Raw Starch Digestion Research Articles

Enhancement of stability and activity of RSD amylase from Paenibacillus lactis OPSA3 for biotechnological applications by covalent immobilization on green silver nanoparticles

The key challenge to the biotechnological applications of amylases is achieving high activity and stability under extreme pH, temperature and often high levels of enzyme denaturants. This study immobilized a novel raw starch-digesting (RSD) amylase from Paenibacillus lactis OPSA3 on glutaraldehyde-activated silver nanoparticles. Effects of time, glutaraldehyde concentration, pH, temperature, and enzyme concentration on immobilization were studied, and the immobilized enzymes were characterized. pH 9.0 was optimum for the enzyme immobilization. The maximum immobilization efficiency of 82.23 ± 7.99 % was achieved at 25 °C for 120 min. After immobilization, the optimum pH and temperature changed from 9.0 to 11.0 and 60 to 70, respectively. Immobilization reduced the amylase's activation energy (KJ/mol) from the initial 58.862 to 45.449 following immobilization. The Km of the amylase decreased after immobilization, while the Vmax increased. The immobilized amylase showed significantly greater storage and thermal stability than the free amylase. At 80, enzyme half-life (min) and D value (min) increased from 12.33 to 179.11 and 40.94 to 594.98, respectively. The immobilized amylase (80–88 %) had more stability to the effects of the studied surfactants than the free enzyme. It also showed improved stability in the presence of commercial detergents compared to the free enzyme. The amylase's enhanced kinetic parameters and stability following successful immobilization on silver nanoparticles indicate its potential for application in the range of biotechnological processes where alkaline- and temperature-stable amylases are employed.

X-ray crystallographic structural studies of α-amylase I from Eisenia fetida.

The earthworm Eisenia fetida possesses several cold-active enzymes, including α-amylase, β-glucanase and β-mannanase. E. fetida possesses two isoforms of α-amylase (Ef-Amy I and II) to digest raw starch. Ef-Amy I retains its catalytic activity at temperatures below 10°C. To identify the molecular properties of Ef-Amy I, X-ray crystal structures were determined of the wild type and of the inactive E249Q mutant. Ef-Amy I has structural similarities to mammalian α-amylases, including the porcine pancreatic and human pancreatic α-amylases. Structural comparisons of the overall structures as well as of the Ca2+-binding sites of Ef-Amy I and the mammalian α-amylases indicate that Ef-Amy I has increased structural flexibility and more solvent-exposed acidic residues. These structural features of Ef-Amy I may contribute to its observed catalytic activity at low temperatures, as many cold-adapted enzymes have similar structural properties. The structure of the substrate complex of the inactive mutant of Ef-Amy I shows that a maltohexaose molecule is bound in the active site and a maltotetraose molecule is bound in the cleft between the N- and C-terminal domains. The recognition of substrate molecules by Ef-Amy I exhibits some differences from that observed in structures of human pancreatic α-amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.

Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius

Amylase capable of raw starch digestion presents a cheap and easier means of reducing sugar generation from various starch sources. Unfortunately, its potential for use in numerous industrial processes is hindered by poor stability. In this work, chemical modification by acylation using citraconic anhydride (CA) and maleic anhydride (MA) was used to stabilize the raw starch saccharifying amylase from A. carbonarius. The effect of the anhydrides on the pH and thermal stability of the free amylase was investigated. Enzyme kinetics and thermodynamic studies of the free and modified amylase were also carried out. Blue shifts in fluorescent spectra were observed after modification with both anhydrides. Citraconylation led to increased affinity of the enzyme for raw potato starch, unlike maleylation. The activation energy (kJ mol−1) for enzyme inactivation was increased by 94.8% after modification with CA while only 17.9% increase was noted after modification with MA. Acylation led to an increase in Gibb's free energy and enthalpy while a reduction in entropy was observed. At 80 °C the half-life (h) was 5.92, 11.18 and 14.74 for free, MA and CA enzyme samples, respectively. These findings have potential value in all industries interested in starch conversion to sugars.

Production of Raw Starch-Digesting Amylolytic Preparation in Yarrowia lipolytica and Its Application in Biotechnological Synthesis of Lactic Acid and Ethanol.

Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.

<p>Molecular dynamics study to improve the substrate adsorption of <em>Saccharomycopsis fibuligera</em> R64 alpha-amylase by designing a new surface binding site</p>

Background: Carbohydrate binding module (CBM) and surface binding site (SBS) are two important parts of amylase which respond to the raw starch digestion. They are related to the enzyme ability to adsorb and to catalyze the starch hydrolysis. However, starch processing is still expensive due to the high temperature in the gelatinization step. Therefore, direct starch digestion is more favorable. One of the solutions is to use α-amylase with high starch adsorptivity, which is expected to be capable of digesting starch below the gelatinization temperature. In Indonesia, Saccharomycopsis fibuligera R64 α-amylase (Sfamy R64) is one of the enzymes with the highest activity on starch. However, its raw starch adsorptivity was low. The aim of this study was to propose an in-silico model of Sfamy R64 mutant by introducing a new SBS using molecular dynamics (MD) simulation.Methods: The structural behavior of Sfamy R64 and positive control were studied using MD simulation. Furthermore, the mutants of Sfamy R64 were designed to have a stable SBS which mimics the positive control. The substrate affinity in all systems was evaluated using the molecular mechanics generalized Born surface area (MM/GBSA) method.Results: The stability of a new SBS constructed by seven substitutions and a loop insertion was improved throughout MD simulation. The substrate was consistently bound to the SBS over 55 ns of simulation, as compared to 14 ns in wild-type. The structural behavior of SBS in mutant and positive control was similar. The interaction energies of the positive control, wild-type, and mutant were −17.6, −5.2, and −8.2 kcal/mol, respectively.Conclusion: The enhanced substrate binding in the mutant, due to the existence of a new SBS, suggests the potential of improving starch adsorptivity of Sfamy R64. This result should be useful in developing an enzyme with better substrate adsorption based on the rational computer-aided molecular design approach.

Improvement of glucoamylase production for raw-starch digestion in Aspergillus niger F-01 by maltose stearic acid ester.

To investigate the role of maltose stearic acid ester (MSAE) for improving raw-starch-digesting glucoamylase (RSDG) production by Aspergillus niger F-01 and its regulatory mechanism. RSDG activity was enhanced 8.3-fold by adding 5g MSAE to 1l basal medium at the beginning of the culture. RSDG biosynthesis nearly ceased by adding 100μg actinomycin D/ml or 50μg cycloheximide/ml in a 48h culture in the basal medium. RSDG biosynthesis was restarted by adding MSAE in the culture repressed by actinomycin D. No revival of RSDG synthesis was observed by adding MSAE in the culture repressed by cycloheximide. MSAE improved RSDG production in A. niger by inducing the transcription of RSDG.

Physicochemical and kinetic properties of a high salt tolerant Aspergillus flavus glucoamylase

Abstract Amylases with excellent biochemical properties, broad starch degradation ability and high glucose yield are ultimate industrial requirements for starch processing. Here, extracellular glucoamylase produced from Aspergillus flavus under submerged fermentation was purified to apparent homogeneity by combination of 0–60% ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. The purified enzyme has a monomeric molecular weight of 63 kDa and exhibits optimum pH and temperature at 5.5 and 50 °C, respectively. The enzyme was acidic thermostable at pH 4.0–7.0 retaining about 62% of initial activity after 1 h of incubation at 60 °C. Using raw cassava starch as substrate, the enzyme has a Km value of 0.72 mg/mL with Vmax of 12.48 μmol/min/mg. This glucoamylase exhibited high degree of salt tolerance with residual activity above 65% after 24 h incubation in 2.0 M NaCl solution. The activity of the purified enzyme was enhanced by Ca2+, Mg2+, Na+ but inhibited by Al3+, Cu2+, Hg2+, Ethylenediamine tetraacetic acid (EDTA) and Urea. The results from this study affirm that this fungal glucoamylase possess good biochemical characteristics for raw-starch digestibility suitable for biotechnological application.

Glucoamylase from the Predacious Fungus Arthrobotrys conoides: a Cationic Enzyme with High Debranching Activity and Raw Starch Digestibility

The extracellular amylolytic activity elaborated by the nematophagous fungus Arthrobotrys conoides was found to resolve into 2 amylolytic peaks when fractionated on Sephadex G-100 column. Around 80% of the eluted glucoamylase activity was attributed to peak I (GA A). GA A being cationic in nature was purified about 70-fold with 57% yield by negative chromatography on DEAE Sephadex at pH 7.0. The enzyme was stable over a broad pH range of 4.8–9.0. KM for the linear polysaccharide amylose was 0.34 mg/mL. Enzyme showed high affinity for the branched polysaccharides as the KM values for amylopectin, glycogen and starch were 0.056, 0.062 and 0.065 mg/mL, respectively. The enzyme clearly demonstrated raw starch digestibility. Probable involvement of Trp and His residues in enzyme catalysis was elucidated using group-specific reagents.

Cloning and extracellular expression of a raw starch digesting α-amylase (Blamy-I) and its application in bioethanol production from a non-conventional source of starch.

The aim of this study was to clone and efficiently express a raw starch-digesting α-amylase enzyme in the culture media and also to investigate the potential application of this recombinant enzyme in the digestion of non-conventional raw starch for bioethanol production. A raw starch digesting α-amylase gene isolated from Bacillus licheniformis strain AS08E was cloned and extracellularly expressed in E. coli cells using the native signal peptide. The mature recombinant α-amylase (Blamy-I) consisting of 483 amino acid residues was found to be homogenous with a mass of 55.3 kDa (by SDS-PAGE analysis) and a predicted pI of 6.05. Structural and functional analysis of Blamy-I revealed the presence of an extra Ca(2+) -binding region between the A and C domains responsible for higher thermostability of this enzyme. The statistical optimization of E. coli culture conditions resulted in an approximately eightfold increase in extracellular expression of Blamy-I as compared to its production under non-optimized conditions. Blamy-I demonstrated optimum enzyme activity at 80 °C and pH 10.0, and efficiently hydrolyzed raw starch isolated from a non-conventional, underutilized jack fruit seeds. Further utilization of this starch for bioethanol production using Blamy-I and Saccharomyces cerevisiae also proved to be highly promising.

Inheritance of low pasting temperature in sweetpotato starch and the dosage effect of wild-type alleles.

Sweetpotato (Ipomoea batatas (L.) Lam.), which is an outcrossing hexaploid, is one of the most important starch-producing crops in the world. During the last decade, new sweetpotato cultivars, e.g. ‘Quick Sweet’, which have approximately 20°C lower pasting temperature, slower retrogradation and higher digestibility of raw starch than ordinary cultivars, have been developed in Japan. Genetic analysis of these variants with low pasting temperature starch was conducted in this study. Using 8 variants and 15 normal clones, 26 families were generated. The results from analyzing these progenies suggested that this trait is a qualitative character controlled by one recessive allele (designated spt), which is inherited in a hexasomic manner. A dosage effect of the wild-type Spt allele was found for starch pasting temperature, although the effect was not linear. These results will aid breeders to develop sweetpotato cultivars with a range of starch pasting temperatures.

The Effect of in Vitro Digestion on the Structural, Morphologic Characteristics and Fermentability of Raw Banana Starch

This study aimed to observe the effect of in vitro digestion on the structural and morphologic characteristics, and find the relationship between in vitro digestion and colonic fermentation of raw banana starch. After digested by artificial gastric juice and artificial intestinal juice, the starch granules were damaged, but not drastic. X-ray diffractograms showed that the crystal polymorph (C-type) was not changed and the degree of crystallinity was increased after digestion. There were no significant differences in chemical structure according to the infrared spectrum analysis. The swelling power and solubility of the digested starches were greater than that of raw banana starch at all temperatures. Banana starches digested by gastric and intestinal juice presented high fermentability, expressed by total short chain fatty acids. The results may be due to the slight changes on structural and morphologic characteristics. To conclude, passage through the stomach and small intestine increased susceptibility of raw banana starch to further fermentation, to increase the production of metabolically active short chain fatty acids.

Cloning and overexpression of raw starch digesting α-amylase gene from Bacillus subtilis strain AS01a in Escherichia coli and application of the purified recombinant α-amylase (AmyBS-I) in raw starch digestion and baking industry

Considering the economic and industrial relevance of α-amylases used in food and starch industries, a raw starch digesting α-amylase gene (amyBS-I) from Bacillus subtilis strain AS01a was cloned and expressed in Escherichia coli BL21 cells. The gene also includes its signal peptide sequence (SPS) for facilitating the efficient extracellular expression of recombinant α-amylase (AmyBS-I) in correctly folded (enzymatically active) form. The native AmyBS-I consists of 659 amino acids with a molecular mass and pI of 72,387 Da and 5.8, respectively. The extracellular secretion of AmyBS-I after response surface optimization of culture conditions was found to be 7-fold higher as compared to its production under non-optimized conditions. Purified AmyBS-I demonstrated optimum activity at 70 °C and pH 6.0. It shows Km and Vmax values toward soluble starch as 2.7 mg/ml and 454 U/ml, respectively. Further, it does not require Ca2+ ion for its α-amylase activity/thermo-stability, which is an added advantage for its use in the starch industry. The AmyBS-I also hydrolyzed a wide variety of raw starches and produced maltose and glucose as main hydrolyzed products. The bread dough supplemented with AmyBS-I showed better amelioration of the bread quality as compared to the bread supplemented with commercial α-amylase.

Electron microscopy and composition of raw acorn starch in relation to in vivo starch digestibility

The structure and composition of starch play an important role as co-factors affecting raw starch digestibility: such features were investigated in raw acorn starch from the most diffused oak trees in the Mediterranean basin. A total of 620 whole ripe acorns from Holm (Quercus ilex L., n = 198), Downy (Quercus pubescens Willd., n = 207) and Cork (Quercus suber L., n = 215) oaks sampled on the Sardinia Isle (40° 56' 0'' N; 9° 4' 0'' E; 545 m above the mean sea level) in the same geographical area, were analyzed for their chemical composition. The starch contents ranged between 51.2% and 53.5% of dry matter. The starch granules displayed a spheroid/ovoid and cylindrical shape; on scanning electron microscopic (SEM) analyses, a bimodal distribution of starch granule size was observed both for Holm and Cork oak acorns, whereas the starch granules of Downy oak acorns showed diameters between 10.2 and 13.8 μm. The specific amylose to amylopectin ratio of acorn starch was 25.8%, 19.5% and 34.0% in the Holm, Downy and Cork oaks, respectively. The (13)C Nuclear Magnetic Resonance (NMR) signal analysis displayed a pivotal spectrum for the identification of the amylose peaks in raw acorn starch, as a basis for the amylose to amylopectin ratio determination.

The Nature of Raw Starch Digestion

The Nature of Raw Starch Digestion

Raw starch-degrading α-amylase fromBacillus aquimarisMKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

The aims were to isolate a raw starch-degrading α-amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli. A 1539 complete open reading frame of a starch-degrading α-amylase gene baqA from B.aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B.aquimaris MKSC 6.2 α-amylase (BaqA) has no starch-binding domain, and together with a few putative α-amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch. A novel raw starch-degrading B.aquimaris MKSC 6.2 α-amylase BaqA is proposed to be a member of new GH13 subfamily. This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.

Preferential and rapid degradation of raw rice starch by an α-amylase of glycoside hydrolase subfamily GH13_37

The α-amylase (AmyP) from a marine metagenomic library belongs to the recently classified glycoside hydrolase subfamily GH13_37. The degradation abilities of AmyP on a broad range of raw starch granules were examined at 40 °C and pH 7.5. It was found that AmyP is a raw starch-degrading enzyme, exhibiting a unique and remarkable ability to preferentially and very rapidly digest raw rice starch. The specific activity of raw rice starch was reached 118.5 ± 0.6 Umg(-1), which was much higher than that of other raw starches. The final hydrolysis degrees were obtained in 4 h for 1 % raw rice starch and 1 h for 8 % concentration, indicating a very rapid speed of hydrolysis. The presence of a starch residue resistant was the main limiting factor for complete hydrolysis, although end product inhibition also existed, especially at high starch concentrations. AmyP randomly attacks unique or susceptible sites on raw rice starch granules, and releases glucose, maltose, and maltotriose as end products. This is the first biochemical characterization of the raw starch-degrading ability of an α-amylase of family GH13_37. The specific ability towards raw rice starch has never been described before, and this makes AmyP a promising candidate for use as a novel enzyme in rice starch processing.

Identification of an acidic α-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry

An acidic α-amylase was purified from thermoacidophilic Alicyclobacillus sp. A4 by ion exchange chromatography with 22% recovery, and showed a molecular mass of 64kDa by SDS–PAGE. Its amino acid sequence was determined by sequencing three internal peptides and the complete genome of strain A4, and shared highest identity (64%) with Alicyclobacillusacidocaldarius α-amylase. Compared with other reported α-amylases, the purified enzyme had some distinct characteristics. The optimal activity was found to occur at 75°C and pH 4.2, similar to the glucamylase widely used in the starch industry. The enzyme was Ca2+ independent, and had strong ability to digest raw starch (96.71%) with commercial glucamylase in one step. These properties of the purified enzyme make up the deficiency of the commercial α-amylases currently used and avoid repeated adjustment of pH and temperature in double-enzymatic sugar-making process. The purified enzyme will be commercially valuable in the starch industry.

Purification and biochemical characterization of a Ca 2+ - independent, thermostable and acidophilic α-amylase from Bacillus sp. RM16

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of α- amylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ-cyclodextrin and pullulan. Key words: Ca 2+ -independent, Bacillus sp, thermostable α-amylases, low pH profile, enzyme, raw starch digestion, HPLC.

Resistant and Digestible Starch Contents in Sweet Potato Cultivars and Lines

The sweet potato is recognized as a health-promoting food, owing to its richness in functional components such as dietary fiber; however, only a few studies have been conducted on resistant starch (RS) levels in the sweet potato. Now, the sweet potato is expected to have potential as a renewable resource for bio-ethanol production. The enzymatic digestibility of raw starch appears to influence the conversion rate of starch to sugar in the first step of ethanol production. In this study, the RS content of gelatinized starch, digestible starch (DS) content of raw starch and other starch properties were investigated using 21 sweet potato cultivars and lines to assess varietal variations and to estimate relationships among these starch properties. The RS content of gelatinized starch ranged between 1.8 and 9.5%. The DS content of raw starch ranged between 69.3 and 98.3%. Analysis of variance indicated that the varietal differences were significant at the 0.1% level for both the RS content and the DS content. Sweet potato starches with high amylose content and low paste viscosity showed high RS content of gelatinized starch. Starches with low pasting temperature and slow retrogradation showed high DS content of raw starch. A new breeding line, Kyukei 03284-156, contained a unique starch having the highest RS content, highest amylose content, lowest paste viscosity and abnormal granule morphology. The results of this study are expected to provide useful information to food industries that utilize sweet potato starch and to breeders that attempt to improve the RS and DS contents in sweet potato cultivars.

Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians

A moderately halophilic bacterium, Kocuria varians, was found to produce active α-amylase (K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus, which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.