Article pubs.acs.org/JAFC
Effects of Granule Swelling on Starch Saccharification by Granular Starch Hydrolyzing Enzyme Zhaofeng Li,‡ Liming Cai,† Zhengbiao Gu,‡ and Yong-Cheng Shi*,† †
Department of Grain Science and Industry, Kansas State University, Manhattan, Kansas 66506, United States School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China
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‡
ABSTRACT: The effects of granule swelling on enzymatic saccharification of normal corn starch by granular starch hydrolyzing enzyme were investigated. After swelling, Km values for the saccharification of granular starch decreased compared with native granular starch, indicating that granule swelling caused granular starch hydrolyzing enzyme to have higher affinity for starch granules. The partial swelling of starch granules enhanced starch saccharification. Furthermore, the enhancement at an earlier stage of enzymatic reaction was much more significant than that at later stages. For granular starch pretreated at 67.5 °C for 30 min, conversions to glucose after incubation with the enzyme at 32 °C for 4 and 24 h were approximately 3-fold and 26% higher than for native granular starch, respectively. As a result, proper heat pretreatment of granular starch before simultaneous saccharification and fermentation has great potential to facilitate industrial production of ethanol by use of granular starch hydrolyzing enzyme. KEYWORDS: corn starch, granule swelling, granular starch hydrolyzing enzyme, saccharification, enhancement
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area of the starch granules.15−19 α-Amylase and glucoamylase are relatively large, several nanometers in size.20−22 Because of their relatively large size, these enzymes have limited access to starch granules. Granule swelling could increase the specific surface area of starch granules (surface area per unit mass of of dry starch) and open up the surface. We postulated that even slight granule swelling would have a great positive impact on enzymatic hydrolysis of granular starch. Thus, the objective of this study was to determine the effects of granule swelling of normal corn starch, the most commonly used starch material in ethanol production, on enzymatic saccharification by a granular starch hydrolyzing enzyme.
INTRODUCTION Ethanol is the primary renewable liquid transportation fuel, with a long history of good performance, and it can be used in over 80% of automobiles and other light-duty transportation vehicles.1 The basic procedures for large-scale production of fuel ethanol from starch are enzymatic conversion of starch into fermentable sugars, microbial fermentation of sugars to ethanol, distillation, dehydration, and denaturing (optional).2 The conventional process for enzymatic hydrolysis of starch into sugars prior to fermentation is to cook a starch slurry of approximately 30% (w/v) in the presence of thermostable αamylase at above 100 °C, hold it at 90 °C for 1−3 h, and then cool it further to 60 °C with added glucoamylase.3 This process has considerable economic drawbacks, mainly due to high energy-consuming cooking and liquefaction steps.4 An alternative process in which granular starch hydrolyzing enzymes are applied to convert granular or uncooked starch to glucose and other fermentable sugars has been suggested.5−7 This process does not require high temperatures during cooking and liquefaction, which could reduce the energy requirements of ethanol production3 and decrease the formation of undesirable byproducts of the Maillard reaction.8 In addition, the process could simplify the operation and eliminate the cooking and cooling equipment required to handle high-viscosity starch slurries. Granular starch hydrolyzing enzymes are commercially available and have been applied in a simultaneous saccharification and fermentation process for ethanol production.9−11 Enzymatic hydrolysis of granular starch is a solid-solution two-phase reaction in which the enzymes need to first diffuse toward and bind to the solid substrate and then cleave the glycosidic linkages.12 Enzyme adsorption onto starch granules is the initial stage for enzymatic hydrolysis of granular starch;13−15 consequently, the initial rate of granular starch hydrolysis is related to the morphology, porosity, particle size, and surface © 2014 American Chemical Society
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MATERIALS AND METHODS
Materials. Normal corn starch was obtained from National Starch LLC (Bridgewater, NJ). Stargen 001, a granular starch hydrolyzing enzyme, was obtained from Genencor International (Palo Alto, CA). Blue dextran, which was prepared from dextran with an average molecular weight of approximately 2.0 × 106, was purchased from Sigma (St. Louis, MO). Other chemicals were purchased from Fisher Scientific (Santa Clara, CA). Determination of Swelling Factor. The swelling factor of the starches was measured according to the blue dextran dye exclusion method of Tester and Morrison.23 Normal corn starch (100 mg, dry basis) was weighed into a 10 mL screw-cap tube, 5.0 mL of water was added, and the sealed tubes were incubated with constant shaking in a water bath at a specific temperature for 30 min. Then 0.5 mL of blue dextran solution (5 mg/mL) was added, and the contents were mixed by gently inverting the closed tubes several times. After centrifugation at 5000g for 5 min, the absorbance of the supernatant was measured at 620 nm. The absorbance of reference tubes that contained no starch Received: Revised: Accepted: Published: 8114
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was also measured. The swelling factor was reported as a ratio of the volume of swollen granules to the volume of dry starch. Light Microscopy. Normal corn starch (100 mg, dry basis) was slurried in water (1.0 mL) and heated in a water bath at a specific temperature for 30 min with constant shaking. The starch slurry without heat pretreatment was used as a control. Starch samples were promptly viewed with an optical microscope with a digital camera (Model BX51, Olympus Co., Japan) and observed under normal visible light by use of a 40× objective. Assay for Glucose-Forming Activity. Glucose-forming activity was measured by incubating 0.05 mL of 100-fold diluted enzyme with 0.95 mL of 1% (dry basis, w/v) normal corn starch slurry in 50 mM citrate buffer (pH 4.2) at 32 °C for 10 min. The reaction was stopped by boiling the mixture for 10 min. Glucose released was measured by high-performance anionic-exchange chromatography (HPAEC) on a Dionex ICS 3000 system equipped with a CarboPac PA-1 column and pulse amperometric detector (PAD, Dionex, Sunnyvale, CA). The mobile phase was 150 mM NaOH at a flow rate of 1 mL/min. One unit of activity was defined as the amount of enzyme that released 1 μmol of glucose/min. Enzyme activity was expressed as units per milliliter of enzyme solution. Enzyme Kinetic Studies. First, normal corn starch slurry in 50 mM citrate buffer (pH 4.2) was heated in a water bath at a specific temperature for 30 min with constant shaking. The starch slurry without heat pretreatment was used as a control. Then 0.05 mL of 100-fold diluted enzyme (Stargen 001) was incubated with 0.95 mL of the preheated starch slurry at 32 °C for 10 min. Kinetic studies were performed by measuring the initial rate of glucose formation at normal corn starch concentrations ranging from 0.05% to 1.0%. The SigmaPlot program (Systat Software Inc., version 10.0 for Windows) was used for curve-fitting of the data to the standard Michaelis− Menten formula: v = Vmax[S]/(Km + [S]), where v is the rate of glucose formation, [S] is the substrate concentration, Vmax is the maximal velocity of the reaction, and Km is the Michaelis−Menten constant. Saccharification Process of Granular Starch. Before enzymatic saccharification, normal corn starch slurry (30%, dry basis, w/v) in 50 mM citrate buffer (pH 4.2) was heated in a water bath at a specific temperature for 30 min with constant shaking. The starch slurry (30%, dry basis, w/v) without heat pretreatment was used as a control. The starch slurry was incubated with Stargen 001 (2.5 units/g dry starch) at 32 °C for 72 h. At regular time intervals, samples were taken and boiled for 10 min to terminate the reaction. Glucose released was measured by HPAEC-PAD. Morphology of the Starch Granule. Starch granule morphology was studied by scanning electron microscopy (SEM). Normal corn starch slurry (30%, dry basis, w/v) in 50 mM citrate buffer (pH 4.2) was heated at 55 or 65 °C for 30 min with constant shaking. The starch slurry (30%, dry basis, w/v) without heat pretreatment was used as a control. Then the above starch slurries were incubated with Stargen 001 (2.5 units/g dry starch) at 32 °C. After 0, 4, or 24 h of incubation, samples were taken, washed with anhydrous ethanol, and dried in a forced-air oven at 30 °C for 24 h. The dried samples were coated with gold−palladium by a sputter coater (Denton Vacuum, LLC, Moorestown, NJ) and viewed at 2000× magnification by SEM (S-3500N, Hitachi Science Systems, Ltd., Japan) operating at an accelerating voltage of 20 kV. Statistical Analysis. Experiments were conducted in triplicate, and average values are reported. Statistical analyses were carried out by SPSS software (SPSS Inc., Chicago, IL). Significant differences (p < 0.05) between means were identified by one-way analysis of variance (ANOVA) using the Student−Newman−Keuls (SNK) procedure.
Figure 1. Swelling curve of normal corn starch granules heated at different temperatures. Each value represents the mean of three independent measurements.
that normal corn starch has an onset gelatinization temperature of about 58 °C.17 Starch granules swell significantly above that temperature, resulting in the loss of crystalline order and birefringence. The swelling factor curve reached a plateau value at 70−80 °C with a swelling factor of 9−11. The swelling factor further increased significantly at temperatures above 80 °C and reached approximately 17 at 90 °C. A similar trend has been observed for waxy corn starch at temperatures below 80 °C; compared with normal corn starch, waxy corn starch showed a higher swelling curve.23,24 The swelling factors of waxy corn starch granules at 75−80 °C reached above 30, which was approximately 2-fold higher than normal corn starch. Previous studies have also shown that the other waxy cereal starches had higher swelling capacities than corresponding normal starches.23,25 The main reason might be that amylose−lipid complexes in normal starches inhibit granule swelling.23 Swelling of starch granules is mainly a property of amylopectin. In addition, when normal corn starch was incubated at 65 or 70 °C for various durations, the period of rapid swelling lasted approximately 10 min (Figure 2). After that, the swelling factor almost plateaued. Thus, incubation at a specific temperature for 30 min could reach a steady state for the swelling of starch granule. The size and shape of normal corn starch with or without heat pretreatment at a specific temperature for 30 min are shown in Figure 3. Normal corn starch granules without heat pretreatment had polygonal and round shapes. After treatment
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RESULTS AND DISCUSSION Swelling of Starch Granules. The swelling factors of normal corn starch granules were investigated in the 40−90 °C temperature range. Very little swelling was observed from 40 to 50 °C; the swelling factor increased only slightly at 50−60 °C but greatly at temperatures above 60 °C (Figure 1). It is known
Figure 2. Effects of different incubation time durations on granule swelling of normal corn starch (2% solids) preheated in a water bath at (◆) 65 or (■) 70 °C with constant shaking. Each value represents the mean of three independent measurements. 8115
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optical microscopy agreed with results of swelling factors of normal corn starch granules (Figure 2). Kinetic Parameters. Heat pretreatment of normal corn starch granules at different temperatures greatly affected the kinetic properties of the granular starch hydrolyzing enzyme. After heat pretreatment, Km values for the saccharification of granular starch decreased compared with those for granular starch without heat pretreatment (Table 1), indicating that granule swelling resulted in higher affinity of the granular starch hydrolyzing enzyme for starch granules. Furthermore, the significant decrease in Km value was not observed until starch granules swelled almost completely, at 70 °C or above (Figure 3). On the other hand, after heat pretreatment at 50−60 °C, Vmax values for the saccharification of granular starch increased only slightly compared with those for granular starch without heat pretreatment (Table 1). After the starch granules began to swell at temperatures above 60 °C, however, the increase in Vmax was significant. Even if the starch granules were pretreated only at 65 or 67.5 °C, at which temperature the starch granules were partly swollen, Vmax values were more than 1.5-fold higher than those of granular starch without heat pretreatment. Catalytic efficiency as expressed by kcat/Km ratios (Table 1) indicated that granular corn starch with heat pretreatment was a better substrate than that without heat pretreatment. It is noteworthy that kcat/Km values for granular starch preheated at temperatures above 60 °C were much higher than those for granular starch without heat pretreatment (Table 1). For granular starch preheated at 65 or 67.5 °C, the value was more than 2-fold higher than for granular starch without heat pretreatment. Saccharification Process of Granular Starch. Because heat pretreatment of 30% normal corn starch slurry (dry basis, w/v) at 70 °C or above resulted in a high slurry viscosity, temperatures below 70 °C (50−67.5 °C) were chosen to treat the normal corn starch granules before enzymatic saccharification. The pretreatment of normal corn starch granules at 50−67.5 °C enhanced starch saccharification by granular starch hydrolyzing enzyme under conditions resembling the industrial yeast fermentation process for ethanol production (Figure 4). The higher the pretreatment temperature, the more significantly starch saccharification was enhanced. In addition, enhancement of starch saccharification at an earlier stage of the enzymatic reaction was much more significant than at later stages. For granular corn starch pretreated at 65 or 67.5 °C before enzymatic saccharification, conversion to glucose after incubation with the enzyme at 32 °C for 4 h was approximately 2- or 3-fold higher, respectively, than for granular starch without heat pretreatment (Figure 4). In contrast, after incubation for 24 h, pretreatment at 65 or 67.5 °C resulted in approximately 18% or 26% increases in starch conversion into glucose, respectively (Figure 4). The significant enhancement in saccharification of heatpretreated granular corn starch at earlier stages might be attributed to the looser surface structure and higher specific surface area of partially swelled starch granules, which give the enzyme greater access to the substrate and allow the enzyme to degrade granular starch more easily and extensively.26 As the sizes of active α-amylase and glucoamylase are several nanometers,20−22 the enzymes can enter into the interior of native starch granule only through the limited number of pores to degrade starch, but granule swelling provides greater enzyme access to the granule interior. However, at later stages of the enzymatic reaction, reduced availability of starch to the enzyme
Figure 3. Light microscopic images of normal corn starch granules heat-treated at a specific temperature for 30 min with constant shaking. The number below each panel represents the treatment temperature. Starch granules without heat pretreatment were used as a control. Starch samples were observed under normal visible light by use of a 40× objective.
at 50−60 °C, the granules began to swell slightly, whereas granule swelling was obvious only at temperatures above 60 °C. Large starch granules appeared to swell first at around 50−60 °C; small granules did not begin to swell obviously until 65 °C. Few starch granules began to be disrupted at 65 or 67.5 °C. More starch granules were disrupted, and the rest swelled at 70−80 °C, indicating partial gelatinization of starch samples. Almost all granules were irreversibly disrupted at 90 °C, indicating complete gelatinization of starch samples. The swelling trend of normal corn starch granules obtained by 8116
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Table 1. Kinetic Parameters for Granular Starch Hydrolyzing Enzyme Acting on Normal Corn Starch Granules Heat-Treated at a Specific Temperature for 30 Min with Constant Shakinga,b temp (°C) native starch 50 55 60 65 67.5 70 75 80 90
Vmaxc (μM/min) 104.2 119.1 126.6 138.9 256.4 285.7 322.6 347.2 355.9 409.8
± ± ± ± ± ± ± ± ± ±
3.5 3.8 4.5 4.3 5.1 5.0 6.2 7.5 6.5 6.8
a b b c d e f g g h
kcatc ( × 105)
Kmc (%) 0.292 0.286 0.241 0.236 0.231 0.229 0.129 0.056 0.053 0.052
± ± ± ± ± ± ± ± ± ±
0.012 d 0.014 d 0.011 c 0.009 c 0.01 c 0.014 c 0.01 b 0.006 a 0.005 a 0.005 a
0.135 0.154 0.164 0.180 0.333 0.371 0.419 0.451 0.462 0.532
± ± ± ± ± ± ± ± ± ±
0.004 0.007 0.008 0.007 0.012 0.014 0.016 0.018 0.021 0.025
a ab ab b c d e f f g
kcat/Km ( × 105) 0.462 0.538 0.680 0.763 1.442 1.620 3.248 8.054 8.717 10.231
a
The starch concentration in reaction mixtures ranged from 0.05% to 1.0%. Stargen 001 concentration [E0] was expressed as enzyme activity units per milliliter of reaction mixture. Stargen 001 was included in reaction mixtures at a constant concentration of 0.077 unit/mL. bKinetic parameters plus standard errors were obtained from fitting of the experimental data to the standard Michaelis−Menten formula by the SigmaPlot program. c Means with different letters within the same column are significantly different (p < 0.05).
resulted in consumption of a small amount of energy, a reduction in the amount of granular starch hydrolyzing enzyme might significantly lower the production cost of ethanol. Heat pretreatment could also result in significant enhancement of the conversion of granular starch into glucose at earlier stages of the enzymatic reaction, which might be beneficial to yeast fermentation for ethanol production because the rapid growth of yeast at the early stage of fermentation (especially the logarithmic growth phase) needs much more glucose as a carbon source than at the later stage of fermentation. Morphology of Starch Granules. During enzymatic saccharification, the morphology of granular corn starch with or without heat pretreatment was observed by SEM (Figure 5A−C). Changes in morphology of normal corn starch before and after digestion by α-amylase alone, glucoamylase alone, and a mixture of α-amylase and glucoamylase have been reported.17 In this study, normal corn starch granules pretreated at 55 or 65 °C were larger and had more irregular shapes and rougher surfaces than starch granules without heat pretreatment (Figure 5A−C), further confirming that larger particle size and corresponding higher specific surface area resulted from heat pretreatment. Enzymatic erosion occurred mainly at the surface of starch granules at earlier stages of the enzymatic reaction (Figure 5D−F). After incubation with granular starch hydrolyzing enzyme at 32 °C for 4 h, the surface of starch granules pretreated at 55 °C, especially relatively large granules (Figure 5E), appeared to have a greater number of small pores than granules without heat pretreatment, which could permit more enzyme penetration.32 As a result, when the external part of the granule was corroded, the enzyme could more easily act internally.33 For starch granules pretreated at a higher temperature (65 °C), many granules swelled to a higher degree. As a result, starches in external parts of the granule could be corroded more easily by the enzyme, and even internal parts of the granule were hydrolyzed and removed (Figure 5F). In contrast, for the normal corn starch granules without pretreatment, only micropores were observed but not the interior of the granules (Figure 5D). After incubation for 24 h, starch granules without heat pretreatment showed more large pores in the surface than those incubated for 4 h, and the internal part of some granules was corroded (Figure 5G). The greater portion of many starch granules, however, was still not hydrolyzed, and some small granules were almost intact. In contrast, starch granules subjected to heat pretreatment
Figure 4. Production of glucose under process conditions resembling the industrial yeast fermentation process for ethanol production. Before enzymatic saccharification, normal corn starch slurry (30%, dry basis, w/v) in 50 mM citrate buffer (pH 4.2) was heated in a water bath at (■) 50, (▲) 55, (×) 60, (*) 65, or (●) 67.5 °C for 30 min with constant shaking. The starch slurry without heat pretreatment (◆) was used as a control. During enzymatic saccharification, the formation of glucose was measured by incubation of Stargen 001 (2.5 units/g dry starch) with a starch sample at 32 °C for 24 h and expressed as starch conversion (percent, dry basis). Each value represents the mean of three independent measurements.
might result in a decreased glucose formation rate, especially in the rigid or tight crystalline region of the starch. Because αamylase binds more slowly to crystalline material,27 the residual starch granules with high crystallinity were resistant to enzymatic hydrolysis. After pretreatment of normal corn starch granules at 55−67.5 °C, which was close to or above the gelatinization temperature, a small portion of the semicrystalline region would be converted into amorphous material,28,29 which is more readily hydrolyzed,30 but the crystalline portion of the starch would not be significantly changed. Thus, partial swelling of starch granules would not significantly enhance starch saccharification at later stages of the enzymatic reaction. During the simultaneous saccharification and fermentation process for ethanol production, improved saccharification of heat-pretreated granular corn starch could reduce the necessary amount of granular starch hydrolyzing enzyme. Granular starch hydrolyzing enzyme is more expensive than conventional enzymes,31 so the amount of granular starch hydrolyzing enzyme is an important factor in the economics of ethanol production. Although heat pretreatment of granular corn starch 8117
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Figure 5. Scanning electron micrographs of normal corn starch granules during enzymatic saccharification. Normal corn starch slurry (30%, dry basis, w/v) without heat pretreatment or pretreated at 55 or 65 °C for 30 min was incubated with Stargen 001 (2.5 units/g dry starch) at 32 °C for (A−C) 0, (D−F) 4, or (G−I) 24 h, respectively.
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exhibited a higher degree of external and internal starch hydrolysis that resulted in the almost complete collapse of large starch granules (Figure 5H,I). For starch granules heatpretreated at 65 °C, the residual portion of large granules was very small, and even small ones were also corroded, resulting in a rough porous surface or collapse of the granule (Figure 5I). Both α-amylase and glucoamylase are able to hydrolyze crystalline as well as amorphous regions of normal corn starch granules, and a synergetic action was observed when both enzymes were used together.17 A side-by-side mechanism has been proposed to explain the enzymatic digestion of amylose and amylopectin, as well as crystalline and amorphous regions in normal corn starch granules.19 In conclusion, the partial swelling of normal corn starch granules greatly enhanced starch saccharification by granular starch hydrolyzing enzymes. Furthermore, enhancement of starch saccharification at an earlier stage of the enzymatic reaction was much more significant than at later stages. Thus, proper heat pretreatment of granular starch before simultaneous saccharification and fermentation has great potential to facilitate industrial production of ethanol by use of granular starch hydrolyzing enzyme.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 785-532-6771; fax 785-532-7010; e-mail ycshi@ ksu.edu. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This is contribution no. 14-137-J from the Kansas Agricultural Experiment Station.
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