Glucoamylase Research Articles

Aromatic residues in N-terminal domain of archaeal trehalase affect the folding and activity of catalytic domain

Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a β-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble β-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity.Key points• Aromatic residues in the BD are involved in BD and CD folding.• Aromatic residues in the BD near the CD active site modulate enzyme activity.• BD interacts with CD and closely modulates enzyme activity.

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.

A Self‐Sustaining Hydrogels with Autonomous Supply of Nutrients and Bioactive Domains for 3D Cell Culture

AbstractPhoto‐crosslinkable platelet lysate (PL)‐based hydrogels have been proven to support human‐derived cell cultures owing to their high content of bioactive molecules, such as cytokines and growth factors. As a unique self‐maintained and biocompatible 3D scaffold, the recently reported self‐feeding hydrogels with enzyme‐empowered degradation capacity have shown high biological performance in vitro and in vivo. To take advantage of all features of both PL and self‐feeding hydrogels, here UV responsive laminaran‐methacrylate (LamMA) and PL‐methacrylate (PLMA) derivatives plus glucoamylase (GA), which significantly improve the overall features of a 3D system, is coupled. This self‐sustaining hybrid hydrogel emerges as a unique scaffold due to the sustained delivery of glucose produced via enzymatic degradation of laminaran while granting the release of growth factors through the presence of PL. This biomaterial is applied to fabricate high‐throughput freestanding microgels with controlled geometric shapes. Furthermore, this multicomponent hybrid hydrogel is successfully implemented as the first reported glucose supplier bioink to manufacture intricate and precisely defined cell‐laden structures using a support matrix. Finally, such hydrogels are utilized as a proof of concept to serve as 3D in vitro cancer models, with the aim of recapitulating the tumor microenvironment.

Microbial glucoamylases: structural and functional properties and biotechnological uses.

Glucoamylases (GAs) are one of the principal groups of enzymes involved in starch hydrolysis and belong to the glycosylhydrolase family. They are classified as exo-amylases due to their ability to hydrolyze α-1,4 glycosidic bonds from the non-reducing end of starch, maltooligosaccharides, and related substrates, releasing β-D-glucose. Structurally, GAs possess a characteristic catalytic domain (CD) with an (α/α)6 foldand exhibit five conserved regions within this domain. The CD may or may not be linked to a non-catalytic domain with variable functions depending on its origin. GAs are versatile enzymes with diverse applications in food, biofuel, bioplastic and other chemical industries. Although fungal GAs are commonly employed for these purposes, they have limitations such as their low thermostability and an acidic pH requirement. Alternatively, GAs derived from prokaryotic organisms are a good option to save costs as they exhibit greater thermostability compared to fungal GAs. Moreover, a group of cold-adapted GAs from psychrophilic organisms demonstrates intriguing properties that make them suitable for application in various industries. This review provides a comprehensive overview of the structural and sequential properties as well as biotechnological applications of GAs in different industrial processes.

Determination of Optimum Reaction Velocity for Glucose Production from Cassava Starch Using Gluco Amylase Sourced from Rice

In an attempt to produce glucose from locally sourced materials to boost glucose syrup production and other applications, the optimum reaction velocity which enzyme becomes saturated with the substrate was investigated. In this study, cassava starch (substrate) and glucoamylase (enzyme) from rice were sourced and prepared. The time course of the enzyme hydrolysis was studied at pH 5 and temperature of 400C as optimum conditions. The hydrolysis was carried out at various substrate concentrations. The time course of the reaction for glucose production was monitored at 10 minutes interval for a period of one hour. The glucose concentration produced was plotted against reaction time to evaluate the optimum glucose amount and its corresponding substrate concentration. The reaction velocity was then computed. Also, the experimental data were fitted into Michaelis-Menten model using Statistical Package for Social Scientists to compare the yield of glucose. Results showed optimum yield of glucose concentration was 0.253 g DE L-1 achieved with 2.5 and 3.0% w/v substrate concentration at 50 and 40 minutes respectively. Further analysis gave the optimum reaction velocity range of 0.00506 to 0.00625 of g DE per litre-min. This range when compared with the model equation result (0.0052 to 0.0068 g DE litre-min) is reasonably close; hence the results from this study are valid. Technical evaluation to standardize the reaction velocity with respect to substrate concentration which may be referred to as specific reaction velocity gave 0.00205 ±0.00005 g D per litre-min per substrate concentration %w/v.

Determination of The Optimum pH and Enzyme Ratio for Starch Hydrolysis Test and Characterization of Steamed, Baked, and Fried Wheat Doughs

Digestibility is the most important nutritional functionality of starchy foods. Unfortunately, in Indonesia, this functionality has not been extensively studied due to the relatively challenging methods involving enzymes. This research aims to determine the optimal pH and enzyme ratio of α-amylase (AA) and glucoamylase (GA) for starch hydrolysis tests and apply them to characterize steamed, baked, and fried wheat doughs. For optimization, hydrolysis was carried out at 37 °C with an enzyme-to-substrate ratio (E/S) of 100 mL/g for 120 min. Samples of wheat dough obtained from three major dough producers in Banyumanik, Semarang, Indonesia was then tested for starch hydrolysis, texture, chemical analyses, sensory, and hedonic test. The collected data were analysed using principal component analysis (PCA). Under the determined conditions, the optimal pH and AA/GA ratio were found to be 6.6 and 0.5. Regarding quality of processed doughs, the steamed dough exhibited a cohesive texture, a soft sensory profile with a pleasant aroma, sweet taste, and was most preferred by the panellists. The baked dough was hard in texture, high in ash content, and brown in colour. The fried dough poses high starch hydrolysis, chewy texture, adhesion, solid particles, protein content, and relatively high lipid. The sensory evaluation indicated that the fried dough poses an oily, rancid, foreign aroma, and bitter and salty taste. It can be concluded that at a temperature of 37 °C, an enzyme-to-substrate ratio of 100 mL/g, and a reaction time of 120 min, the optimal starch hydrolysis of processed wheat dough can be achieved at a pH of 6.6 and an AA/GA ratio of 0.5. Furthermore, steaming resulted in a unique property of dough compared to the baked and fried, while the latter two yielded products with similar properties.

Interfacial enzyme kinetics reveals degradation mechanisms behind resistant starch

The digestive resistance of so-called resistant starch (RS) provides an important nutritional principle. However, as yet the mechanisms underlying digestive resistance of the granular starches are not well described. We address this problem by employing a novel enzyme kinetics approach, which in contrast to conventional enzyme kinetics takes the interfacial nature of the enzyme reaction into account. Structurally different starch granules with different amylose contents were modified by the hydrolytic enzyme glucoamylase (GA). Kinetic-, adsorbtion-, imaging-, particle size-, and spectroscopic data combinedly revealed important origins of hydrolytic resistance of high-amylose starch. Our data demonstrate that amylose restricted the enzymatic catalysis efficiency on starch granules chiefly by providing less hydrolytic effective attack sites for the enzyme at granular surface. Specifically, the total binding sites (adsΓmax) (20.9 nmol/g) on starch with 0% amylose content (AC) were almost all effective attack sites (kinΓmax) (20.2 nmol/g). At 26% AC, the binding sites on the granules decreased to 13.9 nmol/g, and attack sites decreased to 7.4%. At 72% AC, the binding sites were only slightly reduced to 11.2 nmol/g, however, the attack sites were remarkably decreased to 2.6 nmol/g. Beyond the initial catalytic events, i.e., for further degraded granules, the binding and catalysis efficiency differed notably for the three starch types. At 0% AC, both binding and attack increased demonstrating increased hydrolytic susceptibility of the granules. At 26% AC only binding increased, while attack was unchanged. Interestingly, at 72% AC, binding increased, while attack deceased notably with hydrolysis time, demonstrating decreased efficiency of interfacial catalysis during the hydrolysis process.

Efficient cascade conversion of starch to gluconic acid by a chemoenzymatic system with co-immobilized Au nanoparticles and enzymes

A chemoenzymatic cascade system (GA&amp;GOx@Au–SiO2) was fabricated by immobilizing Au nanoparticles onto silica (SiO2) followed by the co-immobilization of glucoamylase (GA) and glucose oxidase (GOx) for the production of gluconic acid from starch.

Starch and chitosan-based antibacterial dressing for infected wound treatment via self-activated NO release strategy

In this work, a positively charged chitosan-grafted-polyarginine (CS-N-PArg) as the macro-molecular NO donor, and a negatively charged acetalated starch (AcSt-O-PAsp) as a glucose donor, have been synthesized. To achieve the multi-enzymatic cascade system for local generation of self-supply glucose to increase the H2O2 concentration for the subsequent oxidization of L-Arg into NO, the designed positively charged CS-N-PArg, negatively charged AcSt-O-PAsp, glucoamylase (GA) and glucose oxidase (GOx) are absorbed and assembled in the pore of the gelatin sponge via electrostatic interaction to establish a smart antibacterial dressings (CS/St + GOx/GA). Once stimulated by Escherichia coli (E. coli)-infected wounds (a slightly acidic environment), the cascade reaction system can sequentially induce to generate glucose, H2O2 and NO, which exhibits a meaningful alternative idea for a high-performance antibacterial therapy.

Sensitive electrochemical sequential enzyme biosensor for glucose and starch based on glucoamylase- and glucose oxidase-controllably co-displayed yeast recombinant

The sequential enzyme biosensors hold significant importance in measuring species which are usually hard to process with single-enzyme-based biosensors. However, sequential enzyme electrodes experience critical issues such as low catalytic efficiency, insensitivity and poor reproducibility. In this work, yeast surface co-displaying sequential enzymes of glucoamylase (GA) and glucose oxidase (GOx) with controllable ratios through the specific cohesion-dockerin protein interaction was explored, by which starch hydrolyzing by GA into glucose is the rate-limiting step. The modified electrodes were prepared by immobilizing yeast-GA&GOx whole-cell and reduced graphene oxide (RGO) on glassy carbon electrode (GCE), for which the direct electron transfer between the electrode and recombinant GOx was arrived. Interestingly, the current responses of sensors to starch and glucose are dependent on the displayed enzyme composition, of which the yeast-GA&GOx (2:1) exhibited the highest current. Thereafter, sequential enzyme sensor of yeast-GA&GOx (2:1)/RGO/GCE was developed. Based on reduction detection at negative potential without interference, the sensor is stable and capable of assaying glucose (linear range: 2.0–100 mg/L) or starch (linear range, 50–3500 mg/L), separately. Coupled with yeast-GOx/RGO/GCE glucose sensor, both glucose and starch in real samples can be detected satisfactorily. This work provides new ideas for the development of other sequential enzyme electrodes for potential applications.

A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD+, which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 μW cm−2, and good operational stability.

Rational reformation of Corynebacterium glutamicum for producing L-lysine by one-step fermentation from raw corn starch.

This article focuses on engineering Corynebacterium glutamicum to produce L-lysine efficiently from starch using combined method of "classical breeding" and "genome breeding." Firstly, a thermo-tolerable L-lysine-producing C. glutamicum strain KT45-6 was obtained after multi-round of acclimatization at high temperature. Then, amylolytic enzymes were introduced into strain KT45-6, and the resultant strains could use starch for cell growth and L-lysine production except the strain with expression of isoamylase. In addition, co-expression of amylolytic enzymes showed a good performance in starch degradation, cell growth and L-lysine production, especially co-expression of α-amylase (AA) and glucoamylase (GA). Moreover, L-lysine yield was increased by introducing AA-GA fusion protein (i.e., strain KT45-6S-5), and finally reached to 23.9 ± 2.3g/L in CgXIIIPM-medium. It is the first report of an engineered L-lysine-producing strain with maximum starch utilization that may be used as workhorse for producing amino acid using starch as the main feedstock. KEY POINTS: • Thermo-tolerable C. glutamicum was obtained by temperature-induced adaptive evolution. • The fusion order between AA and GA affects the utilization efficiency of starch. • C. glutamicum with starch utilization was constructed by optimizing amylases expression.

Potential of Recombinant Raw Starch‐Degrading Enzyme from Escherichia coli for Sugar Syrup and Bioethanol Productions Using Broken Rice Powder as Substrate

AbstractThe recombinant raw starch‐degrading enzyme (LsA175) which harbors the lsa175 gene from Laceyella sacchari LP175 is optimized for expression in Escherichia coli BL21 (DE3). The recombinant strain provides an alternative avenue for enzyme production in a stirrer fermenter with tolerance to shear force and avoids losing stability by protease existing in the native strain. The maximum enzyme production at 4472±112 U g−1 cell dry weight is obtained using the optimum conditions at 22 °C, 1.0 mM IPTG, and 24 h of induction temperature, Isopropyl β‐ d‐1‐thiogalactopyranoside (IPTG) concentration, and induction time, respectively. The enzyme production of 4545 ± 89.8 U g−1 cell dry weight is obtained by batch fermentation in a 2.0 L stirrer fermenter and 5329 ± 86.1 U g−1 cell dry weight by continued glucose feeding with high bacterial cell concentration. The uncooked broken rice powder (200 g L−1) is hydrolyzed by crude recombinant LsA175 (300 U mL−1) at 50 °C for 12 h without the addition of exogenous glucoamylase (GA), yielding 86 ± 1.79 g L−1 reducing sugar. The bioethanol production resulting from modified simultaneous saccharification and fermentation with Kluyveromyces marxianus DMKU‐KS07 at 42 °C gave 71.4±4.35 g L−1 ethanol with 88.24% of theoretical ethanol yield. LsA175 presents engaging performances regarding sugar syrup and bioethanol production, which can diminish the cost of production and energy consumption.

Amylase Production by Aspergillus niger immobilized in Microporous Calcium Alginate Gel Beads

Microporous calcium alginate gel beads were investigated as potential solutions to mass transfer limitations in immobilized cultures. The beads were prepared by gelling mixtures of sodium alginate and fillers (starch or granulated sugars) in calcium chloride solution. The resulting beads were cured in the same solution, during which the fillers leached out of the beads thereby creating micro-pores in the beads (microporous beads). The effectiveness of the microporous beads in improving amylase production by Aspergillus niger LC 269109 was investigated. Spores of A. niger were immobilized in the microporous beads and used for batch alpha amylase and gluco amylase production. Amylase production by the A. niger immobilized in the microporous beads were significantly higher (p&lt;0.01) than the values obtained with the conventional calcium alginate gel beads. Under all the conditions investigated, gluco-amylase activities were significantly (p&lt;0.01) higher than the alpha-amylase activities. Under the optimum conditions of inoculum concentration (1.0 × 105 spores/ml), pH (6), temperature (35°C), bead diameter (3 mm) and calcium chloride concentration (1.5%), the gluco-amylase and alpha amylase activities were 11.98 U/ml and 6.7 U/ml respectively, which were significantly higher (p&lt;0.05) than the 7.8 U/ml and 3.2 U/ml obtained with the conventional gel beads.

Characterization of SdGA, a cold-adapted glucoamylase from Saccharophagus degradans

We investigated the structural and functional properties of SdGA, a glucoamylase (GA) from Saccharophagus degradans, a marine bacterium which degrades different complex polysaccharides at high rate. SdGA is composed mainly by a N-terminal GH15_N domain linked to a C-terminal catalytic domain (CD) found in the GH15 family of glycosylhydrolases with an overall structure similar to other bacterial GAs. The protein was expressed in Escherichia coli cells, purified and its biochemical properties were investigated. Although SdGA has a maximum activity at 39 °C and pH 6.0, it also shows high activity in a wide range, from low to mild temperatures, like cold-adapted enzymes. Furthermore, SdGA has a higher content of flexible residues and a larger CD due to various amino acid insertions compared to other thermostable GAs. We propose that this novel SdGA, is a cold-adapted enzyme that might be suitable for use in different industrial processes that require enzymes which act at low or medium temperatures.

Effect of exogenous glucoamylase inclusion on in vitro fermentation and growth performance of feedlot steers fed a dry-rolled corn-based diet.

The objective was to evaluate the effect of adding an exogenous glucoamylase (GA) enzyme from the fungus Trichoderma reesei on in vitro fermentation, growth performance, and carcass characteristics of feedlot steers fed a dry-rolled corn (DRC)-based diet. Experiment 1 evaluated three levels of added enzyme (0, 0.24, and 0.72 GA enzyme units) and two corn particle sizes (CPS; 2 and 4 mm) in a factorial arrangement using a 7 h in vitro batch culture fermentation. Addition of GA increased (P < 0.01) in vitro dry matter disappearance by 13% and decreased final pH (P < 0.01). Molar proportion of propionate increased with GA inclusion (P < 0.01). A smaller CPS increased (P < 0.01) in vitro dry matter disappearance and total volatile fatty acid and decreased final pH (P < 0.01). A smaller CPS also decreased (P < 0.01) the molar proportion of acetate and increased (P < 0.01) the molar proportion of butyrate. In experiment 2, Angus × Simmental steers (n = 105; initial body weight [BW] = 329 ± 38 kg) were used to evaluate the inclusion of an exogenous GA on growth performance and carcass characteristics. Steers were fed a basal diet consisting of 60% DRC, 17.5% modified distillers grains with solubles, 12.5% corn silage, and 10% dry supplement on a dry matter basis for 136 d. Steers were blocked by weight and allotted to pens. Pens were randomly assigned to one of three treatments (5 pens/treatment): diet with no GA (CON), low inclusion of GA (122 enzyme units/kg dry matter [DM]; LGA), or high inclusion of GA (183 enzyme units/kg DM; HGA). Inclusion of GA did not affect (P ≥ 0.23) final BW, dry matter intake (DMI), or average daily gain (ADG) for the 136-d feeding period. Feed conversion was affected (P = 0.02) by treatment with steers fed HGA having ~8% greater G:F compared with LGA and CON. Treatment did not affect (P = 0.32) fecal starch. Inclusion of GA did not affect (P ≥ 0.19) carcass traits including hot carcass weight, 12th rib fat thickness, yield grade, longissimus muscle area, or marbling score. Overall, results suggest inclusion of exogenous GA enzyme increased in vitro dry matter disappearance in batch culture and improved feed conversion in steers fed 183 enzyme units/kg DM during the finishing phase.

Evaluation of the roles of hydrophobic residues in the N-terminal region of archaeal trehalase in its folding.

Thermoplasma trehalase Tvn1315 is predicted to be composed of a β-sandwich domain (BD) and a catalytic domain (CD) based on the structure of the bacterial GH15 family glucoamylase (GA). Tvn1315 as well as Tvn1315 (Δ5), in which the 5 N-terminal amino acids are deleted, could be expressed in Escherichia coli as active enzymes, but deletion of 10 residues (Δ10) led to inclusion body formation. To further investigate the role of the N-terminal region of BD, we constructed five mutants of Δ5, in which each of the 5th to 10th residues of the N-terminus of Tvn1315 was mutated to Ala. Every mutant protein could be recovered in soluble form, but only a small fraction of the Y9A mutant was recovered in the soluble fraction. The Y9A mutant recovered in soluble form had similar specific activity to the other proteins. Subsequent mutation analysis at the 9th position of Tvn1315 in Δ5 revealed that aromatic as well as bulky hydrophobic residues could function properly, but residues with hydroxy groups impaired the solubility. Similar results were obtained with mutants based on untruncated Tvn1315. When the predicted BD, Δ5BD, Δ10BD, and BD mutants were expressed, the Δ10BD protein formed inclusion bodies, and the BD mutants behaved similarly to the Δ5 and full-length enzyme mutants. These results suggest that the hydrophobic region is involved in the solubilization of BD during the folding process. Taken together, these results indicate that the solubility of CD depends on BD folding. KEY POINTS: • N-terminal hydrophobic region of the BD is involved in the protein folding. • The N-terminal hydrophobic region of the BD is also involved in the BD folding. • BD is able to weakly interact with the insoluble β-glucan.

Effect of enzymes addition on the fermentation of Chinese rice wine using defined fungal starter

A higher fermentation capability and quality of a rice wine was achieved, using mixed fungal (Rhizopus chinonsis R01, Asperigillus niger A20, Mucor pusillus M05 and Saccharomyces cerevisiae S10) starter fermentation of japonica rice supplemented with α-amylase (AM), glucoamylase (GAM) and acid protease (AP). Results showed that alcohol content, free amino acids (FAA) and volatile flavor substances concentrations were increased by 49.22%, 85.54%, and 25.71% compared with the control group respectively. To understand the role of enzymes during rice wine brewing, the effects of hydrolases on chemical and physical properties of cooking rice, on the yeast growth, enzyme productivity of molds were determined. Rice starch was hydrolyzed effectively by AM and GAM, the viscosity reduced significantly, the enzyme contact area expanded, which accelerated the further hydrolysis of japonica rice. Increase of hydrolysate promoted the fungi growth, while improved the ability of molds to produce enzymes. AP promoted the protease activity of M. pusillus M05 and A. niger A20, improved protein utilization, increased the content of FAA and formed higher level of flavor substances in rice wine. The results indicate that hydrolase addition can change the physic-chemical state of the mash to enhance microbial biotransformation and modify flavor metabolism.

Effects of α-amylase and glucoamylase on the characterization and function of maize porous starches

To overcome the limited surface area of starch and improve the adsorption capacity of starch, porous starch has been widely produced in various fields. In this study, the different enzyme activities of α-amylase (AM), glucoamylase (GAM), and mixed enzyme (ME) were used to prepare different types of porous maize starch, and the microstructure, particle morphology, adsorption, and gelatinization characteristics of the porous starch were studied. Scanning electron microscopy showed porous structures with diverse pore size distribution and pore area depending on the enzyme type and its activities; AM produced the largest holes, GAM produced the deepest holes, and ME produced large and deep holes. Adsorptive capacity was significantly affected by enzymatic modification being greater influenced by GAM. The porous starches had higher relative crystallinity as deduced from X-ray diffraction and Fourier transform infrared spectroscopy. The MA-treated starch had higher solubility whereas swelling capacity, peak viscosity, final viscosity, breakdown, and setback of both treatments were decreased compared to the native starch. The results indicate porous maize starch can provide different functions according to the action pattern of enzymes. The type of enzyme and its enzyme activity can be used to produce the ideal cavity and pore size starch granules, which expands its application scope.

Comparison of functional properties of porous starches produced with different enzyme combinations

To obtain porous starch granules with higher absorption capacities, three types of enzyme combinations were adopted to modify wheat and maize starches: (1) sequential α-amylase (AA) → glucoamylase (GA); (2) sequential branching enzyme (BE) → GA; and (3) sequential AA→BE→GA. The results indicated that AA→BE→GA treatment had a most optimal influence on porous starches. Compared to AA→GA and BE→GA, the mesopores in wheat starch granules treated with AA→BE→GA decreased by 52.82 and 48.70%, respectively. Conversely, the macropores increased by 216.68 and 138.18%, respectively. While for maize starch, the percentages of mesopores and macropores hardly changed after three enzyme combinations. Comparing the three enzyme treatments showed that pore volume (0.005 and 0.007 cm3/g) and pore size (36.35 and 26.54 nm) were largest in the AA→BE→GA treated wheat and maize starches, respectively. Compared to the AA→GA and BE→GA, the adsorption capacities for oil, dye and heavy metal ions, wheat starch treated with AA→BE→GA increased by 46.61 and 242.33%, and 44.52 and 134.41%, and 28.83 and 271.72%, respectively. Correspondingly, that of maize starch increased by 29.71 and 133.29%, and 42.92 and 79.93%, and 28.16 and 161.43%, respectively. These results may provide a new and valuable enzyme combination for optimising porous starch granules with higher absorption capacities.