Hydrolysis Of Starch Granules Research Articles

Interfacial kinetics reveal enzymatic resistance mechanisms behind granular starch with smooth surfaces

Heterogeneous (interfacial) enzyme catalyzed hydrolysis of starch granules occurs in various biological systems, including plants, animal/human digestion, and microorganisms. Factors such as granular size, surface area, pores, and smoothness play crucial roles in influencing this process. However, limited understanding persists regarding the high enzymatic resistance of starch granules with smooth surfaces. In this study, we investigated the hydrolysis mechanism of glucoamylase (GA) on three different types of starch granules with smooth surfaces, extracted from Curcuma zedoaria (zedoary) rhizomes, Solanum tuberosum (potato) tubers, and Manihot esculenta (tapioca or cassava) roots. We compared the Langmuir adsorption, interfacial kinetics, and the multi-level structure of the three starches. Our data demonstrate that the lower enzymatic resistance observed in tapioca starch stems from the higher density of enzymatic attack sites (kinΓmax) recognized by GA on tapioca starch (1.0 nmol/g) compared to potato (0.6 nmol/g) and zedoary (0.3 nmol/g) starch granules. The high kinΓmax for tapioca starch was significantly influenced by its relatively lower B-type crystallinity, which is disrupted by the presence of short fa chains (degree of polymerization (DP) < 12) and long amylose chains. Furthermore, the relatively higher proportion of longer chains (fb1 and fb2 chains) on the surface of tapioca starch also contributed to higher kinΓmax for GA, resulting in lower enzymatic resistance. These findings enhance our understanding of how the structure of starch granules affects enzymatic catalysis, particularly in granular starches with smooth surfaces devoid of pores. Such insights are crucial for elucidating the digestion and utilization of starch granules.

Mechanical wounds expedited starch degradation in the wound tissues of potato tubers

Starch degradation occurs rapidly in stressed plants, but it is unclear how starch degradation occurs in potato tubers after they incur mechanical wounding. In this study, we found that wounding significantly upregulated the expression levels of StGWD, StAMY, StBAM, and StISA, and decreased the starch content of potato tubers. Meanwhile, wounding markedly upregulated the expression levels of StSUS, StBG, and StINV genes, and increased the content of sucrose, glucose, and fructose. Furthermore, wounding reduced the proportion of small starch granules and increase that of large as well as medium starch granules, in this way enhancing the average size distribution of starch. Initially, the hard surface layer of starch granules was removed by wounding, but the internal channels and other structures were only slightly affected. Taken together, the results show that wounding can accelerate starch degradation by promoting the accumulation of sucrose, glucose, and fructose, and the hydrolysis of starch granules in potato tubers.

Barley Malt Esterification after Ultrasound and Stearic Acid Treatment: Characterization and Use as Stabilizing Agent in Oil-in-Water Emulsions

This work aimed to obtain novel materials from malt modification for using in oil/water emulsions. Malt was modified using stearic acid (2% w/w) in combination with different energy densities (33.8 × 106, 60 × 106, 72.5 × 106, and 107.5 × 106 J/m3) from ultrasound probe resulting in four materials. These materials were characterized by degree of substitution (DS), amylose content, dextrose equivalent (DE), protein content, solubility, thermal properties, Fourier-transform infrared spectroscopy (FT-IR), crystallinity analysis by X-ray diffraction, and scanning electron microscopy. Emulsions containing 15% total solids (25% canola oil and 75% modified malt as stabilizing agent) were prepared and characterized. Results showed that increasing energy densities produced different effects on malt properties due to an increase in DS, amylose content, and DE values. The sonication process in combination with stearic acid increased malt solubility and the gelatinization temperature of malt, caused modifications of starch crystallinity in the amorphous region and hydrolysis of starch granules, and promoted good stabilizing properties for emulsions containing modified malt.

Investigation on morphological structure and crystal transition of maize starch gelatinized in pure glycerol

The gelatinization phenomena and crystalline structure of maize starch gelatinized in pure glycerol were investigated using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Starch granules were firstly treated in water system, CLSM and SEM micrographs displayed that they were completely broken and the characteristic birefringence of the starch granules disappeared at 70 °C. As for pure glycerol system, the starch granules swelled but maintained granular shape with the increasing of temperature. The crystalline structure of starch granules was partially destroyed at 130 °C and completely destroyed at 140 °C. The DSC thermogram showed that the gelatinization temperature of starch in glycerol started at 123.7 °C, peaked at 128.4 °C, and concluded at 135.2 °C. The X-ray diffractograms indicated that the crystalline structure of maize starch was partially destroyed at 130 °C and completely destroyed at 140 °C. Thus, glycerol served an alternative solvent to destroy crystalline structure of maize starch, which may be helpful for hydrolysis of starch granules by amylase in food industry.

Inhibition of glycosidase by ursolic acid: invitro, in vivo and in silico study.

Controlling the blood glucose level is an effective method to reduce type 2 diabetes and prevent diabetes-related complications. Ursolic acid is a plant extract that can reduce postprandial hyperglycemia effectively. This study aimed to explore the inhibitory effect and interaction mechanism of ursolic acid against α-amylase and α-glucosidase. In this study, the effect of ursolic acid on glycosidase was studied in vitro, in vivo, and in silico. The half-maximal inhibitory concentration (IC50 ) of ursolic acid on α-amylase and α-glucosidase was 0.482 ± 0.12 mg mL-1 and 0.213 ± 0.042 mg mL-1 , respectively. The results of enzymatic kinetics showed that ursolic acid inhibited α-amylase and α-glucosidase activity in a non-competitive manner. The fluorescence spectrum showed that the combination of ursolic acid and glycosidase caused the intrinsic fluorescence quenching of glycosidase. The observation of starch granules revealed that the activity of α-amylase was inhibited and the hydrolysis of starch granules was prevented in the presence of ursolic acid. Molecular docking results showed that ursolic acid bound to the inactive site of α-amylase and α-glucosidase through the formation of ursolic acid-glucosidase complex. Ursolic acid interacted with α-amylase and α-glucosidase mainly through hydrogen bonding. The postprandial hypoglycemic effect of ursolic acid in C57BL/6J mice showed that the high concentration of ursolic acid could quickly reduce postprandial blood glucose level. Ursolic acid can be considered as a natural ingredient in functional foods to control postprandial blood glucose levels and prevent diabetes by delaying the digestion of starch in foods. © 2019 Society of Chemical Industry.

Heat pretreatment improves the enzymatic hydrolysis of granular corn starch at high concentration

Abstract Heat pretreatment was investigated as a way to improve the α-amylase-catalyzed hydrolysis of granular corn starch at high concentration (45%, w/w). Native and preheated starches were hydrolyzed in the granular state for 1 h at 40 °C using a commercially available α-amylase. The initial dextrose equivalent (DE) value of the native granular starch (8.0%) increased significantly (to 12.6%) after pretreatment at 60 °C for 15 min. Microscopic analysis of the starch granules indicated that heat pretreatment increased the number and size of the pores and pinholes on the granule surface, which facilitated the adsorption and subsequent penetration of enzyme molecules. Furthermore, the amylose present in amorphous regions leached rapidly when corn starch was incubated at 60 °C, suggesting weaker interactions within the granules. The DE value of the hydrolysate showed a linear dependence on the amount of amylose that leached during the course of heat pretreatment (R 2 = 0.9771). Mobilization of amylose chains within the granule, caused by the heat pretreatment, may have allowed the enzyme to make greater use of amylose, which is its primary substrate during starch granule hydrolysis. In addition, a 35% reduction in the K m value showed that heat pretreatment increased the affinity of α-amylase for the starch granules. This can be attributed to the mobilization of amylose chains within the granule and expansion of the surface pinholes. These effects demonstrate that proper heat pretreatment of granular starch before amylolysis has great potential in industry.

Amylase binding to starch granules under hydrolysing and non-hydrolysing conditions

Although considerable information is available about amylolysis rate, extent and pattern of granular starches, the underlying mechanisms of enzyme action and interactions are not fully understood, partly due to the lack of direct visualisation of enzyme binding and subsequent hydrolysis of starch granules. In the present study, α-amylase (AA) from porcine pancreas was labelled with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye with maintenance of significant enzyme activity. The binding of FITC/TRITC-AA conjugate to the surface and interior of granules was studied under both non-hydrolysing (0°C) and hydrolysing (37°C) conditions with confocal microscopy. It was observed that enzyme binding to maize starch granules under both conditions was more homogenous compared with potato starch. Enzyme molecules appear to preferentially bind to the granules or part of granules that are more susceptible to enzymic degradation. The specificity is such that fresh enzyme added after a certain time of incubation binds at the same location as previously bound enzyme. By visualising the enzyme location during binding and hydrolysis, detailed information is provided regarding the heterogeneity of granular starch digestion.

Morphological changes of starch granules during grain filling and seed germination in wheat

AbstractIn this study, the morphological changes of wheat starch granules during grain filling and seed germination were observed under scanning electron microscopy. Until 3 days post anthesis (DPA), the pericarp tissue was the major site of starch deposited. From 6 DPA, the size and the number of granules in endosperm gradually increased. The “double disk structure”, “pin holes”, and “equatorial groove” at the surfaces of the granules were observed during grain filling, these micro‐structural features might aid the enzymatic hydrolysis of starch granules during seed germination. The equatorial groove of granule was more susceptible to enzymatic hydrolysis than the flat surface.

Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.

Two Secondary Carbohydrate Binding Sites on the Surface of Barley α-Amylase 1 Have Distinct Functions and Display Synergy in Hydrolysis of Starch Granules

Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.

Processing and Characterization of Waxy Maize Starch Films Plasticized by Sorbitol and Reinforced with Starch Nanocrystals

Nanocomposites films have been processed from a filler and a matrix having the same nature, i.e. waxy maize starch. The filler consists of nanoplatelet-like starch particles obtained as an aqueous suspension by acid hydrolysis of starch granules and the matrix was prepared by plasticization and disruption of starch granules with water and sorbitol. Nanocomposite films were obtained by casting and evaporating the mixture of the aqueous suspension of starch nanocrystals with the gelatinized starch. The resulting films were conditioned before testing and the effect of accelerated ageing in moist atmosphere was investigated. The thermal properties of the nanocomposite films were determined from DSC measurements and the mechanical characterization was performed in both the linear and nonlinear range.

Kinetic Analysis of Glucoamylase-Catalyzed Hydrolysis of Starch Granules from Various Botanical Sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.

Synergistic Action of Recombinant α-Amylase and Glucoamylase on the Hydrolysis of Starch Granules

Barley alpha-amylase 1 mutant (AMY) and Lentinula edodes glucoamylase (GLA) were cloned and expressed in Saccharomyces cerevisiae. The purified recombinant AMY hydrolyzed corn and wheat starch granules, respectively, at rates 1.7 and 2.5 times that of GLA under the same reaction conditions. AMY and GLA synergistically enhanced the rate of hydrolysis by approximately 3x for corn and wheat starch granules, compared to the sum of the individual activities. The exo-endo synergism did not change by varying the ratio of the two enzymes when the total concentration was kept constant. A yield of 4% conversion was obtained after 25 min 37 degrees C incubation (1 unit total enzyme, 15 mg raw starch granules, pH 5.3). The temperature stability of the enzyme mixtures was < or =50 degrees C, but the initial rate of hydrolysis continued to increase with higher temperatures. Ca(++) enhanced the stability of the free enzymes at 50 degrees C incubation. Inhibition was observed with the addition of 10 mM Fe(++) or Cu(++), while Mg(++ )and EDTA had lesser effect.

Sedimentation field flow fractionation monitoring of bimodal wheat starch amylolysis

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this study, we investigated the capacity of sedimentation field flow fractionation (SdFFF) to monitor the amylolysis of a bimodal starch population: native wheat starch. Results demonstrated a correlation between fractogram changes and enzymatic hydrolysis. Furthermore, SdFFF was used to sort sub-populations which enhanced the study of granule size distribution changes occurring during amylolysis. These results show the interest in coupling SdFFF with particle size measurement methods to study complex starch size/density modifications associated to hydrolysis. These results suggested different applications such as the association of SdFFF with structural investigations to better understand the specific mechanisms of amylolysis or starch granule structure.

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH4)2SO4 fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4-6.4 stability range. The temperature optimum was 50 degrees C with stability <or=50 degrees C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn++ activated the enzyme by 45%, while Li+, Zn++, Mg++, Cu+, Ca++, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10x the amount of GLA. Apparent Km and Vmax for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40 degrees C, pH 5.3), with an apparent kcat of 2.9 x 10(5) min(-1).

Structure of lintnerized starch is related to X-ray diffraction pattern and susceptibility to acid and enzyme hydrolysis of starch granules

Acid-resistant residues (lintnerized starches, Ls) were prepared from starches showing A-, B- and C- X-ray diffraction patterns. Ls retained the same X-ray crystalline type as their native counterparts with an improvement in diffraction intensity. Fluorophore-assisted capillary electrophoresis (FACE) study indicated that structural characteristics of Ls were associated with X-ray diffraction patterns. Double helices originated from linear chains with an approximate average degree of polymerisation (DP) 14, 16, and 15 would span the entire length of crystalline lamellae of A-, B-, and C-type starches, respectively. The proportion of singly branched materials (SB) with DP 25 protected in Ls was higher for A-type Ls (10–17%) than for B-type Ls (4–6%) and C-type Ls (8%). The structures of SB were similar in which branched chain (DP 13–15) was longer than main chain (DP 10–12). The structural characteristics of Ls are discussed in relation to acid and enzymatic degradations of starch granules.

Sedimentation field flow fractionation monitoring of rice starch amylolysis

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.

Use of tannic acid to protect barley meal against ruminal degradation

AbstractThe inefficiency of starch utilisation by ruminants fed readily fermentable cereal grains, such as barley, decreases the metabolic potential of such feeds and can cause serious dysfunctions related to acidosis or pre‐acidosis status in animals with higher energy requirements. The rate and extent of ruminal disappearance of grain dry matter (DM) are determined largely by the morphological features of the seed endosperm, but the presence of polyphenols may also constitute a limiting factor in ruminal hydrolysis. To assess the impact of tannins on the rate and extent of ruminal fermentation of barley grain, commercial grade tannic acid (TA) was included at 0, 1.0, 2.5 or 5.0% (wt/wt, DM basis) with ground barley grain in incubation in situ, and disappearance of DM and crude protein were monitored over 48 h. A dose‐dependent effect of TA on in situ degradation of barley meal was observed; significance was attained (p &lt; 0.05) at the 5% treatment level. Scanning electron microscopy revealed limited microbial hydrolysis of endosperm cell walls in TA‐treated samples, although TA did not prevent microbial attachment to or hydrolysis of starch granules. Tannins may be effective for slowing ruminal disappearance of barley to improve starch utilisation by ruminants. Copyright © 2005 Society of Chemical Industry

Engineering of Barley α-Amylase

Protein engineering of barley α-amylase addressed the roles of Ca2+ in activity and inhibition by barley α-amylase/subtilisin inhibitor (BASI), multiple attach in polysaccharide hydrolysis, secondary starch binding sites, and BASI hot spots in AMY2 recognition. AMY1/AMY2 isozyme chimeras faciliatated assignment of function to specific regions of the structure. An AMY1 fusion with starch binding domain and AMY1 mutants in the substrate binding cleft gave degree of multiple attack of 0.9–3.3, compared to 1.9 for wild-type. About 40% of the secondary attacks, succeeding the initial endo-attack, produced DP5-10 maltooligosaccharides in similar proportion for all enzyme variants, whereas shorter products, comprising about 25%, varied depending on the mutation. Secondary binding sites were important in both multiple attack and starch granule hydrolysis. Surface plasmon resonance and inhibition analyses indicated the importance of fully hydrated Ca2+ at the AMY2/BASIinterface to strengthen the complex. Engineering of intermolecular contacts in BASI modulated the affinity for AMY2 and the target enzyme specificity.

The single screw extruder as a bioreactor for sago starch hydrolysis

Simultaneous pregelatinization and preliquefaction of sago starch with a thermostable α-amylase was carried out in a Brabender single screw extruder. Response surface methodology was employed to study the effects of processing conditions; feed moisture content (21–38%), enzyme concentrations (1.48–6.52%) and mass temperatures in the compression and die zones (70.5–97.5 °C), on the properties of the extrudates. Twenty runs were performed based on a multifactorial composite rotatable design. Changes in the dextrose equivalent (DE), water solubility index (WSI), water absorption index (WAI), degree of gelatinization (DG) and high performance size exclusion chromatography (HPSEC) profiles were assessed. From the HPSEC profiles, the degree of degradation (DGR) and oligosaccharide content were calculated. Feed moisture content and enzyme concentration were found to be the most significant variables affecting most of the measured physicochemical properties. Hydrolysis of starch granules was the fundamental reaction occurring during the extrusion process as indicated by the higher DE, WSI, oligosaccharide content and correspondingly lower WAI, DG and DGR. Product spectra from HPSEC showed that the amylopectin (Ap) was preferentially degraded and amylose (Am) was apparently protected. The predominant oligosaccharide species were G3, G5 and G6.