Starch Liquefaction Research Articles

Computer‐Aided Protein Surface Modification Strategy to Improve the Thermostability of α‐Amylase

AbstractStability under high‐temperature environments is crucial for amylase to function in starch‐based industries. This study develops a method of modifying protein surfaces by combining computer‐aided tools including FoldX, PoPMuSiC, Discovery Studio, and I‐Mutant 2.0 with conserved sequence analysis. A truncated α‐amylase ∆AmyPTG from Parageobacillus thermoglucosidasius DSMZ 2542 is rationally designed through this method to improve its thermostability. Seven single‐site variants are constructed and five of them displayed enhanced thermostability. Next, three double‐site variants are constructed with one particularly successful double‐site variant N31RT213R, exhibiting a 4.3‐fold longer half‐life at 80 °C. Notably, the specific activity of N31RT213R reaches 10 567.16 U mg−1, higher than ∆AmyPTG (6645.43 U mg−1). When applied to the corn starch liquefaction reaction, the mutant N31RT213R gets a higher yield of product concentration of about 255.70 µg mL−1, compared to 190.72 µg mL−1 for ∆AmyPTG. Intramolecular forces analysis and surface electrostatic charges analysis are conducted to determine possible causes for the improvement. Also, molecular dynamics simulation is used to analyze the flexibility shifts of the entire protein. This innovative rational engineering approach has proven to be a successful strategy for the selection of hot spots for protein thermostability evolution and has the potential to be applied to other enzymes.

Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement.

α-Amylase (EC 3.2.1.1) from Geobacillus stearothermophilus (generally recognized as safe) exhibited thermal inactivation, hampering its further application in starch-based industries. To address this, we performed structural analyses based on molecular dynamics targeting the flexible regions of α-amylase. Subsequently, we rationally designed a thermostable mutant, AmyS1, by introducing disulfide bonds to stabilize the flexible regions. AmyS1 showed excellent thermostability without any stability-activity trade-off, giving a 40-fold longer T1/2 (1359 min) at 90 °C. Thermostability mechanism analysis revealed that the introduction of disulfide bonds in AmyS1 refined weak spots and reconfigured the protein's force network. Moreover, AmyS1 exhibited improved pH compatibility and enhanced corn starch liquefaction at 100 °C with a 5.1-fold increased product concentration. Baking tests confirmed that AmyS1 enhanced bread quality and extended the shelf life. Therefore, mutant AmyS1 is a robust candidate for the starch-based industry.

Catalytic liquefaction of starch by sulfamic acid and its curing kinetics for preparing polyurethane

Catalytic liquefaction of starch by sulfamic acid and its curing kinetics for preparing polyurethane

Exploiting loop-grafting strategy resorting on computer-aided design to improve the thermostability of alpha-amylase from Geobacillus stearothermophilus

Starch-based industries require a robust alpha-amylase capable of withstanding elevated temperatures and long incubation periods. In this study, a loop-grafting strategy based on a computer-aided design was proposed to engineer a thermostable alpha-amylase from Geobacillus stearothermophilus. Molecular dynamics (MD) simulations and trajectory analyses were employed to target the weak spots responsible for thermal inactivation, and three loop regions were identified as weak spots requiring improvement. Subsequently, substitute candidates from the thermophilic orthologous library were selected to refine these weak spots and the most promising mutant was screened using thermal unfolding and MD simulations. Expectedly, an alpha-amylase variant was constructed efficiently and specifically, which can withstand high-temperatures up to 100 °C without any stability-activity trade-off, giving an 8.0-fold longer half-life at 100 °C. Notably, the variant exhibited excellent thermotolerance during corn starch liquefaction at 100 °C, giving a 3.3-fold increased product concentration. Furthermore, the origin of the enhanced thermostability based on the dynamic trajectory of the variant was revealed. Successful attempts to tailor the thermal resistance and catalytic activity of alpha-amylase can further fuel the food industry, particularly high-temperature starch-based industries.

Hydrothermal liquefaction of starch using homogeneous and heterogeneous co-catalysts

We examined the hydrothermal liquefaction (HTL) of potato starch with K2HPO4, which dissolves in hot, compressed water, along with metal-containing co-catalysts, which present a separate solid phase in the reaction system. Co-catalytic HTL with K2HPO4 and either Mo2C or Co-Mo/γ-Al2O3 produced more biocrude than did HTL with no catalyst or either catalyst alone. The synergistic effect of co-catalysis was greatest when the masses of K2HPO4 and Co-Mo/γ-Al2O3 were equal. At this condition, HTL with the two catalysts produced a biocrude that contained more than 90% of the chemical energy originally in the biomass feedstock. This biocrude also had a larger portion (61 wt%) of desirable lighter components (volatilized below 343 ℃) than did the biocrudes from HTL with either catalyst individually at the same conditions (38 and 41 wt%). With the addition of catalysts, there was less aliphatic content in the biocrude, and more ethers and aromatics appeared. The use of K2HPO4 and Co-Mo/γ-Al2O3 together for HTL led to greater fractions of alcohols, aldehydes/ketones, hydrocarbons, and phenolic compounds in the biocrude. In addition, less of the organic carbon in the feedstock migrated to the aqueous products. We identify catalysis of different HTL pathways to account for the experimental observations.

Introducing hydroxyl-containing POSS to liquefaction of biomass: An alternative route to bio-based polyurethane nanocomposites for coated fertilizers

A new route, introducing POSS-OH to liquefaction of biomass, is developed for bio-based polyurethane nanocomposites and applied in the controlled release of chemicals. The effect of POSS-OH on the chemical structures and properties of biopolyols and their coatings was characterized by GC–MS, FTIR and others. The results indicated that after adding POSS-OH to liquefaction of starch, the hydroxyl content of the biopolyol synthesized was higher than that of the biopolyol from sole starch, and the resultant nanocomposite owned good thermal stability, water resistance and, importantly, non-toxicity to plants. Meanwhile, after coating the urea granules, the controlled release ability of the nanocomposite coating with 3% POSS-OH was the best, and in comparison with the coating without POSS-OH the nitrogen release duration was prolonged by a month. Overall, POSS-OH was used not only as both co-liquefier of biomass instead of the commonly used glycerol to affect the liquefaction process, but also as filler for bio-based polyurethane nanocomposite to improve the interaction. Thus, the substitution of petroleum-based polyols can be expected in synthesizing the green and bio-based coatings for controlled release of chemicals.

High Production of Maltooligosaccharides in the Starch Liquefaction Process: A Study on the Hyperthermophilic Mechanism of α-Amylase.

The efficient production of high-value-added bioproducts from starchy substances requires α-amylases with hyperthermophilic properties for industrial starch liquefaction. In this study, two hyperthermophilic α-amylases with significant differences in thermostability, PfAmy and TeAmy, were comparatively studied through structural analysis, domain swapping, and site-directed mutagenesis, finding that three residues, His152, Cys166, and His168, located in domain B were the main contributors to hyperthermostability. The effects of these three residues were strongly synergistic, causing the optimum temperature for the mutant K152H/A166C/E168H of TeAmy to shift to 95-100 °C and stabilize at 90 °C without Ca2+. Compared to PfAmy and TeAmy, the mutant K152H/A166C/E168H, respectively, exhibited 1.7- and 2.5-times higher starch hydrolysis activity at 105 °C and pH 5.5 (10411 ± 70 U/mg) and released 1.1- and 1.7-times more maltooligosaccharides from 1% starch. This work has interpreted the hyperthermophilic mechanism of α-amylase and thereby providing a potential candidate for the efficient industrial conversion of starch to bioproducts.

Fabrication of 1,4-alpha-D-glucan glucanohydrolase holding Gel-Scaffolds using Agar-Agar, a natural polysaccharide and Polyacrylamide, a synthetic organic polymer for continuous liquefaction of starch

1,4-alpha-D-glucan glucanohydrolase is among the most widely used commercial hydrolytic enzymes acting randomly on the glycosidic linkages of starch resulting in its saccharification and liquefaction. Its applicability in different industries can be improved by enhancing its stability and reusability. Therefore, in the present study attempts have been made to enhance the industrial applicability of 1,4-alpha-D-glucan glucanohydrolase from Bacillus subtilis KIBGE-HAR by adapting immobilization technology. The study developed mechanically stable, enzyme containing gel-frameworks using two support matrices including agar-agar, a natural polysaccharide and polyacrylamide gel, a synthetic organic polymer. These catalytic gel-scaffolds were compared with each other in terms of kinetics and stability of entrapped 1,4-α-D-glucan glucanohydrolase. In case of polyacrylamide gel, Km value for immobilized enzyme increased to 7.95 mg/mL, while immobilization in agar-agar resulted in decreased Km value i.e 0.277 mg/mL as compared to free enzyme. It was found that immobilized enzyme showed maximum activity at 70 °C in both the supports as compared to free enzyme having maximum activity at 60 °C. Immobilized 1,4-α-D-glucan glucanohydrolase exhibited no change in optimal pH 7.0 before and after entrapment in polyacrylamide gel and agar-agar. The enzyme containing gel-scaffold was found suitable for repeated batches of starch liquefaction in industrial processes. Agar-agar entrapped 1,4-α-D-glucanglucanohydrolase was capable to degrade starch up to seven repeated operational cycles whereas polyacrylamide entrapped enzyme conserved its activity up to sixth operational cycle.

Antibiofilm Potential of Alpha-Amylase from a Marine Bacterium, Pantoea agglomerans.

Bacterial biofilms are a big menace to industries and the environment and also in the health sector, accumulation of which is a major challenge. Despite intensive efforts to curb this issue, a definitive solution is yet to be achieved. Enzyme-templated disruption of the extracellular matrix of biofilm and its control and elimination are emerging as an efficient and greener strategy. The study describes the antibiofilm potential of alpha-amylase from the marine microorganism Pantoea agglomerans PCI05, against food-borne pathogens. Amylase exhibited stability in a wide pH range and retained 50% of its activity at temperatures as high as 100°C. Thermal analysis of the enzyme produced showed thermal stability, up to 130°C. From these findings, it can be envisaged that the alpha-amylase produced from P. agglomerans can be used for starch liquefaction; it was also evaluated for antibiofilm activity. Amylase from this marine bacterium was found to efficiently disrupt the preformed biofilms of food-borne pathogens such as Bacillus cereus, Serratia marcescens, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella enterica enterica serotype Typhi based on the value of biofilm inhibitory concentrations.

Hydrothermal liquefaction of polysaccharide feedstocks with heterogeneous catalysts

Hydrothermal liquefaction (HTL) of starch, cellulose, pectin, and chitin with Pd/C, Co-Mo/γ-Al2O3, and zeolite was investigated at 320 °C for 30 min. Using Co-Mo/γ-Al2O3 at 5 wt% loading led to the highest biocrude yields from starch (25 wt%) and cellulose (23 wt%). The yields from cellulose are more than twice those from noncatalytic HTL (11 wt%). Co-Mo/γ-Al2O3 was also the only catalyst (25 wt% loading) to increase biocrude yields (by 1.6 – 2.6 wt%) from HTL of chitin and pectin. The biocrudes were characterized by elemental analysis, TGA, FT-IR and GC–MS. Catalytic HTL with Co-Mo/γ-Al2O3 had little effect on the elemental composition of the biocrudes. The presence of Co-Mo/γ-Al2O3 increased the low-boiling portion of biocrude from<30% to over 50% for HTL of starch. Finally, a component additivity model that accurately predicts biocrude yields from catalytic HTL of a mixture is presented.

Review of Strategies for the Industrial Production of α-amylase by Bacillus subtilis

Bacillus subtilis can secrete industrially important proteins such as proteases and α-amylases and used on industrial scale. α-Amylase enzyme has market demands due to its applications in food, bakery, detergent industries, starch liquefaction, pre-digestion of the animal feed to enhance its quality, sizing of the fibres in textiles. The safety issues associated with the use of this bacteria for industrial applications are studied and it has been observed that the products obtained from it are having GRAS status of US Food and Drug Administration. Bacillus subtilis is considered the most widely experimental organism to conduct the genetic modification studies due to its properties which make it a suitable host for biosynthesis of the products. The genomic structure of Bacillus subtilis can be modified with the help of high quality genomic sequences. The genetic strategies for such modifications include the use of mutagenic treatments, screening of better expression systems, use of better promoters and high secretion level peptides. Another aspect of these strategies to enhance the enzyme yield includes the application of different fermentation methods and use of different substrates. Present review article summarizes some of such strategies applied for obtaining higher yields of α-amylase enzyme using Bacillus subtilis.

Conversion of corn fiber into fuel ethanol

Corn fiber due to its chemical composition (up to 20% starch, 50 - 60% non-starch polysaccharides) and availability has potential to serve as a substrate for manufacture of various products, including fuel ethanol. This paper deals with assessment of fiber-to-ethanol conversion. The water/dry fiber ratio in suspensions was 10/1. Enzyme liquefaction and saccharification of residual starch in corn fiber was carried out in two steps with thermostable α-amylase (20 min, 120°C) and mixture of pullulanase and glucomalyse (24 hours, 60°C). Procedures resulted in release of 57.7±1.6 mg of glucose per gram of dry fiber basis. It responds to the dextrose equivalent expression to 96.7±2.2%. By fermentation of the starch hydrolysates by yeasts Saccharomyces cerevisiae CCY-11-3 (5% v/v inoculum, 28°C, 72 hours) 0.48 g of ethanol per gram of glucose in hydrolysates was obtained. The solids after starch hydrolysis were separated by filtration and processed by acid pretreatment (0.1 g of conc. HCl/g of biomass/5 ml of water, 120°C, 20 min) with subsequent enzyme hydrolysis (24 hours, 60°C) by the multienzyme preparations containing cellulases and hemicellulases. Overall yield of reducing sugars after these two steps was&#x0D; 740.7±3.9 mg/gram of dry corn fiber basis. Fermentation of lignocellulosic hydrolysates by yeasts Pichia stipitis CCY-39-50-1 and Candida shehatea CCY-29-68-4 (in both cases 5% v/v inoculum, 28°C, 72 hours) resulted in 0.38 and 0.12 g of ethanol per gram of reducing sugars. The results indicate that applied pretreatment methods and used microorganisms are able to produce ethanol from corn fiber.

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization.

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100°C), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90°C for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

Fine structure impacts highly concentrated starch liquefaction process and product performance

Designing a highly concentrated (45 %, w/w) starch liquefaction process is a green method to enhance the productivity of starch syrup and related fermentation products. Previous studies mainly focused on handling highly concentrated normal corn starch slurries, but the production efficiency and product performance cannot perfectly match the conventional liquefaction process (30 %, w/w). In the present research, four starches from various botanical sources were selected with an objective to accelerate highly concentrated starch liquefaction process. The results demonstrated that with potato starch or tapioca starch as a substrate, liquefaction process was more feasible as observed from the obvious reduction in paste viscosity and acceleration in amylolysis. To clarify the mechanism of these differences, changes in the fine structure during liquefaction were further characterized. The long external chains (16.2 glucose units on average) in potato starch and long internal chains (5.1 glucose units on average) in tapioca starch, which indicated high proportion of consecutive α-1,4 linkages, seemed more susceptible to enzymatic attack under highly concentrated substrate condition. This caused rapid degradation of starch molecules. The liquefied products were suitable for glucose syrup production. By comparison, normal corn starch and waxy corn starch, which contain relatively shorter linear fragments, were less accessible to α-amylase. This suppressed liquefaction process led to the survival of large molecules, thereby being unsuitable for subsequent saccharification process. The results suggest that selecting an appropriate substrate is an effective strategy to accelerate highly concentrated starch liquefaction and improve product performance.

Preparation, characterization and stability studies of cross-linked α-amylase aggregates (CLAAs) for continuous liquefaction of starch

In current study, α-amylase of fungal origin was immobilized using cross-linking strategy. The influence of precipitant (ammonium sulphate) and cross-linker (glutaraldehyde) concentration revealed that 60% (w/v) precipitant and 1.5% (v/v) cross-linker saturation was required to attain optimum activity. Cross-linked amylase aggregates (CLAAs) were characterized and 10-degree shift in optimum temperature (soluble enzyme: 50 °C; cross-linked: 60 °C) and 1-unit shift in pH (soluble enzyme: pH -6; cross-linked: pH -7) was observed after immobilization. The Vmax for soluble α-amylase and its cross-linked form was 1225 U ml−1 and 3629 U ml−1, respectively. The CLAAs was more thermostable than its soluble form and retained its 30% activity even after 60 min of incubation at 70 °C. Moreover, cross-linked amylase retained its activity after two months while its soluble counterpart lost its complete activity after 10 and 20 days at 30 °C and 4 °C storage, respectively. Reusability test showed that cross-linked amylase could retain 13% of its residual activity after 10 repeated cycles. Therefore, 10 times more glucose was produced after cross-linking than soluble amylase when it was utilized multiple times. This study indicates that amylase aggregates are highly effective for continuous liquefaction of starch, hence have strong potential to be used for different industrial processes.

Production of Bacterial Amylase and Evaluation for Starch Hydrolysis

In starch degrading industry, the first step to be performed is gelatinization of starch granules at elevated temperature. This process tends to form a viscose suspension, thus making mixing and pumping a huge challenge. Therefore, to lower the viscosity of starch paste thermostable enzymes are added for breaking down the polysaccharides into oligosaccharides by a process known as liquefaction. Therefore, the availability of these enzymes can be considered a prerequisite to start successful starch liquefaction and saccharification industry. The main objective of this study was, therefore, to produce efficient microbial amylolytic enzymes for starch hydrolysis. One bacterial isolate designated as BACC 2107 was isolated from locally collected samples. The enzyme has optimum activity at pH of 5.0 and 70°C. The addition of calcium in the reaction mixture was shown to stabilize the enzyme. The isolate BACC 2107 was able to grow under solid-state fermentation (SSF) using wheat bran as a solid substrate. Optimum enzyme production was recorded at 32°C, and at 120 h of incubation and at a moisture content of 75%. Addition of peptone to the SSF medium as an organic nitrogen supplement enhanced enzyme production. The enzyme was used to hydrolyze a 25% (w/v) of starch at 70°C and pH 5.0, and the resulting hydrolysate was analyzed on TLC plates. After 4 h hydrolysis, the TLC chromatogram showed the release of glucose, maltose, maltotriose, and other oligosaccharides, indicating that the enzyme is endo-acting alpha-amylase. The results of this study show that amylase from BACC 2107 has a good potential to be used in starch liquefaction and saccharification processes. The ability of the isolate to grow and produce the enzyme under SSF using cheap agro-industrial wastes could help to greatly reduce the production cost of the enzyme.

Liquefaction of starch using solid-acid catalysts derived from spent coffee for the production of plasticized poly (vinyl alcohol) films

This paper reports a strategy for preparing polyether polyols from corn starch, with (i) a mixture of polyethylene glycol 400 and glycerin (7:3, w/w) as the liquefying solvent and (ii) a spent-coffee-derived solid-acid catalyst (SC-SAC) (1:10, w/w, SC-SAC/starch) at 433 K for 1.5 h, under which conditions the liquefaction yield exceeded 99 %. The SC-SAC was prepared via hydrothermal carbonization at 453 K for 12 h, followed by sulfonation with H2SO4 at 343 K for 10 h. The liquefied starch product (SLP) was then used to plasticize poly(vinyl alcohol) (PVA) films with various mixing ratios. The optimal 0.4 SLP/PVA blend film exhibited good mechanical properties (tensile strength 38.07 MPa, elongation at break 1199 %), good transparency, and excellent flexibility. The results highlight the possibility of using SLP/PVA films in the development of degradable packaging materials.

Fine regulation of the starch liquefaction process and its application in the production of citric acid

Citric acid (CA), generally fermented from the starchy material, has attracted intense attention because of its wide applications in many aspects. However, the traditional starchy-liquefaction process based on the dextrose equivalent value and iodine-testing method is inappropriate as it hinders the subsequent CA fermentations. Here, a novel method of evaluating the starch liquefaction in the CA production process was established. Firstly, dextrin samples with the molecular weight (Mw) narrow distribution were prepared by alcohol fractional precipitation. Glucoamylase (GM) from the culture of CA-producing strain Aspergillus niger was purified. Then, the structure-activity relationship between the dextrin and GM was analyzed. Results demonstrated that the Mw of liquefied components aggregated in the range of 1.4–1.9 kDa could improve the efficiency of simultaneous saccharification and fermentation process. CA production rate and total sugar uptake rate were evidently improved with residual total sugar decreasing by 10.8% and fermentation efficiency enhanced by 21.1% in 9 h shorter fermentation time. All these results confirmed that fine regulation of the starch liquefaction based on the Mw characteristics was feasible and effective to improve the CA fermentation. Our proposed strategy could also be useful for other fermentations and sugar industry involving the starchy material.

Life cycle assessment for bioethanol production from whole plant cassava by integrated process

A new integrated process is proposed to convert whole plant cassava to bioethanol. It could improve the utilization of straw and contribute to the industrial production of cellulose ethanol. The key technologies were the by-products organic acids enhanced pretreatment of straw, and the mixed liquefaction and fermentation of cassava root starch and cassava straw sugar. Process simulation technology and life cycle assessment were employed to simulate and analyze the bioethanol processes from the cassava root, cassava straw and whole plant cassava, respectively. The results showed that cassava root ethanol had the lowest process energy consumption as 4186 MJ/1000L ethanol. Cassava straw bioethanol had a negative net energy value as −1329 MJ/1000L ethanol. The whole plant cassava bioethanol showed a competitive net energy ratio = 1.45, the highest reproducibility = 2.35–2.55, and the lowest environmental emissions. The effects of fermentation efficiency and straw utilization rate on the integrated process were further investigated, which would help the practical application of the integrated process. When the fermentation efficiency was maintained at more than 80%, the straw utilization rate was more conducive to improving the renewability and environmental friendliness of bioethanol. Moreover, the influence of the process simulation deviation on life cycle assessment was determined by Monte Carlo Analysis. The net energy analysis and environmental impact assessment proved that the proposed integrated process was beneficial for energy utilization and cleaner production.

Relationship between starch liquefaction behavior and properties of polymer coated urea from liquefied starch

The relationship between starch liquefaction behavior and properties of coated urea was explored to give some insights into the optimized technical selection of bio-polyol for polymer coated urea. The liquefied corn starch was monitored for insoluble residue content, viscosity, acid value and hydroxyl value. Nitrogen release, water permeability and biodegradation properties of coating material were also studied. FTIR and 1H-NMR results revealed that alcoholysis, hydroxyalkylation and side reactions co-existed during the liquefaction process, and side reactions predominated as the process continued. It was found that re-condensation leading to the increase of polyester polyols was beneficial for subsequent coating of urea according to the nitrogen release and biodegradation performance. The selected bio-polyols derived from starch can be available to prepare polymer coated urea being more accordant with the nutrient requirement of plants at growth stages.