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Inhibition of α-amylase activity by cellulose: Kinetic analysis and nutritional implications
Carbohydrate Polymers 123 (2015) 305–312
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Inhibition of ␣-amylase activity by cellulose: Kinetic analysis and nutritional implications Sushil Dhital a , Michael J. Gidley a,∗ , Frederick J. Warren b a ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Qld 4072, Australia b Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Qld 4072, Australia
a r t i c l e
i n f o
Article history: Received 3 October 2014 Received in revised form 22 January 2015 Accepted 23 January 2015 Available online 2 February 2015 Keywords: Cellulose Starch Inhibition Alpha-amylase Adsorption isotherm Michaelis–Menten kinetics
a b s t r a c t We report on inhibition of ␣-amylase activity by cellulose based on in vitro experiments. The presence of cellulose in the hydrolysing medium reduced the initial velocity of starch hydrolysis in a concentration dependent manner. ␣-Amylase adsorption to cellulose was reversible, attaining equilibrium within 30 min of incubation, and showed a higher affinity at 37 ◦ C compared to 20 and 0 ◦ C. The adsorption was almost unchanged in the presence of maltose (2.5–20 mM) but was hindered in the presence of excess protein, suggesting non-specific adsorption of ␣-amylase to cellulose. Kinetic analyses of ␣-amylase hydrolysis of maize starch in the presence of cellulose showed that the inhibition is of a mixed type. The dissociation constant (Kic ) of the EI complex was found to be ca. 3 mg/mL. The observed inhibition of ␣-amylase activity suggests that cellulose in the diet can potentially attenuate starch hydrolysis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Starch and cellulose are two of the most abundant biopolymers in nature. Although both are primarily (1 → 4)-linked polymers of glucose, they play strikingly different roles in terms of human and animal nutrition. Starch is composed of amylose, a mostly linear polymer of ␣(1 → 4)-linked anhydroglucose unit and amylopectin, a highly branched polymer via additional ␣(1 → 6)-linkages. Cellulose, in contrast, has linear (1 → 4)-linked anhydroglucose chains with strong inter- and intra-molecular hydrogen bonding. The reduced availability of hydroxy groups due to their involvement in hydrogen bonding makes cellulose water insoluble and limits processability and functionality in its native forms (Klemm, Heublein, Fink, & Bohn, 2005). In herbivorous ruminants, a considerable part of the caloric intake comes from bacterial fermentation of cellulosic residues in the rumen. Humans and non-ruminant animals such as pigs, however, are unable to extract energy from cellulose directly due to the absence of  glucanases and glucosidases in their intestinal tracts. Undigested cellulosic material may be partially hydrolysed by bacterial enzymes into small soluble oligomers which are either absorbed, or are fermented primarily to gases and
∗ Corresponding author. Tel.: +61 7 3365 2145; fax: +61 7 3365 1177. E-mail address: [email protected] (M.J. Gidley). http://dx.doi.org/10.1016/j.carbpol.2015.01.039 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
short chain fatty acids in the large intestine (Cummings & Branch, 1986; Williams, Olmsted, Hamann, Fiorito, & Duckles, 1936). Unfermented residues, due to their water holding capacity, increase faecal weight and shorten transit time through the large intestine (Cummings et al., 1978). Less frequent and concentrated small volume defecation can increase the risk of developing diverticular disease and large-bowel cancer (Glober, Kamiyama, Nomura, Shimada, & Abba, 1977). Starch, on the other hand, is a major energy source in the human diet, irrespective of culture or socio-economic status. The worldwide scenario of over-nutrition and associated metabolic disorders such as diabetes has prompted intense interest in the study of digestion of starches as a major glycaemic carbohydrate. It is now well understood that the ␣-amylase susceptibility of starch is highly variable and careful selection of starch source and food preparation techniques can lead to controlled release of glucose in the intestinal lumen (Dhital, Warren, Butterworth, Ellis, & Gidley, 2015). In the early 1980’s, several authors (Dunaif & Schneeman, 1981; Hansen & Schulz, 1982; Isaksson, Lundquist, & Ihse, 1982) investigated the effect of dietary fibres on both human and porcine pancreatic amylase activity and provided evidence of inhibition of amylase activity by both soluble and insoluble fibre, however, the mechanism or mechanisms by which dietary fibre inhibits ␣amylase activity was not determined. Similarly, several studies, both in vitro and in vivo, have shown that soluble fibres such as
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guar gum, (1–3,1–4)--d-glucan and arabinoxylan increase the viscosity of the hydrolysing medium, slowing down hydrolysis of macronutrients by impeding the enzyme-substrate interaction (Dhital, Dolan, Stokes, & Gidley, 2014; Dunaif & Schneeman, 1981; Ellis et al., 2001; Hansen & Schulz, 1982; Leeds, 1979; Montagne, Pluske, & Hampson, 2003; Taylor, 1979; Tharakan, Norton, Fryer, & Bakalis, 2010). Although Dunaif and Schneeman (1981) indicated the potential interaction between fibre source and human pancreatic enzymes, Slaughter, Ellis, Jackson and Butterworth (2002) provided the first evidence for binding of ␣-amylase to guar galactomannan and of inhibition of ␣-amylase activity through a non-competitive mechanism. However, the mechanism behind insoluble fibre inhibiting the activity of pancreatic enzymes is still not well understood. ␣-Amylase interacts with its substrate, starch, through a specific binding mechanism, involving five contiguous ␣ (1 → 4) linked glucose residues, a high affinity and highly specific interaction that may be blocked by active site specific inhibitors, e.g. maltose or acarbose (Seigner, Prodanov, & Marchis-Mouren, 1987; Warren, Butterworth, & Ellis, 2012). In the current paper, we studied the activity of ␣-amylase in the presence of cellulose (or wheat bran) as an exemplar of insoluble fibre, using different kinetics techniques, and show that the presence of even small amounts of cellulose can significantly inhibit ␣-amylase activity. The mode of interaction of ␣-amylase and cellulose is also investigated and discussed. 2. Materials and methods Maize starch (MS) was obtained from Penford Australia Ltd. (Lane Cove, Sydney, Australia), and alpha-cellulose (Sigma C8002) was purchased from Sigma-Aldrich, Australia. Wheat bran (Natural Bran, Woolworths, Australia) was purchased locally and was purified (de-proteinised and de-starched) as described in supplementary information. 2.1. Methods 2.1.1. In vitro hydrolysis of starch in presence of cellulose In vitro hydrolysis of starch dispersed in 10 mL acetate buffer (0.2 M, pH 6, containing 0.02% (w/v) sodium azide) was carried out with 0.4 unit porcine pancreatic ␣-amylase (A6255, Sigma) per mg of starch in the presence of 0, 10, 50, 100, 200, 300 and 500% (w/w compared with starch) cellulose at 37 ◦ C under constant stirring (350 rpm). At set times between 0 and 1440 min the reducing sugar content of the incubate was analysed using the PAHBAH assay as described by Moretti and Thorson (2008). The values were expressed as % starch hydrolysis obtained through multiplying the reducing sugar value with a factor of 0.9. In another similar experiment, cellulose was substituted with wheat bran; experimental details and results are shown in the supplementary section. 2.1.2. Visualization of ˛-amylase bound to cellulose Forty units of TRITC-␣-amylase conjugate (Dhital, Warren, Zhang, & Gidley, 2014) was incubated with 100 mg of cellulose in acetate buffer (10 mL, 0.2 M, pH 6) at 37 ◦ C with constant mixing. After 5 min, an aliquot of 500 L was transferred to a microcentrifuge tube, centrifuged at 4000 g for 1 min and the residue was observed under the confocal microscope as described by Dhital et al. (2014). In a separate experiment, the enzyme-bound cellulose residue was washed twice with 1 mL of acetate buffer at room temperature before being observed by confocal microscopy. 2.1.3. Enzyme activity assay by solution depletion method ␣-Amylase (80 units with 200 mg cellulose in 10 mL of acetate buffer) was incubated for 5 and 30 min with mixing at 350 rpm
at 37 ◦ C. After each incubation time, an aliquot (250 L) was transferred into a micro-centrifuge tube, centrifuged at 4000 g for 60 s and 50 L of supernatant (containing unbound enzyme) was removed to a fresh tube. This enzyme was used to hydrolyse 2 mL of cooked starch (1% maize starch dispersed in acetate buffer and cooked at 95 ◦ C for 15 min with mixing). This equated to 0.02 unit of ␣-amylase per mg of cooked starch, assuming none of the enzyme had bound to the cellulose. The slope of the hydrolysis curve, expressed as digestion rate, was determined from a plot of % starch hydrolysis (as described in the previous section) versus hydrolysis time. Adsorption isotherms for ␣-amylase binding to cellulose were obtained using a modification of a method devised for determining ␣-amylase binding to starch (Warren, Royall, Gaisford, Butterworth & Ellis, 2011). A range of cellulose concentrations were prepared from 0.15 to 30 mg/mL in acetate buffer (0.2 M, pH 6). One micro litre of cellulose suspension was placed in a micro-centrifuge tube and equilibrated at 37 ◦ C, before the addition of 50 L of (∼100 nM) ␣-amylase. The mixture was kept at 37 ◦ C for 30 min to allow binding of ␣-amylase to cellulose to reach equilibrium with intermittent (every 5 min) vortex mixing to keep the cellulose in suspension. The tubes were then centrifuged at 4000 g for 60 s and the supernatant (containing unbound enzyme) was analysed for ␣-amylase activity using an Invitrogen Enzcheck® Ultra ␣-amylase assay kit. The amount of bound enzyme was calculated by subtraction and plotted as % bound enzyme versus cellulose concentration. All data represents the average of triplicate determinations. Data are presented as binding isotherms and Scatchard plots. Analysis is carried out following the method of Warren et al. (2012, 2011). The effect of temperature on ␣-amylase binding to cellulose starch was investigated by repeating the experiment at two additional temperatures; 20 ◦ C and 0 ◦ C. The experiment was also repeated in the presence of up to 20 mM maltose to study the role of maltose on ␣-amylase binding to cellulose. Furthermore, in order to understand the specificity of ␣amylase binding to cellulose, binding experiments were conducted in the presence of 10 mg/mL of bovine serum albumin (BSA, Sigma A2153) mixed with cellulose concentrations ranging from 0.15 to 30 mg/mL as described previously, prior to amylase addition. 2.1.4. Kinetics of enzyme inhibition Michaelis–Menten kinetic parameters were determined using maize starch granules as a substrate in the presence of cellulose based on the method described by Dhital et al. (2014). Briefly, 4 mL of various concentrations of starch (5–40 mg/mL) in acetate buffer (0.2 M, pH 6) were incubated at 37 ◦ C in a water bath in 15 mL polypropylene tubes with mixing (350 rpm, using a magnetic stirrer bar). At time 0, enzyme was added to a concentration of 1.5 nM. After 3, 6, 9, 12 and 15 min, 200 L of starch suspension was removed and immediately added to 200 L of 0.3 M Na2 CO3 in a micro-centrifuge tube to stop the reaction. These samples were then centrifuged at 4000 g for 1 min to sediment the unreacted starch and cellulose. The reducing sugar content in the supernatant was assayed by the PAHBAH method as described previously. Kinetic parameters were obtained from non-linear regression analysis using Sigmaplot® 12.5. All kinetic analysis was carried out in triplicate. 3. Results 3.1. Effect of cellulose on ˛-amylolysis of maize starch granules The presence of cellulose reduces the hydrolysis of starch by ␣amylase in a concentration dependent manner as seen in Fig. 1a. While inhibition of ␣-amylase activity was seen at all cellulose
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Fig. 1. (a) Progress curves of in vitro hydrolysis of maize starch by ␣-amylase in the presence of cellulose. The numerical ratio refers to the relative amounts of starch and cellulose used during hydrolysis. A control was performed in the absence of cellulose. (1b) Initial progress curve of in vitro starch hydrolysis of maize starch in the presence of cellulose. A plot of rate against starch: cellulose ratio is shown in the inset and the IC50, calculated by fitting to a semi-log model, is 1.09 starch: cellulose ratio.
concentrations, at a starch to cellulose ratio of 1:0.1 inhibition was barely detectable, whereas at ratios of 1:0.5 and higher there was a significant deviation from the cellulose free control. The inhibiting effect of cellulose on starch digestion was quantified by obtaining the reaction rate from the initial linear part of the digestion curve (Fig. 1b). The initial rate of reaction decreases rapidly with an increase in the starch to cellulose ratio from 1:0.1 to 1:1, and the effect plateaus at higher cellulose concentrations. The same effect is seen in the proportion of starch digested at later digestion times. At 300 min, the extent of starch digestion is dependent upon the cellulose concentration, decreasing rapidly with increasing cellulose, but the effect again plateaus at higher (1:3 or 1:5 starch to cellulose ratio) concentrations of cellulose.
binding interaction must be strong enough to resist the mixing and centrifugation conditions used during the incubation and separation processes. Initial rates of starch hydrolysis were taken to be indicative of the enzyme concentration remaining in solution
3.2. Activity assay of non-cellulose-bound ˛-amylase on cooked maize starch The activity of ␣-amylase that had been previously incubated with cellulose acting on cooked starch is shown in Fig. 2. The enzyme activity of the control (enzyme in the absence of cellulose) is considerably higher than that of samples that had been previously incubated with cellulose, suggesting significant binding of ␣-amylase to cellulose which is in accordance with previous results for human and porcine pancreatic amylases (Dunaif & Schneeman, 1981; Hansen & Schulz, 1982; Isaksson et al., 1982). The
Fig. 2. Activity assay of non-bound ␣-amylase on cooked maize starch. The legends 5 min and 30 min refers to the incubation time of ␣-amylase and cellulose before isolating the non-bound ␣-amylase. Note the line break corresponding to a change of scale on the Y-axis.
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Fig. 5. Effect of maltose on binding of ␣-amylase on cellulose.
Fig. 3. ␣-Amylase adsorption isotherms at different temperatures.
following incubation with cellulose. Following 5 min incubation, the enzyme activity subsequently measured was reduced by 90%, and after 30 min the activity was reduced by 95% relative to the control, suggesting that the enzyme was capable of binding to cellulose. 3.3. ˛–Amylase adsorption isotherm The binding affinity of ␣-amylase for cellulose shows a temperature dependency as seen in Fig. 3. At low temperatures (0 ◦ C) ␣-amylase has a much lower affinity for cellulose, and the binding curve only reaches saturation at a high (30 mg/mL) cellulose concentration, whereas at higher temperatures (20 or 37 ◦ C) the binding curve reaches saturation at a much lower cellulose concentration. This behaviour is unusual, as generally it would be expected that binding affinity would increase at lower temperatures, and requires further investigation. Scatchard plots obtained following the method of Warren et al. (2011), a modification of Cornish-Bowden (2004), are shown in Fig. 4. The Scatchard plots obtained for ␣-amylase binding to cellulose are non-linear, unlike those observed for ␣-amylase binding to starch (Warren et al., 2011), suggesting that there are at least two distinct modes of binding, with different binding affinities. The presence of at least two distinct binding modes makes obtaining independent values for the binding affinities challenging, especially as cellulose suspensions of less than 0.125 mg/mL proved difficult to accurately prepare due to the relatively large cellulose particle size, limiting the extent to which the high affinity interaction could be probed, but binding curves still prove useful for comparisons of relative affinity (Warren et al., 2011). The reason for the presence of multiple binding affinities for ␣-amylase binding to cellulose is as
yet unclear, but may reflect the heterogeneity of the cellulose surface, presenting multiple binding sites for ␣-amylase with differing affinities. Fig. 5 shows the effect of maltose on binding of ␣-amylase on cellulose at 37 ◦ C. Maltose is a competitive inhibitor of ␣-amylase binding to starch. As a product of the enzyme it occupies the active site, with two molecules of maltose being accommodated. The first molecule binds with an affinity of around 10 mM, and the second with an affinity of around 90 mM (Elödi, Móra, & Krysteva, 1972; Seigner et al., 1987; Warren et al., 2012). If the interaction between ␣-amylase and cellulose is specific and active site mediated, then one would expect the binding curve to shift significantly in the presence of maltose at mM concentrations, as is observed for starch (Fig. 3). Fig. 5, however, shows that there is no significant effect of maltose on the cellulose binding curve, suggesting that the amylase-cellulose binding interaction is not active site mediated. To further probe the specificity of the ␣-amylase-cellulose binding interaction, a binding curve was obtained in the presence of 10 mg/mL BSA, corresponding to ca 5 × 104 times the protein concentration of ␣-amylase. BSA would be expected to have no particular specificity of binding towards cellulose, so if ␣-amylase binding to cellulose was a specific interaction, the presence of BSA in the solution would not be expected to interfere with the binding. It was found that the presence of BSA completely obviated any binding interaction between ␣-amylase and cellulose, with 100% of the ␣-amylase remaining active in solution at all cellulose concentrations tested. 3.4. Kinetics of cellulose inhibition of ˛-amylase activity on maize starch granules The inhibition of ␣-amylase activity on starch by cellulose was investigated in more detail through the application of Michaelis–Menten kinetics. The Michaelis–Menten plot of ␣amylase hydrolysis of starch concentrations from 5 to 40 mg per mL in the presence of 0 to 30 mg per mL of cellulose in 0.2 M, pH 6 acetate buffer is shown in Fig. 6. The Vmax and Km values in the absence of inhibitor (i, cellulose) are 0.125 mM/min and 75 mg/mL respectively. Visual inspection of the Michaelis–Menten plot (Fig. 6) clearly indicates, in support of the previous findings, that cellulose does indeed inhibit ␣-amylase activity over a range of concentrations. In order to determine the mechanism of inhibition, Dixon (Fig. 7a) and modified Dixon (Fig. 7b) plots were produced. If a general mechanism for reversible inhibition is taken to be:
v= Fig. 4. Scatchard plot, plotted from the adsorption isotherm data shown in Fig. 3.
Vmax s Km (1 + i/Kic ) + s(1 + i/Kiu )
(1)
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sides of Eq. (1) yields: 1
v
=
i( KKm + Ks ) Km + s ic iu + Vmax s Vmax s
(2)
Such that a plot of 1/v against i is linear (a Dixon plot). Plotting at more than one s yields multiple straight lines, which in the case of mixed inhibition will intercept so that i = Kic and 1/v = 1/(1 − Kic /Kiu )Vmax at the point of intersection. An alternative rearrangement of Eq. (1) yields: s
v Fig. 6. Decrease in rate of starch hydrolysis by ␣-amylase in the presence of cellulose. A Michaelis–Menten plot of hydrolysis rate (v) against starch substrate (s) concentration (mg/mL) at a range of cellulose concentrations. Concentrations of added cellulose are indicated in the figure.
where the initial reaction rate is denoted v (mM/min), substrate concentration s (mg/mL), inhibitor (cellulose) concentration i (mg/mL), competitive inhibitor constant Kic (mg/mL) and uncompetitive inhibitor constant Kiu (mg/mL). Takingreciprocals of both
=
Km Vmax
1+
i Kic
+
s Vmax
1+
i Kiu
(3)
Such that a plot of s/v against i is linear (a modified Dixon plot). Plotting at more than one s yields multiple straight lines, which in the case of mixed inhibition will intercept so that i = Kiu and s/v = Km (1 − Kiu /Kic )/Vmax at the point of intersection. The inhibition was shown to be of a mixed linear type, as the inhibition curves cross in the fourth quadrant of the Dixon plot (Fig. 7a) and the third quadrant of the modified Dixon plot (Fig. 7b) (Cornish-Bowden, 2004), with a Kic value estimated from the Dixon plot from a median value of the intercepts to be 2.8 mg/mL, and a
Fig. 7. a Inhibition of ␣-amylase activity in the presence of cellulose shown by a Dixon plot of 1/v against inhibitor (cellulose) (i) at a range of starch concentrations (mg/mL). Concentrations of starch are indicated in the figure. (7b) Inhibition of ␣-amylase activity in the presence of cellulose shown by a modified Dixon plot of inhibitor (cellulose) [i] against substrate/reaction rate (s/v) at a range of starch concentration (mg/mL). Concentrations of starch are indicated in the figure.
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Fig. 8. Representative confocal microscopy images of TRITC-amylase conjugate bound on to the cellulose surface. (A) Before washing; (B) after two successive washing. Pictures were taken under similar microscopic settings.
Kiu estimated from the modified Dixon plot from a median value of the intercepts to be 7.9 mg/mL. A full list of the intercepts used to calculate Kic and Kiu are shown in supplementary information, Table S1. 3.5. Microscopic observation of ˛-amylase binding on cellulose Representative confocal microscopy images of TRITC-amylase conjugate in the presence of cellulose are shown in Fig. 8. It was observed that the labelled ␣-amylase was apparently homogenously bound to the cellulose surface (Fig. 8A). Fig. 8B shows the ␣-amylase-cellulose complex after two successive washing steps. Fluorescence was mostly retained following washing, showing that the interaction between ␣-amylase and cellulose is sufficiently strong to survive both washing and centrifugation processes. 3.6. ˛-Amylase binding to wheat bran In order to test whether binding of ␣-amylase is specific to isolated cellulose, a typical food component containing cellulose (wheat bran) was tested. Results of overall starch digestion progress curves (Fig. S1) and the initial linear portion of progress curves (Fig. S2) are similar to those of pure cellulose, and show that pure cellulose has a slightly more pronounced effect on enzyme activity relative to the non-purified fibre source (bran) over the same range of bran/cellulose concentrations which is in agreement with the findings of Dunaif and Schneeman (1981). This suggests that amylase binding to cellulosic substrates is a general property. 4. Discussion 4.1. ˛-Amylase binding to cellulose: nutritional implications Consumption of plants and plant foods are vital for the nutritional health and well-being of humans. Among many nutrients, cellulose is an important and abundant insoluble dietary fibre (IDF) found in e.g. cereals, fruits and vegetables. Nutritionally, cellulose has an important role as a bulking agent, resulting in shorter transit times and increased faecal mass (Slavin & Marlett, 1980) with associated beneficial effects in the treatment of constipation, diverticular disease, haemorrhoids and in the prevention of colonic cancer (Jensen et al., 2004). In this paper, we describe a potential additional nutritional role for cellulose. In agreement with Hansen and Schulz (1982), our results show that the presence of small amounts of cellulose in an in vitro digestion medium has an
inhibitory effect on the hydrolysis of starch. The progress curve is analysed in two time frames. The first 40 min digestion data (Fig. 1b and S2) is analysed to address the acute or rapid effect of cellulosic materials (pure cellulose and wheat bran) on starch hydrolysis considering the fact that most of the post-prandial blood glucose rise (and therefore initial insulin demand) occurs within 30–60 min from the ingestion of a starchy meal (Ellis, Roberts, Low, & Morgan, 1995). Prolonged digestion up to 5 h (Fig. 1a and S1) was also carried out to observe the effects over longer times. From both observations, it was found that even small amounts of cellulosic materials (pure cellulose or wheat bran) have inhibitory effects on starch digestion. 4.2. ˛-Amylase binding to cellulose: possible binding mechanisms Solution depletion experiments clearly demonstrate that ␣amylase binds to cellulose, and that the binding interaction is very rapid, relative to ␣-amylase binding to granular starch (Warren et al., 2011), approaching equilibrium within 5 min (Fig. 3). This suggests that cellulose may be able to act as an inhibitor of ␣amylase activity on starch, although the binding mechanism of ␣-amylase to cellulose appears to be complex, with multiple binding modes (Fig. 4). ␣-Amylase is unable to hydrolyse the  (1 → 4) glucan linkages of cellulose, so cellulose cannot be degraded by the enzyme, but, being a glucose polymer, cellulose bears some structural similarities to starch. An interesting question raised by these solution depletion assays is whether there is a specific, active site mediated, binding interaction between ␣-amylase and cellulose, suggested by the apparently high affinity of ␣-amylase for cellulose, or whether the interaction is non-specific in nature as postulated by Dunaif and Schneeman (1981). To explore this further we devised a series of experiments to explore the influence of the addition of maltose (which binds to the active site of ␣amylase with high specificity, blocking other interactions) and BSA (a protein with no known carbohydrate binding sites) on ␣-amylase binding to cellulose. Maltose is known to be a competitive inhibitor of ␣-amylase. The inhibition is due to occupancy of one or two maltose molecules in the active site of amylase (Elödi et al., 1972; Warren et al., 2012). In the presence of a range of maltose concentrations (0–20 mM), there was a slight downward shift in the absorption isotherm, but there was no concentration dependence observed. From these experiments (Fig. 5) it is apparent that maltose does not inhibit ␣-amylase binding to cellulose, strongly suggesting that the binding between ␣-amylase and cellulose is not active site mediated.
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A binding isotherm for ␣-amylase binding to cellulose was obtained in the presence of BSA, a protein with no known affinity for carbohydrates. It was found that the addition of excess BSA completely blocked ␣-amylase binding to cellulose. Therefore, we hypothesise that both proteins are non-specifically adsorbed onto cellulose, and that the ca 5 × 104 fold excess BSA protein occupied all available protein binding sites on the cellulose surface, saturating the cellulose with protein and blocking binding by ␣-amylase. The combined results of the maltose and BSA experiments indicate that the binding interaction between ␣-amylase and cellulose is non-specific in nature. It appears that ␣-amylase is adsorbed to the surface of cellulose, depleting the protein from solution, hence allowing cellulose to act as an inhibitor of ␣-amylase activity in a simplified starch-cellulose-␣-amylase system. In more complex, multi-component systems such as the human intestine, however, it is possible that additional inhibitory and other effects of cellulose on ␣-amylase activity may occur, and will require study. 4.3. ˛-Amylase binding to cellulose: inhibition mechanism In order to determine the mode of inhibition of ␣-amylase action on starch by cellulose, the initial velocity (v) at a range of inhibitor concentrations (i) across a range of substrate concentrations (S) were used to plot Dixon and modified Dixon plots (Cornish-Bowden, 1974). A competitive inhibitor, [i] binds to the active site of the enzyme to form an enzyme–inhibitor complex (EI). As seen in Fig. 7a, when [i] is plotted against 1/v, at a range of S, the intersect is in the fourth quadrant, indicating that the Km increases with increasing inhibitor concentration. This is typical of competitive or mixed inhibition and rules out the possibility of uncompetitive and noncompetitive inhibition where lines respectively intersect exactly at a negative x-axis or run parallel to each other without intersecting (Cornish-Bowden, 1974). The dissociation constant of the EI complex for competitive inhibition (Kic ) was found to be ca. 3 mg/mL (Fig. 4). To distinguish between competitive or mixed inhibition, the data was also plotted as a modified Dixon plot (s/v versus [i]) as presented in Fig. 7b. In the case of competitive inhibition, the lines for each starch concentration will run parallel to each other without intersecting (Cornish-Bowden, 1974) however as seen in Fig. 7b, the lines intersect in the third quadrant, suggesting a mixed mode of inhibition. The dissociation constant for EIS complex (Kiu ) derived from a modified Dixon plot is 6 mg/mL. Data suggesting mixed inhibition brings up the question of the possible mechanism by which mixed inhibition kinetics occur in the present system. In competitive inhibition, the inhibitor cannot bind if the substrate has already bound to the enzyme, whereas in uncompetitive inhibition, the inhibitor can only bind if the substrate has already bound to the enzyme. However in mixed inhibition, the inhibitor can bind whether or not the substrate has already bound to the enzyme. In the case of classical reversible mixed type inhibition as presented in Fig. 9a, inhibitor (I) can interact with both free ␣-amylase (E) and ␣-amylase-starch complex (EP) at a site other than the active site. The scheme presented in Fig. 9a seems unlikely, however, as is difficult to conceive how two large, insoluble substrates such as starch granules and cellulose could both be bound to ␣-amylase simultaneously. An alternative mechanism is suggested in Fig. 9b. The data presented in Fig. 5 show that enzyme with maltose (the product of starch hydrolysis) bound in the enzyme active site could still bind to cellulose. In the mechanism suggested in Fig. 9b, enzyme with maltose bound in the active site following hydrolysis can bind to cellulose, reducing the overall turnover of the enzyme. This mechanism is, however, only speculative and clearly requires further investigation.
Fig. 9. (a) Schematic diagram of classical linear mixed inhibition. In this type of reversible inhibition, an inhibitor can interact with both the free enzyme and the enzyme–substrate complex at a site other than the active site. This results in an apparent decrease in Vmax (apparent enzyme maximum velocity in the presence of an inhibitor) and an apparent increase in Ks (apparent enzyme–substrate dissociation constant) in the presence of an inhibitor. (9b) Schematic diagram of linear mixed inhibition of amylase activity by cellulose in the presence of maltose.
4.4. Inhibition of amylase activity, soluble versus insoluble fibres These findings may be contrasted with the inhibition of ␣-amylase activity by another dietary fibre, the soluble polysaccharide guar gum (Slaughter et al., 2002). Guar gum was found to inhibit ␣-amylase activity in a non-competitive mechanism with a ki value of between 3 and 5 mg/mL. This is a similar value to the ki determined in the present study, although a different mechanism of inhibition. While we do not presently have sufficient data to determine the reasons for differences in inhibition mechanism between these two forms of fibre, it could be speculated that the soluble galactomannan may exert effects such as reducing mass transfer through viscosity effects. Interestingly, the ELISA assay reported by Slaughter et al. (2002) suggested that, unlike the results presented here for cellulose, there was a degree of specificity for ␣-amylase binding to guar gum.
5. Conclusions Starch is a major source of energy in the human diet, whereas cellulose cannot be degraded in the upper digestive tract, and is often considered to have a nutritional role limited to its action as an inert bulking agent in the diet. In the present study we demonstrate that in vitro cellulose can bind ␣-amylase, and inhibit the activity of the enzyme through a mixed type inhibition mechanism. The binding of ␣-amylase to cellulose was found to be reversible and non-specific. The binding was not affected by the addition of maltose, a competitive inhibitor that binds to the enzymes active site, but was completely obviated by the addition of an additional protein, BSA, which was presumably also adsorbed to the surface of the cellulose in a competitive manner. Taken together, these results indicate that cellulose (either purified or as a component of wheat bran) can have a significant inhibitory effect on ␣-amylase, that may have relevance in vivo during gastrointestinal digestion, as well as to industrial processing systems involving ␣-amylase and plant matter. More generally, the non-specific binding mechanism suggests that a range of proteins such as other digestive enzymes are also likely to bind to cellulosic components in digesta.
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Acknowledgements FJW acknowledges the support of a University of Queensland Postdoctoral Fellowship. This work was supported by the Australian Research Council Centre of Excellence in Plant Cell Walls CE110001007 and Discovery Project DP 130102461. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol. 2015.01.039. References Cornish-Bowden, A. (1974). A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochemical Journal, 137(1), 143–144. Cornish-Bowden, A. (2004). Fundamentals of enzyme kinetics. London: Portland Press. Cummings, J. H., & Branch, W. J. (1986). Fermentation and the production of shortchain fatty acids in the human large intestine. In G. V. Vahouny, & D. Kritcgevsky (Eds.), Dietary fiber–Basic and clinical aspects (pp. 131–149). New York: Plenum Press. Cummings, J., Branch, W., Jenkins, D., Southgate, D., Houston, H., & James, W. (1978). Colonic response to dietary fibre from carrot, cabbage, apple, bran, and guar gum. The Lancet, 311(8054), 5–9. Dhital, S., Dolan, G., Stokes, J. R., & Gidley, M. (2014). Enzymatic hydrolysis of starch in the presence of cereal soluble fibre polysaccharides. Food & Function, 5, 579–586. Dhital, S., Warren, F. J., Butterworth, P. J., Ellis, P. R., & Gidley, M. J. (2015). Mechanisms of starch digestion by ␣-amylase—Structural basis for kinetic properties. Critical Reviews in Food Science and Nutrition, http://dx.doi.org/10.1080/10408398.2014.922043 (accepted manuscript) Dhital, S., Warren, F. J., Zhang, B., & Gidley, M. J. (2014). Amylase binding to starch granules under hydrolysing and non-hydrolysing conditions. Carbohydrate Polymers, 113, 97–107. Dunaif, G., & Schneeman, B. (1981). The effect of dietary fiber on human pancreatic enzyme activity in vitro. The American Journal of Clinical Nutrition, 34(6), 1034–1035. Ellis, P., Qi, W., Rayment, P., YiLong, R., Ross-Murphy, S., Cho, S., et al. (2001). Guar gum: Agricultural and botanical aspects, physicochemical and nutritional properties and its use in the development of functional foods. In S. S. Cho, & M. L. Dreher (Eds.), Handbook of dietary fiber (pp. 613–657). New York: Marcel Dekker. Ellis, P., Roberts, F., Low, A., & Morgan, L. (1995). The effect of high-molecularweight guar gum on net apparent glucose absorption and net apparent insulin and gastric inhibitory polypeptide production in the growing pig: Relationship to rheological changes in jejunal digesta. British Journal of Nutrition, 74(04), 539–556.
Elödi, P., Móra, S., & Krysteva, M. (1972). Investigation of the active center of porcine-pancreatic amylase. European Journal of Biochemistry, 24(3), 577–582. Glober, G., Kamiyama, S., Nomura, A., Shimada, A., & Abba, B. (1977). Bowel transittime and stool weight in populations with different colon-cancer risks. The Lancet, 310(8029), 110–111. Hansen, W., & Schulz, G. (1982). The effect of dietary fiber on pancreatic amylase activity in vitro. Hepato-gastroenterology, 29(4), 157–160. Isaksson, G., Lundquist, I., & Ihse, I. (1982). Effect of dietary fiber on pancreatic enzyme activity in vitro. Gastroenterology, 82(5), 918–924. Jensen, M. K., Koh-Banerjee, P., Hu, F. B., Franz, M., Sampson, L., Grønbæk, M., et al. (2004). Intakes of whole grains, bran, and germ and the risk of coronary heart disease in men. The American Journal of Clinical Nutrition, 80(6), 1492–1499. Klemm, D., Heublein, B., Fink, H. P., & Bohn, A. (2005). Cellulose: fascinating biopolymer and sustainable raw material. Angewandte Chemie International Edition, 44(22), 3358–3393. Leeds, A. R. (1979). Gastric-emptying, fiber, and absorption. The Lancet, 1(8121), 872–873. Montagne, L., Pluske, J., & Hampson, D. (2003). A review of interactions between dietary fibre and the intestinal mucosa, and their consequences on digestive health in young non-ruminant animals. Animal Feed Science and Technology, 108(1), 95–117. Moretti, R., & Thorson, J. S. (2008). A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay. Analytical Biochemistry, 377(2), 251–258. Seigner, C., Prodanov, E., & Marchis-Mouren, G. (1987). The determination of subsite binding energies of porcine pancreatic ␣-amylase by comparing hydrolytic activity towards substrates. Biochimica et Biophysica Acta (BBA)—Protein Structure and Molecular Enzymology, 913(2), 200–209. Slaughter, S. L., Ellis, P. R., Jackson, E. C., & Butterworth, P. J. (2002). The effect of guar galactomannan and water availability during hydrothermal processing on the hydrolysis of starch catalysed by pancreatic ␣-amylase. Biochimica et Biophysica Acta (BBA)—General Subjects, 1571(1), 55–63. Slavin, J., & Marlett, J. (1980). Influence of refined cellulose on human bowel function and calcium and magnesium balance. The American Journal of Clinical Nutrition, 33(9), 1932–1939. Taylor, R. H. (1979). Gastric-emptying, fiber, and absorption. The Lancet, 1(8121), 872-872 Tharakan, A., Norton, I. T., Fryer, P. J., & Bakalis, S. (2010). Mass transfer and nutrient absorption in a simulated model of small intestine. Journal of Food Science, 75(6), 339–346. Warren, F. J., Butterworth, P. J., & Ellis, P. R. (2012). Studies of the effect of maltose on the direct binding of porcine pancreatic ␣-amylase to maize starch. Carbohydrate Research, 358, 67–71. Warren, F. J., Royall, P. G., Gaisford, S., Butterworth, P. J., & Ellis, P. R. (2011). Binding interactions of ␣-amylase with starch granules: The influence of supramolecular structure and surface area. Carbohydrate Polymers, 86(2), 1038–1047. Williams, R. D., Olmsted, W. H., Hamann, C., Fiorito, J., & Duckles, D. (1936). The effect of cellulose, hemicellulose and lignin on the weight of the stool: A contribution to the study of laxation in man. The Journal of Nutrition, 11(5), 433–449.
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Inhibition of ␣-amylase activity by cellulose: Kinetic analysis and nutritional implications Sushil Dhital a , Michael J. Gidley a,∗ , Frederick J. Warren b a ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Qld 4072, Australia b Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Qld 4072, Australia
a r t i c l e
i n f o
Article history: Received 3 October 2014 Received in revised form 22 January 2015 Accepted 23 January 2015 Available online 2 February 2015 Keywords: Cellulose Starch Inhibition Alpha-amylase Adsorption isotherm Michaelis–Menten kinetics
a b s t r a c t We report on inhibition of ␣-amylase activity by cellulose based on in vitro experiments. The presence of cellulose in the hydrolysing medium reduced the initial velocity of starch hydrolysis in a concentration dependent manner. ␣-Amylase adsorption to cellulose was reversible, attaining equilibrium within 30 min of incubation, and showed a higher affinity at 37 ◦ C compared to 20 and 0 ◦ C. The adsorption was almost unchanged in the presence of maltose (2.5–20 mM) but was hindered in the presence of excess protein, suggesting non-specific adsorption of ␣-amylase to cellulose. Kinetic analyses of ␣-amylase hydrolysis of maize starch in the presence of cellulose showed that the inhibition is of a mixed type. The dissociation constant (Kic ) of the EI complex was found to be ca. 3 mg/mL. The observed inhibition of ␣-amylase activity suggests that cellulose in the diet can potentially attenuate starch hydrolysis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Starch and cellulose are two of the most abundant biopolymers in nature. Although both are primarily (1 → 4)-linked polymers of glucose, they play strikingly different roles in terms of human and animal nutrition. Starch is composed of amylose, a mostly linear polymer of ␣(1 → 4)-linked anhydroglucose unit and amylopectin, a highly branched polymer via additional ␣(1 → 6)-linkages. Cellulose, in contrast, has linear (1 → 4)-linked anhydroglucose chains with strong inter- and intra-molecular hydrogen bonding. The reduced availability of hydroxy groups due to their involvement in hydrogen bonding makes cellulose water insoluble and limits processability and functionality in its native forms (Klemm, Heublein, Fink, & Bohn, 2005). In herbivorous ruminants, a considerable part of the caloric intake comes from bacterial fermentation of cellulosic residues in the rumen. Humans and non-ruminant animals such as pigs, however, are unable to extract energy from cellulose directly due to the absence of  glucanases and glucosidases in their intestinal tracts. Undigested cellulosic material may be partially hydrolysed by bacterial enzymes into small soluble oligomers which are either absorbed, or are fermented primarily to gases and
∗ Corresponding author. Tel.: +61 7 3365 2145; fax: +61 7 3365 1177. E-mail address: [email protected] (M.J. Gidley). http://dx.doi.org/10.1016/j.carbpol.2015.01.039 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
short chain fatty acids in the large intestine (Cummings & Branch, 1986; Williams, Olmsted, Hamann, Fiorito, & Duckles, 1936). Unfermented residues, due to their water holding capacity, increase faecal weight and shorten transit time through the large intestine (Cummings et al., 1978). Less frequent and concentrated small volume defecation can increase the risk of developing diverticular disease and large-bowel cancer (Glober, Kamiyama, Nomura, Shimada, & Abba, 1977). Starch, on the other hand, is a major energy source in the human diet, irrespective of culture or socio-economic status. The worldwide scenario of over-nutrition and associated metabolic disorders such as diabetes has prompted intense interest in the study of digestion of starches as a major glycaemic carbohydrate. It is now well understood that the ␣-amylase susceptibility of starch is highly variable and careful selection of starch source and food preparation techniques can lead to controlled release of glucose in the intestinal lumen (Dhital, Warren, Butterworth, Ellis, & Gidley, 2015). In the early 1980’s, several authors (Dunaif & Schneeman, 1981; Hansen & Schulz, 1982; Isaksson, Lundquist, & Ihse, 1982) investigated the effect of dietary fibres on both human and porcine pancreatic amylase activity and provided evidence of inhibition of amylase activity by both soluble and insoluble fibre, however, the mechanism or mechanisms by which dietary fibre inhibits ␣amylase activity was not determined. Similarly, several studies, both in vitro and in vivo, have shown that soluble fibres such as
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guar gum, (1–3,1–4)--d-glucan and arabinoxylan increase the viscosity of the hydrolysing medium, slowing down hydrolysis of macronutrients by impeding the enzyme-substrate interaction (Dhital, Dolan, Stokes, & Gidley, 2014; Dunaif & Schneeman, 1981; Ellis et al., 2001; Hansen & Schulz, 1982; Leeds, 1979; Montagne, Pluske, & Hampson, 2003; Taylor, 1979; Tharakan, Norton, Fryer, & Bakalis, 2010). Although Dunaif and Schneeman (1981) indicated the potential interaction between fibre source and human pancreatic enzymes, Slaughter, Ellis, Jackson and Butterworth (2002) provided the first evidence for binding of ␣-amylase to guar galactomannan and of inhibition of ␣-amylase activity through a non-competitive mechanism. However, the mechanism behind insoluble fibre inhibiting the activity of pancreatic enzymes is still not well understood. ␣-Amylase interacts with its substrate, starch, through a specific binding mechanism, involving five contiguous ␣ (1 → 4) linked glucose residues, a high affinity and highly specific interaction that may be blocked by active site specific inhibitors, e.g. maltose or acarbose (Seigner, Prodanov, & Marchis-Mouren, 1987; Warren, Butterworth, & Ellis, 2012). In the current paper, we studied the activity of ␣-amylase in the presence of cellulose (or wheat bran) as an exemplar of insoluble fibre, using different kinetics techniques, and show that the presence of even small amounts of cellulose can significantly inhibit ␣-amylase activity. The mode of interaction of ␣-amylase and cellulose is also investigated and discussed. 2. Materials and methods Maize starch (MS) was obtained from Penford Australia Ltd. (Lane Cove, Sydney, Australia), and alpha-cellulose (Sigma C8002) was purchased from Sigma-Aldrich, Australia. Wheat bran (Natural Bran, Woolworths, Australia) was purchased locally and was purified (de-proteinised and de-starched) as described in supplementary information. 2.1. Methods 2.1.1. In vitro hydrolysis of starch in presence of cellulose In vitro hydrolysis of starch dispersed in 10 mL acetate buffer (0.2 M, pH 6, containing 0.02% (w/v) sodium azide) was carried out with 0.4 unit porcine pancreatic ␣-amylase (A6255, Sigma) per mg of starch in the presence of 0, 10, 50, 100, 200, 300 and 500% (w/w compared with starch) cellulose at 37 ◦ C under constant stirring (350 rpm). At set times between 0 and 1440 min the reducing sugar content of the incubate was analysed using the PAHBAH assay as described by Moretti and Thorson (2008). The values were expressed as % starch hydrolysis obtained through multiplying the reducing sugar value with a factor of 0.9. In another similar experiment, cellulose was substituted with wheat bran; experimental details and results are shown in the supplementary section. 2.1.2. Visualization of ˛-amylase bound to cellulose Forty units of TRITC-␣-amylase conjugate (Dhital, Warren, Zhang, & Gidley, 2014) was incubated with 100 mg of cellulose in acetate buffer (10 mL, 0.2 M, pH 6) at 37 ◦ C with constant mixing. After 5 min, an aliquot of 500 L was transferred to a microcentrifuge tube, centrifuged at 4000 g for 1 min and the residue was observed under the confocal microscope as described by Dhital et al. (2014). In a separate experiment, the enzyme-bound cellulose residue was washed twice with 1 mL of acetate buffer at room temperature before being observed by confocal microscopy. 2.1.3. Enzyme activity assay by solution depletion method ␣-Amylase (80 units with 200 mg cellulose in 10 mL of acetate buffer) was incubated for 5 and 30 min with mixing at 350 rpm
at 37 ◦ C. After each incubation time, an aliquot (250 L) was transferred into a micro-centrifuge tube, centrifuged at 4000 g for 60 s and 50 L of supernatant (containing unbound enzyme) was removed to a fresh tube. This enzyme was used to hydrolyse 2 mL of cooked starch (1% maize starch dispersed in acetate buffer and cooked at 95 ◦ C for 15 min with mixing). This equated to 0.02 unit of ␣-amylase per mg of cooked starch, assuming none of the enzyme had bound to the cellulose. The slope of the hydrolysis curve, expressed as digestion rate, was determined from a plot of % starch hydrolysis (as described in the previous section) versus hydrolysis time. Adsorption isotherms for ␣-amylase binding to cellulose were obtained using a modification of a method devised for determining ␣-amylase binding to starch (Warren, Royall, Gaisford, Butterworth & Ellis, 2011). A range of cellulose concentrations were prepared from 0.15 to 30 mg/mL in acetate buffer (0.2 M, pH 6). One micro litre of cellulose suspension was placed in a micro-centrifuge tube and equilibrated at 37 ◦ C, before the addition of 50 L of (∼100 nM) ␣-amylase. The mixture was kept at 37 ◦ C for 30 min to allow binding of ␣-amylase to cellulose to reach equilibrium with intermittent (every 5 min) vortex mixing to keep the cellulose in suspension. The tubes were then centrifuged at 4000 g for 60 s and the supernatant (containing unbound enzyme) was analysed for ␣-amylase activity using an Invitrogen Enzcheck® Ultra ␣-amylase assay kit. The amount of bound enzyme was calculated by subtraction and plotted as % bound enzyme versus cellulose concentration. All data represents the average of triplicate determinations. Data are presented as binding isotherms and Scatchard plots. Analysis is carried out following the method of Warren et al. (2012, 2011). The effect of temperature on ␣-amylase binding to cellulose starch was investigated by repeating the experiment at two additional temperatures; 20 ◦ C and 0 ◦ C. The experiment was also repeated in the presence of up to 20 mM maltose to study the role of maltose on ␣-amylase binding to cellulose. Furthermore, in order to understand the specificity of ␣amylase binding to cellulose, binding experiments were conducted in the presence of 10 mg/mL of bovine serum albumin (BSA, Sigma A2153) mixed with cellulose concentrations ranging from 0.15 to 30 mg/mL as described previously, prior to amylase addition. 2.1.4. Kinetics of enzyme inhibition Michaelis–Menten kinetic parameters were determined using maize starch granules as a substrate in the presence of cellulose based on the method described by Dhital et al. (2014). Briefly, 4 mL of various concentrations of starch (5–40 mg/mL) in acetate buffer (0.2 M, pH 6) were incubated at 37 ◦ C in a water bath in 15 mL polypropylene tubes with mixing (350 rpm, using a magnetic stirrer bar). At time 0, enzyme was added to a concentration of 1.5 nM. After 3, 6, 9, 12 and 15 min, 200 L of starch suspension was removed and immediately added to 200 L of 0.3 M Na2 CO3 in a micro-centrifuge tube to stop the reaction. These samples were then centrifuged at 4000 g for 1 min to sediment the unreacted starch and cellulose. The reducing sugar content in the supernatant was assayed by the PAHBAH method as described previously. Kinetic parameters were obtained from non-linear regression analysis using Sigmaplot® 12.5. All kinetic analysis was carried out in triplicate. 3. Results 3.1. Effect of cellulose on ˛-amylolysis of maize starch granules The presence of cellulose reduces the hydrolysis of starch by ␣amylase in a concentration dependent manner as seen in Fig. 1a. While inhibition of ␣-amylase activity was seen at all cellulose
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Fig. 1. (a) Progress curves of in vitro hydrolysis of maize starch by ␣-amylase in the presence of cellulose. The numerical ratio refers to the relative amounts of starch and cellulose used during hydrolysis. A control was performed in the absence of cellulose. (1b) Initial progress curve of in vitro starch hydrolysis of maize starch in the presence of cellulose. A plot of rate against starch: cellulose ratio is shown in the inset and the IC50, calculated by fitting to a semi-log model, is 1.09 starch: cellulose ratio.
concentrations, at a starch to cellulose ratio of 1:0.1 inhibition was barely detectable, whereas at ratios of 1:0.5 and higher there was a significant deviation from the cellulose free control. The inhibiting effect of cellulose on starch digestion was quantified by obtaining the reaction rate from the initial linear part of the digestion curve (Fig. 1b). The initial rate of reaction decreases rapidly with an increase in the starch to cellulose ratio from 1:0.1 to 1:1, and the effect plateaus at higher cellulose concentrations. The same effect is seen in the proportion of starch digested at later digestion times. At 300 min, the extent of starch digestion is dependent upon the cellulose concentration, decreasing rapidly with increasing cellulose, but the effect again plateaus at higher (1:3 or 1:5 starch to cellulose ratio) concentrations of cellulose.
binding interaction must be strong enough to resist the mixing and centrifugation conditions used during the incubation and separation processes. Initial rates of starch hydrolysis were taken to be indicative of the enzyme concentration remaining in solution
3.2. Activity assay of non-cellulose-bound ˛-amylase on cooked maize starch The activity of ␣-amylase that had been previously incubated with cellulose acting on cooked starch is shown in Fig. 2. The enzyme activity of the control (enzyme in the absence of cellulose) is considerably higher than that of samples that had been previously incubated with cellulose, suggesting significant binding of ␣-amylase to cellulose which is in accordance with previous results for human and porcine pancreatic amylases (Dunaif & Schneeman, 1981; Hansen & Schulz, 1982; Isaksson et al., 1982). The
Fig. 2. Activity assay of non-bound ␣-amylase on cooked maize starch. The legends 5 min and 30 min refers to the incubation time of ␣-amylase and cellulose before isolating the non-bound ␣-amylase. Note the line break corresponding to a change of scale on the Y-axis.
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Fig. 5. Effect of maltose on binding of ␣-amylase on cellulose.
Fig. 3. ␣-Amylase adsorption isotherms at different temperatures.
following incubation with cellulose. Following 5 min incubation, the enzyme activity subsequently measured was reduced by 90%, and after 30 min the activity was reduced by 95% relative to the control, suggesting that the enzyme was capable of binding to cellulose. 3.3. ˛–Amylase adsorption isotherm The binding affinity of ␣-amylase for cellulose shows a temperature dependency as seen in Fig. 3. At low temperatures (0 ◦ C) ␣-amylase has a much lower affinity for cellulose, and the binding curve only reaches saturation at a high (30 mg/mL) cellulose concentration, whereas at higher temperatures (20 or 37 ◦ C) the binding curve reaches saturation at a much lower cellulose concentration. This behaviour is unusual, as generally it would be expected that binding affinity would increase at lower temperatures, and requires further investigation. Scatchard plots obtained following the method of Warren et al. (2011), a modification of Cornish-Bowden (2004), are shown in Fig. 4. The Scatchard plots obtained for ␣-amylase binding to cellulose are non-linear, unlike those observed for ␣-amylase binding to starch (Warren et al., 2011), suggesting that there are at least two distinct modes of binding, with different binding affinities. The presence of at least two distinct binding modes makes obtaining independent values for the binding affinities challenging, especially as cellulose suspensions of less than 0.125 mg/mL proved difficult to accurately prepare due to the relatively large cellulose particle size, limiting the extent to which the high affinity interaction could be probed, but binding curves still prove useful for comparisons of relative affinity (Warren et al., 2011). The reason for the presence of multiple binding affinities for ␣-amylase binding to cellulose is as
yet unclear, but may reflect the heterogeneity of the cellulose surface, presenting multiple binding sites for ␣-amylase with differing affinities. Fig. 5 shows the effect of maltose on binding of ␣-amylase on cellulose at 37 ◦ C. Maltose is a competitive inhibitor of ␣-amylase binding to starch. As a product of the enzyme it occupies the active site, with two molecules of maltose being accommodated. The first molecule binds with an affinity of around 10 mM, and the second with an affinity of around 90 mM (Elödi, Móra, & Krysteva, 1972; Seigner et al., 1987; Warren et al., 2012). If the interaction between ␣-amylase and cellulose is specific and active site mediated, then one would expect the binding curve to shift significantly in the presence of maltose at mM concentrations, as is observed for starch (Fig. 3). Fig. 5, however, shows that there is no significant effect of maltose on the cellulose binding curve, suggesting that the amylase-cellulose binding interaction is not active site mediated. To further probe the specificity of the ␣-amylase-cellulose binding interaction, a binding curve was obtained in the presence of 10 mg/mL BSA, corresponding to ca 5 × 104 times the protein concentration of ␣-amylase. BSA would be expected to have no particular specificity of binding towards cellulose, so if ␣-amylase binding to cellulose was a specific interaction, the presence of BSA in the solution would not be expected to interfere with the binding. It was found that the presence of BSA completely obviated any binding interaction between ␣-amylase and cellulose, with 100% of the ␣-amylase remaining active in solution at all cellulose concentrations tested. 3.4. Kinetics of cellulose inhibition of ˛-amylase activity on maize starch granules The inhibition of ␣-amylase activity on starch by cellulose was investigated in more detail through the application of Michaelis–Menten kinetics. The Michaelis–Menten plot of ␣amylase hydrolysis of starch concentrations from 5 to 40 mg per mL in the presence of 0 to 30 mg per mL of cellulose in 0.2 M, pH 6 acetate buffer is shown in Fig. 6. The Vmax and Km values in the absence of inhibitor (i, cellulose) are 0.125 mM/min and 75 mg/mL respectively. Visual inspection of the Michaelis–Menten plot (Fig. 6) clearly indicates, in support of the previous findings, that cellulose does indeed inhibit ␣-amylase activity over a range of concentrations. In order to determine the mechanism of inhibition, Dixon (Fig. 7a) and modified Dixon (Fig. 7b) plots were produced. If a general mechanism for reversible inhibition is taken to be:
v= Fig. 4. Scatchard plot, plotted from the adsorption isotherm data shown in Fig. 3.
Vmax s Km (1 + i/Kic ) + s(1 + i/Kiu )
(1)
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sides of Eq. (1) yields: 1
v
=
i( KKm + Ks ) Km + s ic iu + Vmax s Vmax s
(2)
Such that a plot of 1/v against i is linear (a Dixon plot). Plotting at more than one s yields multiple straight lines, which in the case of mixed inhibition will intercept so that i = Kic and 1/v = 1/(1 − Kic /Kiu )Vmax at the point of intersection. An alternative rearrangement of Eq. (1) yields: s
v Fig. 6. Decrease in rate of starch hydrolysis by ␣-amylase in the presence of cellulose. A Michaelis–Menten plot of hydrolysis rate (v) against starch substrate (s) concentration (mg/mL) at a range of cellulose concentrations. Concentrations of added cellulose are indicated in the figure.
where the initial reaction rate is denoted v (mM/min), substrate concentration s (mg/mL), inhibitor (cellulose) concentration i (mg/mL), competitive inhibitor constant Kic (mg/mL) and uncompetitive inhibitor constant Kiu (mg/mL). Takingreciprocals of both
=
Km Vmax
1+
i Kic
+
s Vmax
1+
i Kiu
(3)
Such that a plot of s/v against i is linear (a modified Dixon plot). Plotting at more than one s yields multiple straight lines, which in the case of mixed inhibition will intercept so that i = Kiu and s/v = Km (1 − Kiu /Kic )/Vmax at the point of intersection. The inhibition was shown to be of a mixed linear type, as the inhibition curves cross in the fourth quadrant of the Dixon plot (Fig. 7a) and the third quadrant of the modified Dixon plot (Fig. 7b) (Cornish-Bowden, 2004), with a Kic value estimated from the Dixon plot from a median value of the intercepts to be 2.8 mg/mL, and a
Fig. 7. a Inhibition of ␣-amylase activity in the presence of cellulose shown by a Dixon plot of 1/v against inhibitor (cellulose) (i) at a range of starch concentrations (mg/mL). Concentrations of starch are indicated in the figure. (7b) Inhibition of ␣-amylase activity in the presence of cellulose shown by a modified Dixon plot of inhibitor (cellulose) [i] against substrate/reaction rate (s/v) at a range of starch concentration (mg/mL). Concentrations of starch are indicated in the figure.
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Fig. 8. Representative confocal microscopy images of TRITC-amylase conjugate bound on to the cellulose surface. (A) Before washing; (B) after two successive washing. Pictures were taken under similar microscopic settings.
Kiu estimated from the modified Dixon plot from a median value of the intercepts to be 7.9 mg/mL. A full list of the intercepts used to calculate Kic and Kiu are shown in supplementary information, Table S1. 3.5. Microscopic observation of ˛-amylase binding on cellulose Representative confocal microscopy images of TRITC-amylase conjugate in the presence of cellulose are shown in Fig. 8. It was observed that the labelled ␣-amylase was apparently homogenously bound to the cellulose surface (Fig. 8A). Fig. 8B shows the ␣-amylase-cellulose complex after two successive washing steps. Fluorescence was mostly retained following washing, showing that the interaction between ␣-amylase and cellulose is sufficiently strong to survive both washing and centrifugation processes. 3.6. ˛-Amylase binding to wheat bran In order to test whether binding of ␣-amylase is specific to isolated cellulose, a typical food component containing cellulose (wheat bran) was tested. Results of overall starch digestion progress curves (Fig. S1) and the initial linear portion of progress curves (Fig. S2) are similar to those of pure cellulose, and show that pure cellulose has a slightly more pronounced effect on enzyme activity relative to the non-purified fibre source (bran) over the same range of bran/cellulose concentrations which is in agreement with the findings of Dunaif and Schneeman (1981). This suggests that amylase binding to cellulosic substrates is a general property. 4. Discussion 4.1. ˛-Amylase binding to cellulose: nutritional implications Consumption of plants and plant foods are vital for the nutritional health and well-being of humans. Among many nutrients, cellulose is an important and abundant insoluble dietary fibre (IDF) found in e.g. cereals, fruits and vegetables. Nutritionally, cellulose has an important role as a bulking agent, resulting in shorter transit times and increased faecal mass (Slavin & Marlett, 1980) with associated beneficial effects in the treatment of constipation, diverticular disease, haemorrhoids and in the prevention of colonic cancer (Jensen et al., 2004). In this paper, we describe a potential additional nutritional role for cellulose. In agreement with Hansen and Schulz (1982), our results show that the presence of small amounts of cellulose in an in vitro digestion medium has an
inhibitory effect on the hydrolysis of starch. The progress curve is analysed in two time frames. The first 40 min digestion data (Fig. 1b and S2) is analysed to address the acute or rapid effect of cellulosic materials (pure cellulose and wheat bran) on starch hydrolysis considering the fact that most of the post-prandial blood glucose rise (and therefore initial insulin demand) occurs within 30–60 min from the ingestion of a starchy meal (Ellis, Roberts, Low, & Morgan, 1995). Prolonged digestion up to 5 h (Fig. 1a and S1) was also carried out to observe the effects over longer times. From both observations, it was found that even small amounts of cellulosic materials (pure cellulose or wheat bran) have inhibitory effects on starch digestion. 4.2. ˛-Amylase binding to cellulose: possible binding mechanisms Solution depletion experiments clearly demonstrate that ␣amylase binds to cellulose, and that the binding interaction is very rapid, relative to ␣-amylase binding to granular starch (Warren et al., 2011), approaching equilibrium within 5 min (Fig. 3). This suggests that cellulose may be able to act as an inhibitor of ␣amylase activity on starch, although the binding mechanism of ␣-amylase to cellulose appears to be complex, with multiple binding modes (Fig. 4). ␣-Amylase is unable to hydrolyse the  (1 → 4) glucan linkages of cellulose, so cellulose cannot be degraded by the enzyme, but, being a glucose polymer, cellulose bears some structural similarities to starch. An interesting question raised by these solution depletion assays is whether there is a specific, active site mediated, binding interaction between ␣-amylase and cellulose, suggested by the apparently high affinity of ␣-amylase for cellulose, or whether the interaction is non-specific in nature as postulated by Dunaif and Schneeman (1981). To explore this further we devised a series of experiments to explore the influence of the addition of maltose (which binds to the active site of ␣amylase with high specificity, blocking other interactions) and BSA (a protein with no known carbohydrate binding sites) on ␣-amylase binding to cellulose. Maltose is known to be a competitive inhibitor of ␣-amylase. The inhibition is due to occupancy of one or two maltose molecules in the active site of amylase (Elödi et al., 1972; Warren et al., 2012). In the presence of a range of maltose concentrations (0–20 mM), there was a slight downward shift in the absorption isotherm, but there was no concentration dependence observed. From these experiments (Fig. 5) it is apparent that maltose does not inhibit ␣-amylase binding to cellulose, strongly suggesting that the binding between ␣-amylase and cellulose is not active site mediated.
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A binding isotherm for ␣-amylase binding to cellulose was obtained in the presence of BSA, a protein with no known affinity for carbohydrates. It was found that the addition of excess BSA completely blocked ␣-amylase binding to cellulose. Therefore, we hypothesise that both proteins are non-specifically adsorbed onto cellulose, and that the ca 5 × 104 fold excess BSA protein occupied all available protein binding sites on the cellulose surface, saturating the cellulose with protein and blocking binding by ␣-amylase. The combined results of the maltose and BSA experiments indicate that the binding interaction between ␣-amylase and cellulose is non-specific in nature. It appears that ␣-amylase is adsorbed to the surface of cellulose, depleting the protein from solution, hence allowing cellulose to act as an inhibitor of ␣-amylase activity in a simplified starch-cellulose-␣-amylase system. In more complex, multi-component systems such as the human intestine, however, it is possible that additional inhibitory and other effects of cellulose on ␣-amylase activity may occur, and will require study. 4.3. ˛-Amylase binding to cellulose: inhibition mechanism In order to determine the mode of inhibition of ␣-amylase action on starch by cellulose, the initial velocity (v) at a range of inhibitor concentrations (i) across a range of substrate concentrations (S) were used to plot Dixon and modified Dixon plots (Cornish-Bowden, 1974). A competitive inhibitor, [i] binds to the active site of the enzyme to form an enzyme–inhibitor complex (EI). As seen in Fig. 7a, when [i] is plotted against 1/v, at a range of S, the intersect is in the fourth quadrant, indicating that the Km increases with increasing inhibitor concentration. This is typical of competitive or mixed inhibition and rules out the possibility of uncompetitive and noncompetitive inhibition where lines respectively intersect exactly at a negative x-axis or run parallel to each other without intersecting (Cornish-Bowden, 1974). The dissociation constant of the EI complex for competitive inhibition (Kic ) was found to be ca. 3 mg/mL (Fig. 4). To distinguish between competitive or mixed inhibition, the data was also plotted as a modified Dixon plot (s/v versus [i]) as presented in Fig. 7b. In the case of competitive inhibition, the lines for each starch concentration will run parallel to each other without intersecting (Cornish-Bowden, 1974) however as seen in Fig. 7b, the lines intersect in the third quadrant, suggesting a mixed mode of inhibition. The dissociation constant for EIS complex (Kiu ) derived from a modified Dixon plot is 6 mg/mL. Data suggesting mixed inhibition brings up the question of the possible mechanism by which mixed inhibition kinetics occur in the present system. In competitive inhibition, the inhibitor cannot bind if the substrate has already bound to the enzyme, whereas in uncompetitive inhibition, the inhibitor can only bind if the substrate has already bound to the enzyme. However in mixed inhibition, the inhibitor can bind whether or not the substrate has already bound to the enzyme. In the case of classical reversible mixed type inhibition as presented in Fig. 9a, inhibitor (I) can interact with both free ␣-amylase (E) and ␣-amylase-starch complex (EP) at a site other than the active site. The scheme presented in Fig. 9a seems unlikely, however, as is difficult to conceive how two large, insoluble substrates such as starch granules and cellulose could both be bound to ␣-amylase simultaneously. An alternative mechanism is suggested in Fig. 9b. The data presented in Fig. 5 show that enzyme with maltose (the product of starch hydrolysis) bound in the enzyme active site could still bind to cellulose. In the mechanism suggested in Fig. 9b, enzyme with maltose bound in the active site following hydrolysis can bind to cellulose, reducing the overall turnover of the enzyme. This mechanism is, however, only speculative and clearly requires further investigation.
Fig. 9. (a) Schematic diagram of classical linear mixed inhibition. In this type of reversible inhibition, an inhibitor can interact with both the free enzyme and the enzyme–substrate complex at a site other than the active site. This results in an apparent decrease in Vmax (apparent enzyme maximum velocity in the presence of an inhibitor) and an apparent increase in Ks (apparent enzyme–substrate dissociation constant) in the presence of an inhibitor. (9b) Schematic diagram of linear mixed inhibition of amylase activity by cellulose in the presence of maltose.
4.4. Inhibition of amylase activity, soluble versus insoluble fibres These findings may be contrasted with the inhibition of ␣-amylase activity by another dietary fibre, the soluble polysaccharide guar gum (Slaughter et al., 2002). Guar gum was found to inhibit ␣-amylase activity in a non-competitive mechanism with a ki value of between 3 and 5 mg/mL. This is a similar value to the ki determined in the present study, although a different mechanism of inhibition. While we do not presently have sufficient data to determine the reasons for differences in inhibition mechanism between these two forms of fibre, it could be speculated that the soluble galactomannan may exert effects such as reducing mass transfer through viscosity effects. Interestingly, the ELISA assay reported by Slaughter et al. (2002) suggested that, unlike the results presented here for cellulose, there was a degree of specificity for ␣-amylase binding to guar gum.
5. Conclusions Starch is a major source of energy in the human diet, whereas cellulose cannot be degraded in the upper digestive tract, and is often considered to have a nutritional role limited to its action as an inert bulking agent in the diet. In the present study we demonstrate that in vitro cellulose can bind ␣-amylase, and inhibit the activity of the enzyme through a mixed type inhibition mechanism. The binding of ␣-amylase to cellulose was found to be reversible and non-specific. The binding was not affected by the addition of maltose, a competitive inhibitor that binds to the enzymes active site, but was completely obviated by the addition of an additional protein, BSA, which was presumably also adsorbed to the surface of the cellulose in a competitive manner. Taken together, these results indicate that cellulose (either purified or as a component of wheat bran) can have a significant inhibitory effect on ␣-amylase, that may have relevance in vivo during gastrointestinal digestion, as well as to industrial processing systems involving ␣-amylase and plant matter. More generally, the non-specific binding mechanism suggests that a range of proteins such as other digestive enzymes are also likely to bind to cellulosic components in digesta.
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