube and immediately spun in a microfuge for 30 s. to sediment undigested starch. The supernatant was then transferred to a new microfuge tube and placed in boiling water for 2 min to inactivate the enzyme. Preliminary work established that this exposure to boiling water inactivated the enzyme completely. The samples were then frozen for later analysis of reducing sugar content. Unless otherwise indicated, the concentration of porcine pancreatic amylase in reaction mixtures was maintained at 0.033 units per ml where 1 unit, as de¢ned by the suppliers, will liberate 1 mg of maltose from starch in 3 min. This unit is equivalent to 16.2 nanokatal. Enzyme concentrations [E0 ] were calculated using a molecular weight of 56 000 for amylase and the supplier's estimate of a mean activity of 1000 units per mg protein. This means that for routine assays the concentration was assumed to be 0.59 nM. Measured activities are expressed as WM maltose formed per min. Our units can be converted to nanokatal by multiplying by the factor 0.05. 2.8. Determination of reducing sugar Frozen samples were allowed to thaw on ice and mixed thoroughly once they were fully defrosted before assay using a slight modi¢cation of a method that depends on the production of Prussian Blue [29,30]. Equal portions (0.5 ml) of sample (diluted with distilled water if necessary), of solution A (16 mM KCN, 0.19 M Na2 CO3 ) and of solution B (1.18 mM K3 Fe (CN)6 ) were mixed in glass test tubes capped with glass marbles. The tubes were placed into a boiling water bath for exactly 15 min. and then removed and allowed to cool at room temperature for approximately 5 min. Once cool, 2.5 ml of solution C (3.11 mM NH4 Fe(SO4 )2 , 0.1% sodium dodecyl sulphate, 0.42% v/v H2 SO4 ) was added to the tubes which were then left for at least 2 h to allow colour development to occur. The absorbance was then read on a Cecil CE 2041 spectrophotometer at 690 nm. Maltose standards covering the range 0^10 WM in the ¢nal assay solution were assayed by the same procedure. Reducing sugar concentrations are expressed as maltose equivalents. Preliminary experiments

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established that the presence of protein, which probably contaminates some starch preparations, contributes to the colour yield and so slightly increases both blank and test values in the assay. The linearity or sensitivity of standard curves up to a level of 8 WM maltose are not a¡ected by the presence of protein however. Therefore all samples were routinely diluted to fall within this range and readings were taken using blanks containing amounts of inactivated enzyme and starch equivalent to that present in test assays. 2.9. Kinetic parameters Data obtained from measurements of initial reaction rates at various substrate concentrations were treated by weighted non-linear regression [31] for the determination of values for Km , Vmax and kcat /Km . The initial rates were calculated from analysis of samples taken at 30 s and then at intervals of 2 min up to 10.5 min. 2.10. Kinetics of reactions involving insoluble substrates As pointed out many years ago [32], the reaction between K-amylase and starch is a two-phase system and if the reaction mechanism involves a kinetically signi¢cant absorption step, the relationship between reaction rate and enzyme concentration is not exactly 1:1. Following the argument set out in [32], the number of enzyme molecules bound per unit of surface area (Es /As ) is related to the concentration of the enzyme in the aqueous or bulk phase (Eb ) by the Freundlich equation: Es =As ˆ K Eb n where K is a partition coe¤cient and n equals 2/3 for enzyme adsorbed on a perfectly smooth surface. For adsorption on edges and/or into cracks in the surface, n is predicted to lie between 1/3 and 2/3 ([32] and citations therein). Equating Es /As with EAs , which is the `concentration' of adsorbed enzyme and is analogous to an enzyme^substrate complex: the initial rate v is given by the equation: v ˆ kcat EAs i:e: v ˆ kcat K Eb n The total enzyme concentration E0 = [EAs +Eb ] so that: v ˆ kcat K ‰E0 3EAs Šn If the fraction bound is very small i.e. EAsIE0 : logv ˆ nlogE0 ‡ log…kcat K† Thus for enzymes acting on insoluble substrates the rate is not directly proportional to the total enzyme concentration assuming that n is less than 1. A corollary is that a linear plot of log v against E0 with a slope n of less than one shows that the fraction of enzyme molecules bound productively is small compared with the total amount in the system.

Fig. 1. Freundlich plot for pancreatic amylase acting on 1% native wheat starch. Samples were taken from the reaction mixture at 30 s intervals up to 10.5 min before determination of the amount of reducing sugar formed and the calculation of activity. The concentration of enzyme in the reaction mixtures ranged from 0.59 to 59.0 nM. The reaction velocity v is given as WM maltose formed per min and [E0 ] is expressed in nM terms. The plot is linear with a slope of 0.69. The correlation coe¤cient for y on x is 0.98

3. Results The rate of amylolysis of native and heat-treated starch suspensions was essentially linear for up to 2 h but the rate over the ¢rst 60 s or so was often higher than over the remaining time course. Thus in some of the experiments described below, data are presented both for measurements made over the ¢rst 30 s of reaction time to catch this faster rate and for results recorded over a standard period of 0.5^10.5 min during which the reaction settled to a constant rate. Measurements were not extended beyond 20 min so as to avoid complications arising from inhibition of the reaction by the products, principally maltose. Fig. 1 shows a Freundlich logarithmic plot for the action of a range of 0.59^59.0 nM concentrations of amylase on native wheat starch. The plot is linear with a slope of 0.69 that is signi¢cantly di¡erent from unity. These data refer to rates determined over 0.5^10.5 min of reaction but Table 2 summarises the results obtained for several di¡erent botanical starches both for the initial 30 s measurements and for the 10.5 min ones. It is noticeable that with the exception of native waxy rice and the amylopectin suspension, the rate during the ¢rst 30 s. provides a value for n of unity, i.e. a kinetically signi¢cant adsorption step is not involved. For the longer time period n values signify adsorption and in most cases the values are less than 2/3. This is probably to be expected since the granules are likely to be cracked and cratered and edge e¡ects come into play [32]. When the starch has been fully gelatinised by pre-treatment at 100³C the n values determined from a 10.5 min assay approximate more closely to the theoretical one of 2/3.

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S.L. Slaughter et al. / Biochimica et Biophysica Acta 1525 (2001) 29^36 Table 2 Values of n obtained from the slopes of Freundlich plots Starch source and type

A (n)

Bn

Wheat (native) Wheat (gelatinised) Potato (native) Potato (gelatinised) Normal rice (native) Normal rice (gelatinised) Waxy rice (native) Waxy rice (gelatinised) Amylopectin suspension Amylopectin solution

0.9 þ 0.027 0.84 þ 0.01 1.00 þ 0.08 0.86 þ 0.02 1.00 þ 0.03 0.94 þ 0.02 0.48 þ 0.03 0.98 þ 0.01 0.62 þ 0.03 0.99 þ 0.02

0.68 þ 0.007 0.73 þ 0.011 0.49 þ 0.009 0.71 þ 0.008 0.51 þ 0.005 0.71 þ 0.002 0.50 þ 0.024 0.72 þ 0.024 0.62 þ 0.006 0.91 þ 0.005

Rate^enzyme concentration data for amylase acting on various starches were analysed. Starch was used both in native form and after gelatinisation. The values in A refer to rate measurements made over an initial 30 s assay period and those in B are calculated from catalytic rates determined over the following 10.0 min. All values represent the mean and S.E.M. of four determinations in each case.

Product formed during the early stages of the reaction probably arises from fragments of amylose (and possibly small amounts of low molecular weight amylopectin) that are leached from starch granules during preparation of the suspension and/or from polysaccharide chains of relatively short length that protrude from the mass of the compact granule [33]. Thus at this stage, amylase is acting on soluble substrate and `normal' enzyme kinetics apply. Waxy rice starch granules are compact structures containing mainly amylopectin (i.e. 6 1% (w/w) amylose) and therefore produce very little amylose by leaching. Therefore the amount of solubilised amylose in the preparation is small (Table 1) so that an adsorption step is involved even during the earliest stages of the reaction. Thus n is less than unity for the early reaction also. In experiments where catalytic properties of di¡erent starches were being compared, the enzyme concentration was carefully adjusted to ensure that the same concentration was used throughout so as to minimise anomalies arising from the Freundlich relationship between v and [E0 ]. It is well known that amylase action on starch granules obeys Michaelis^Menten kinetics [24] and so di¡erences in measured Vmax values re£ect true di¡erences in

33

kcat provided that [E0 ] is constant. The values for the kinetic parameters calculated from non-linear regression ¢tting of data obtained in experiments conducted with varying substrate concentrations are shown in Table 3. All reaction rates were determined over an incubation period of 0.5^10.5 min. Km is expressed in terms of percentage because starch is of unknown molecular mass and so kcat / Km values are of non-standard physical dimensions but are still useful for comparing the e¤ciency of the amylolytic process. The Km values for native starches from the four di¡erent sources tested are all very similar and in the range 0.5^ 0.7%. Heat treatment at 100³C decreases Km markedly and suggests that enzyme accessibility to the starch is greatly improved by the order^disorder transition induced by heating. Vmax data are more di¤cult to interpret in that heating of wheat and potato starches increases Vmax but rice starches are a¡ected very little, if at all. The relative catalytic e¤ciencies of the native starches, as assessed by kcat /Km , di¡er signi¢cantly, with the rice starches appearing to be better substrates for amylase. Following heat treatment of the same starches, the kcat /Km ratios increase enormously, with increases ranging from 13-fold for waxy rice to 239-fold for potato, relative to the native samples. The relatively smaller increase seen for waxy rice may be connected with the low amylose content of this starch. Native amylopectin is a good substrate (Table 3) and heat treatment seems to bring about little enhancement of its susceptibility to amylolysis. The marked increase in catalytic e¤ciency for heat-treated potato starch is particularly interesting. One possible explanation for this is that phosphorus, which is present in the highest amounts in potato starch compared with the others tested, a¡ects the properties of starch. Phosphate ester groups are attached to O-6 and O-3 positions of the K-D-glucopyranosyl units of the amylopectin fraction of potato giving starch a slight negative charge, which may contribute to rapid swelling of the granules during hydrothermal treatment [34]. The phosphate may also inhibit the rate of starch retrogradation [34]. This e¡ect on retrogradation may be trivial however, given that the phosphate is associated with the amylopectin, rather than the amylose fraction

Table 3 Kinetic parameters for porcine pancreatic K-amylase acting on a range of starches Substrate

Km (%)

Vmax (WM min31 )

kcat (WM min31 U1035 )

kcat /Km U1035

Wheat (native) Wheat (100³C) Potato (native) Potato (100³C) Normal rice (native) Normal rice (100³C) Waxy rice (native) Waxy rice (100³C)

0.51 þ 0.08 0.13 þ 0.00 0.58 þ 0.09 0.04 þ 0.00 0.70 þ 0.02 0.03 þ 0.00 0.68 þ 0.04 0.07 þ 0.01

11.03 þ 1.3 135.6 þ 4.1 7.48 þ 0.8 129.3 þ 2.0 73.6 þ 1.5 72.3 þ 0.2 104.7 þ 5.2 136.9 þ 5.0

0.14 þ 0.02 1.66 þ 0.05 0.09 þ 0.01 1.59 þ 0.03 0.9 þ 0.02 0.9 þ 0.00 1.35 þ 0.06 1.68 þ 0.06

0.27 12.5 0.16 38.2 1.3 34 1.9 25.5

The starch concentration in reaction mixtures ranged from 0.05 to 1.0% and K-amylase was included in reaction mixtures at a constant concentration within the range of 0.75^0.81 nM. The kinetic parameters plus standard errors were obtained from ¢tting of experimental data to the Michaelis^Menten equation by weighted regression [31].

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Fig. 2. E¡ect of heat treatment of starch on susceptibility to amylolysis. Suspensions of native wheat starch (1% w/v) were heated at the indicated temperatures for 20 min with constant swirling. The starch was then used as substrate in concentrations ranging from 0.05^1.0 in reaction mixtures at 37³C. Kinetic parameters were obtained by weighted regression analysis.

and it is the latter which retrogrades relatively rapidly to produce a product that is resistant both to solubilisation in water and to the action of amylase (the so-called `resistant starch') [33^35]. It seems that the polysaccharide can adopt an altered secondary structure consequent to the heating^cooling cycle, which makes the starch more resistant to amylolysis. Over the time frame of the experiments, retrogradation of amylopectin would be expected to be much less than that of the amylose fraction. The e¡ect on catalytic e¤ciency of pre-heating wheat starch is presented in more detail in Fig. 2. The kcat /Km value rises sharply above 65³C but then decreases again if the pre-treatment process is conducted above 75³C. The

Fig. 3. E¡ect of heat on amylose leaching. Suspensions of wheat starch were treated as described in the legend to Fig. 2 and then samples were analysed for total soluble carbohydrate and for amylose. a, Total soluble carbohydrate; O, solubilised amylose (note that above 75³C, leached amylose represents 90^99% of the total soluble carbohydrate).

value of kcat increased approximately 11^12-fold with pretreatment at temperatures up to 75³C but then remained relatively unchanged. The fall in catalytic e¤ciency that was associated with treatment at the higher temperatures came about through a 4-fold rise in Km (0.03% and 0.13% for starch treated at 75 and 100³ respectively). Over this temperature range, the swelling power increased to a value of 23 g/g approximately. Fig. 3 shows the soluble material leached from starch resulting from heat treatment and indicates that almost of the material is amylose. As mentioned previously, retrograded amylose is a poor substrate for amylase action [33]. Under the conditions of bu¡er composition and pH used in the pre-heating processes, To , Tp , and Tc were 61.2, 65.9 and 70.9³C respectively. Leaching occurs only to a very limited extent before the temperature reaches the Tc point (Fig. 3) but then there is a dramatic increase in amylose release once Tc (the `melting temperature' of the starch) is exceeded. Fig. 2 indicates that the marked rise in kcat /Km spans the temperature region of Tc . The increased amount of leached amylose resulting from starch subjected to temperatures in excess of 71³C almost certainly becomes retrograded however [35] and is therefore associated with the sharp fall in kcat /Km . Fig. 4 shows that an inverse relationship exists between catalytic e¤ciency and the solubility of the di¡erent starches after gelatinisation. At ¢rst sight this result seems counter to common sense, but taking into account the raised probability of retrogradation accompanying the rise in solubility, the fall in kcat /Km can be accounted for. A similar explanation can be applied to the results obtained with waxy rice; although this starch consists mainly of amylopectin which retrogrades slowly, the solu-

Fig. 4. Relationship between the solubility of fully gelatinised starch and susceptibility to amylolysis. Catalytic e¤ciencies (kcat /Km values) for the several starches investigated in this paper are plotted against the measured solubility of the fully gelatinised materials. In order of solubility the plotted points represent starches from potato, normal rice, waxy rice and wheat. The error bars indicate S.E.M. values determined from four separate experiments.

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bilised fraction in our experiment is likely to consist of material with relatively short chain lengths that is known to retrograde more rapidly than bulk amylopectin [36]. 4. Discussion Studies of the catalytic properties of K-amylase are sometimes conducted using small-molecular weight arti¢cial substrates such as p-nitrophenyl K-D-maltoside [37] but while such measurements can provide extremely valuable information, we decided to use the natural substrate starch to model, very simply, the action of the enzyme in vivo. The arti¢cial substrates derived from maltose have relatively large Km values (typically 15 mM approximately) [37]. The 3-D structures reveal the presence of 7^11 subsites for sugar residues in the active site cleft [22,23] and so the small substrates probably lack the structures required to ensure maximal binding. The K-amylase of Bacillus subtilis has been shown to adsorb to crystalline starchy materials and the binding is a prerequisite of catalysis [25]. Acid treatment of the starch particles prevented amylase adsorption; a ¢nding that was interpreted by assuming that the acid degraded amorphous zones of semi-crystalline starch, thus leaving only highly ordered material to which the enzyme binds poorly [25]. Fungal cellulases possess characteristic cellulose binding domains that seem to anchor the enzyme to the cellulose ¢bres so that the cellulose chain can be threaded into the active site for hydrolysis [38]. The cellulases seem to be a useful model for carbohydrases in general because those K-amylases for which 3-D structures are available [22,23,39] are seen to possess a domain that is probably important for polysaccharide binding. Glucoamylase 1 from Aspergillus niger also possesses a starch binding domain [40,41]. A kinetically important adsorption step is suggested by our results where it is shown that the relationship between reaction velocity and enzyme concentration is described by the Freundlich equation. The binding step is only of kinetic signi¢cance when the enzyme is acting on particulate starch however. When acting on soluble starch fragments, as for instance during the very early stages of our assays, the reaction can be described by conventional Michaelis^Menten kinetics i.e. the rate is directly proportional to the enzyme concentration. Systems where small arti¢cial substrates are employed behave in the same way [37]. Values for Km and for kcat are greatly in£uenced by both the starch source and by treatments to gelatinise the starch. There have been many reasons proposed for the di¡erences in susceptibility to amylolysis of di¡erent starch foodstu¡s [33]. The degree to which amylose leaching occurs during gelatinisation is important, as discussed above, but other physical-chemical properties are also of likely signi¢cance. For instance, restrictions in the rates of di¡usion of substrate and products because of variations

35

in viscosity associated with di¡erent starch materials together with limitations on accessibility of amylase to the starch itself because of particular structural features are undoubtedly important. The number of systematic enzyme studies to test these suggestions seems to be rather few however and this paucity no doubt re£ects the inherent di¤culties presented by the complexity of the system. In conclusion, despite the very high catalytic activity demonstrable in digestive £uid, the enzymic hydrolysis of starch by K-amylase may be a limiting factor in the digestion and absorption of dietary carbohydrate because of the many factors related to the physico-chemical properties of starchy foods. Acknowledgements The authors acknowledge the ¢nancial support of the Ministry of Agriculture, Fisheries and Food (studentship for S.S.) and SmithKline Beecham. Special thanks are extended to Professor J.D. Scho¢eld of the Department of Food Science and Technology, University of Reading, and Dr R. Angold and P. Bowler of RHM Technology Ltd., High Wycombe for providing facilities and advice for methods to characterise starch samples. We also thank Drs C Hedley and T.Ya Bogracheva of the John Innes Research Centre, Norwich for the helpful and encouraging discussions of starch properties that can a¡ect amylolysis.

References [1] British Nutrition Foundation. Complex Carbohydrates in Foods, The Report of the British Nutrition Foundation's Task Force, Chapman and Hall, London, 1991. [2] National Food Survey Committee 1997/1998, The National Food Survey ^ Annual Report on Food Expenditure, Consumption and Nutrient Intake, H.M. Stationery O¤ce, London, 1998. [3] P.A. Crapo, G. Reaven, J. Olefsky, Postprandial plasma glucose and insulin responses to di¡erent carbohydrates, Diabetes 26 (1977) 1178^ 1183. [4] D.J.A. Jenkins, T.M.S. Wolever, D.J.A. Jenkins, L.U., Thompson, A.V. Rao, T. Francis, in: G.V. Vahouny, D. Kritchevsky (Eds.), Dietary Fibre and Clinical Aspects, Plenum, New York, 1986, pp. 167^179. [5] T.M.S. Wolever, The glycaemic index, World Rev. Nutr. Diet. 62 (1990) 120^185. [6] Y. Granfeldt, I. Bjo«rk, A. Drews, J. Tovar, An in vitro procedure based on chewing to predict metabolic responses to starch in cereal and legume products, Eur. J. Clin. Nutr. 46 (1992) 649^660. [7] L. Noah, F. Guillon, B. Bouchet, A. Bule¨on, C. Molis, M. Gratas, M. Champ, Digestion of carbohydrate from white beans (Phaseolus vulgaris L.) in healthy humans, J. Nutr. 128 (1998) 977^985. [8] A.M. Stephen, A.C. Haddad, S.F. Phillips, Passage of carbohydrate into the colon. Direct measurements in humans, Gastroenterol. 85 (1983) 589^595. [9] K.R. Silvester, H.N. Englyst, J.H. Cummings, Ileal recovery of starch from whole diets containing resistant starch measured in vitro and fermentation of ileal e¥uent, Am. J. Clin. Nutr. 62 (1995) 403^411. [10] K.N. Englyst, H.N. Englyst, G.J. Hudson, T.J. Cole, J.H. Cum-

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36

[11] [12]

[13] [14]

[15]

[16] [17]

[18]

[19]

[20] [21]

[22]

[23]

[24] [25]

S.L. Slaughter et al. / Biochimica et Biophysica Acta 1525 (2001) 29^36 mings, Rapidly available glucose in foods: an in vitro measurement that re£ects the glycaemic response, Am. J. Clin. Nutr. 69 (1999) 448^ 454. P. Resmini, M.A. Pagani, Ultrastructure studies of pasta: A review, Food Microstruct. 2 (1983) 1^12. K. O'Dea, P.J. Nestel, L. Antono¡, Physical factors in£uencing postprandial glucose and insulin responses to starch, Am. J. Clin. Nutr. 33 (1980) 760^765. M.A. Pagani, D.J. Gallant, B. Bouchet, P. Remini, Ultrastructure of cooked spaghetti, Food Microstruct. 5 (1986) 111^129. S.M. Kingman, H.N. Englyst, The in£uence of food preparation methods on the in vitro digestibility of starch in potatoes, Food Chem. 49 (1994) 181^186. M. Goddard, G. Young, R. Marcus, The e¡ect of amylose content on insulin and glucose responses to ingested rice, Am. J. Clin. Nutr. 39 (1984) 388^392. B. Juliano, M. Goddard, Qual. Plant Hum. Nutr. 36 (1986) 35^41. P.R. Ellis, The e¡ects of ¢bre on diabetes, in: M. Hill (Ed.), The Right Fibre for the Right Disease, Royal Society of Medicine Press, 1999, pp. 33^42. P.R. Ellis, P. Rayment, Q. Wang, A physico-chemical perspective of plant polysaccharides in relation to glucose absorption, insulin secretion and the entero-insular axis, Proc. Nutr. Soc. 55 (1996) 881^898. C.S. Brennan, D.E. Blake, P.R. Ellis, J.D. Scho¢eld, E¡ect of guar galactomannan on wheat bread microstructure and on the in vitro and in vivo digestibility of starch in bread, J. Cereal Sci. 24 (1996) 151^160. A. Dahlqvist, B. Borgstrom, Digestion and absorption in man, Biochem. J. 81 (1961) 411^418. M.R. Fogel, G.M. Gray, Starch hydrolysis in man: an intraluminal process not requiring membrane digestion, J. Appl. Physiol. 35 (1973) 236^267. G.D. Brayer, L. Yaoguang, S.G. Withers, The structure of human î resolution and comparisons with repancreatic K-amylase at 1.8 A lated enzymes, Protein Sci. 4 (1995) 1730^1742. S.B. Larson, A. Greenwood, D. Casio, J. Day, A. McPherson, Reî ¢ned molecular structure of pig pancreatic alpha-amylase at 2.1 A resolution, J. Mol. Biol. 235 (1994) 1560^1584. G.J. Walker, P.M. Hope, The action of some K-amylases on starch granules, Biochem. J. 86 (1963) 452^462. M. Leloup, P. Colonna, S.G. Ring, K-Amylase adsorption on starch crystallites, Biotechnol. Bioeng. 38 (1991) 127^134.

[26] M.A. Harrison, I.F. Rae, General Techniques of Cell Culture, Cambridge University Press, Cambridge, 1997, p. 40. [27] H.W. Leach, L.D. McCowen, T.J. Schoch, Structure of the starch granule. I. Swelling and solubility patterns of various starches, Cereal Chem. 36 (1959) 534^544. [28] D. Chrastil, Improved colorimetric determination of amylose in starches or £ours, Carbohydr. Res. 159 (1987) 154^158. [29] J.T. Park, M.J. Johnson, A submicrodetermination of glucose, J. Biol. Chem. 181 (1949) 149^151. [30] F. Schinner, W. von Mersi, Xylanase-, CM-cellulase- and invertase activity in soil: an improved method, Soil Biol. Biochem. 22 (1990) 511^515. [31] G.G. Wilkinson, Statistical estimations in enzyme kinetics, Biochem. J. 80 (1961) 324^332. [32] A.D. McLaren, Enzyme reactions in structurally restricted systems IV. The digestion of insoluble substrates by hydrolytic enzymes, Enzymologia 26 (1963) 237^246. [33] P. Colonna, V. Leloup, A. Buleon, Limiting factors of starch hydrolysis, Eur. J. Clin. Nutr. 46 (Suppl. 2) (1992) S17^S32. [34] J.N. BeMiller, R.L. Whistler, in: O.R. Fennema (Ed.), Food Chemistry, 3rd. Edn., Marcel Dekker, New York, 1996, pp. 157^223. [35] P. Cairns, V.M. Leloup, M.J. Miles, S.G. Ring, V.J. Morris, Resistant starch: an X-ray scattering study of starch gelatinization in excess and limiting water, J. Cereal Sci. 12 (1991) 203^206. [36] J.-O. Kim, W.-S. Kim, M.-S. Shin, A comparative study on retrogradation of rice starch gels by DSC, X-ray and K-amylase methods, Starch/Sta«rke 49 (1997) 71^75. [37] E.R. Wilcox, J.R. Whitaker, Some aspects of the mechanism of complexation of red kidney bean K-amylase inhibitor and K-amylase, Biochemistry 23 (1984) 1783^1791. [38] M. Claeyssens, W. Nerinckx, K. Piens, Carbohydrases from Trichoderma reesi and other Microorganisms : Structures, Biochemistry, Genetics and Applications, Royal Society of Chemistry, Cambridge, 1998. [39] G. Buisson, E. Duce, F. Payan, R. Haser, Three dimensional strucî resolution ^ role of ture of porcine pancreatic K-amylase at 2.9 A calcium in structure and activity, Food EMBO J. 6 (1987) 3909^3916. [40] N.J. Belshaw, G. Williamson, Production and puri¢cation of a granular-starch-binding domain of glucoamyalse 1 from Aspergillus niger, FEBS Lett. 269 (1990) 350^353. [41] N.J. Belshaw, G. Williamson, Speci¢city of the binding domain of glucoamylase 1, Eur. J. Biochem. 211 (1993) 717^724.

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