Aspergillus Niger Glucoamylase Research Articles

Improved Hydrolysis of Granular Starches by a Psychrophilic α-Amylase Starch Binding Domain-Fusion.

Degradation of starch granules by a psychrophilic α-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was facilitated by C-terminal fusion to a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) via a decapeptide linker. Depending on the waxy, normal or high-amylose starch type and the botanical source, the AHA-SBD fusion enzymes showed up to 3 times higher activity than AHA wild-type. The SBD-fusion thus increased the density of enzyme attack-sites and binding-sites on the starch granules by up to 5- and 7-fold, respectively, as measured using an interfacial catalysis approach that combined conventional Michaelis-Menten kinetics, with the substrate in excess, and inverse kinetics, having enzyme in excess, with enzyme-starch granule adsorption isotherms. Higher substrate affinity of the SBDGA compared to SBDGWD3 was accompanied by the superior activity of AHA-SBDGA in agreement with the Sabatier principle of adsorption limited heterogenous catalysis.

Research progress of glucoamylase with industrial potential.

Glucoamylase is one of the most widely used enzymes in industry, but the development background and existing circumstances of industrial glucoamylase were not described by published articles. CiteSpace, a powerful tool for bibliometric, was used to analyze the past, existing circumstances, and trends of a professional field. In this study, 1820 Web-of-Science-indexed articles from 1991 to 2021 were collected and analyzed by CiteSpace. The research hotspots of industrial glucoamylase, like glucoamylase strain directional improvement, Aspergillus niger glucoamylase, glucoamylase immobilization, application of glucoamylase in ethanol production, and "customized production" of porous starch, were found by analyzing countries, institutions, authors, keywords, and references of articles. PRACTICAL APPLICATIONS: The research progress of glucoamylase with industrial potential was analyzed by CiteSpace, and a significant research direction of glucoamylase with industrial potential was found. This is helpful for academic and corporate audiences to understand the current situation of glucoamylase with industrial potential and carry out follow-up works.

Production and optimization of Aspergillus niger glucoamylase using amylopectin from guinea corn starch as the sole carbon source

A Fourteen day experimental study was carried out to determine the day of highest glucoamylase activity using amylopectin from guinea corn starch as the sole carbon source. Two peaks of high activity were observed on the fifth and twelveth days, and were thus mass produced. Specific activities for crude enzymes were found to be 729.45 U/mg and 1046.82 U/mg for day five and twelve harvested enzymes respectively. Ammonium sulphate saturations, 70% and 20%, were found suitable to precipitate proteins with highest glucoamylase activity for day five and twelve harvested enzymes respectively. After ammonium sulphate precipitation and gel filtration, specific activities were found to be 65.98 U/mg and 180.52 U/mg respectively for day five harvested enzyme and 61.51 U/mg and 272.81 U/mg for day twelve harvested enzyme. The pH optimum for day five harvested enzyme were found to be 7.5, 7.5 and 6.0 using tiger nut, cassava and guinea corn starches as substrates respectively, also, the pH optimum for day twelve harvested enzyme were found to be 5.0, 8.5 and 7.0 using tiger nut, cassava and guinea corn starches as substrate, respectively. Optimum temperatures were found to be 50˚C and 45˚C for day five and twelve harvested enzymes, respectively. Km and Vmax, of day five harvested enzyme were found to be 770.75 mg/ml and 2500 μmol/min, 158.55 mg/ml and 500 μmol/min and 46.23 mg/ml and 454.53 μmol/min using cassava, guinea corn and tiger nut starches as substrate respectively. Km and Vmax of day twelve harvested enzyme were found to be 87.1 mg/ml and 384.61 μmol/min, 29.51 mg/ml and 243.90 μmol/min, and 2364 mg/ml and 2500 μmol/min, using cassava, guinea corn and tiger nut starches as substrate respectively.Bangladesh J. Sci. Ind. Res. 52(4), 263-272, 2017

Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization

Aspergillus niger glucoamylase (GLA) was covalently immobilized on 1,3,5-triazine-functionalized chitosan coated superparamagnetic nanoparticles (MNPCh-CC). The morphology, structure and properties of functionalized nanocomposite were investigated, as well as GLA immobilization process, through different analytical tools. Different experimental parameters such as optimum temperature, pH, reaction time and enzyme concentration were studied for free and immobilized enzyme. The GLA immobilized on nanocarrier exhibited excellent catalytic activity at pH 4.5 and 60°C. Notably, the immobilized GLA showed quite impressive stability, even after 10 reaction cycles, it could still retain about 70% of the initial activity. The results showed that immobilization process couldn’t significantly inhibit enzyme-substrate interaction and subsequently retained its effective catalytic activity. The substantial improvement of reactivity, reusability, and stability of this biocatalyst system may confer it a wider range of applications in industrial processes.

Immobilisation of Aspergillus oryzae α-amylase and Aspergillus niger glucoamylase enzymes as cross-linked enzyme aggregates

AbstractCross-linked enzyme aggregates (CLEA) of Aspergillus oryzea α-amylase (AoAA) and Aspergillus niger glucoamylase (AnGA) were prepared using glutaraldehyde and dextran polyaldehyde as crosslinkers. The maximum activity recoveries for glutaraldehyde cross-linking were 21.8 % and 41.2 %, respectively. The addition of a proteic feeder (bovine serum albumin) exhibited a negative effect on the activity recoveries for both enzymes. Dextran polyaldehyde was used as a cross-linking agent instead of glutaraldehyde to reduce the activity losses. As a result, an activity recovery of 60.0 % was obtained for Aspergillus oryzea α-amylase. On the other hand, no activity recovery was observed for Aspergillus niger glucoamylase due to the latter enzyme’s affinity for dextran.

Kinetic and thermodynamic parameters of immobilized glucoamylase on different mesoporous silica for starch hydrolysis: A comparative study

Abstract Large ordered mesoporous silica materials having different pore diameters were synthesized by hydrothermal method functionalized with 3-APTES and bifuctional agent glutaraldehyde. Aspergillus niger glucoamylase was immobilized onto mesosilica via simple adsorption technique and covalent binding. The bare and enzyme immobilized supports were characterized by low angle XRD, nitrogen adsorption studies, FT-IR spectroscopy, thermogravimetric analysis (TG) and scanning electron microscopy (SEM). Kinetic and thermodynamic parameters were evaluated for soluble starch hydrolysis. Kinetic parameters were calculated according to Lineweaver–Burk plot and Km value was found to increase and Vmax was found to decrease after immobilization. Activation energy for free enzyme was found to 20.43 kJ mol−1. The enthalpy of activation (ΔH), Gibbs free energy (ΔG substrate binding) and entropy of activation (ΔS) for soluble starch hydrolysis by glucoamylase are reported.

Dietary Fiber Content of Cross-linked Phosphorylated Resistant Starch (RS4) Determined by the Prosky and McCleary Methods. Part I. Factors Affecting In Vitro Digestion of Starch in a Food Sample

Resistant starch (RS), a component of dietary fiber in the human diet, occurs in five classes (RS1–RS5); each class exhibits a different barrier to digestion by amylolytic enzymes. Various in vitro assays for determining total dietary fiber utilize different conditions to remove digestible starch from a food sample. Because the five classes of RS react differently under different assay conditions, the in vitro levels of RS vary by method. This article highlights important parameters that impact the digestibility of starch when using porcine pancreatic alpha-amylase and Aspergillus niger glucoamylase.

Halogenol- versus alkanol-induced structural transitions of acid-denatured glucoamylase: Characterization of alcohol-induced states

Different probes such as far- and near-UV CD spectral signals, ANS binding, Trp fluorescence and three-dimensional fluorescence were used to study halogenol- versus alkanol-induced conformational transitions in the acid-denatured state (pH 1.0) of Aspergillus niger glucoamylase (GA). These alcohols showed significant retrieval of the protein structure, inducing both secondary and tertiary structural changes, as evident from the increase in the α-helix and decrease in ANS binding, respectively. However, halogenols were found more competent than alkanols, requiring lesser alcohol concentration to induce similar spectral change. The effectiveness of these alcohols showed the order: HFIP>TFE>2-chloroethanol for halogenols while tert-butanol>1-propanol>2-propanol>ethanol>methanol for alkanols. Both Trp fluorescence and near-UV CD spectra showed anomalous pattern, though the order of effectiveness remained the same as found with far-UV CD spectra and ANS fluorescence. Three-dimensional fluorescence results of the acid-denatured state (pH 1.0) of GA in the presence of 5.5M tert-butanol agreed well with the data obtained from far-UV CD and Trp fluorescence. All these results suggested the formation of partially folded states of GA obtained in the presence of these alcohols, being more effective with halogenols than alkanols.

Regioselective glucosylation of inositols catalyzed by Thermoanaerobacter sp. CGTase

Monoglucosylated products of l-chiro-, d-chiro-, muco-, and allo-inositol were synthesized by regioselective α-d-glucosylation with cyclodextrin glucosyl transferase from Thermoanaerobacter sp. after hydrolysis of by products with Aspergillus niger glucoamylase. While the reactions carried out with d-chiro-, muco-, and allo-inositol resulted in the regioselective formation of monoglucosylated products, two products were obtained in the reaction with l-chiro-inositol. Through the structural characterization of the glucosylated inositols here we demonstrated that the selectivity observed in the glucosylation of several inositols by Thermoanaerobacter sp. CGTase, is analogous to the specificity observed for the glucosylation of β-d-glucopyranose and equivalent glucosides.

Study on the reduction of acrylamide in mixed rye bread by fermentation with bacteriocin-like inhibitory substances producing lactic acid bacteria in combination with Aspergillus niger glucoamylase

This study mainly focuses on a strategy for reducing of acrylamide in cereal products, predominantly in bakery products. The effect of mesophilic lactic acid bacteria (LAB) strains as well as a novel fermentation media on the bases of extruded rye wholemeal on the acrylamide formation in mixed rye bread of different weight of loaf was studied. The LC–MS/MS method for acrylamide determination in bread crumb has been applied. Addition of 15% low pH (pH 3.4–4.3) fermented product to bread dough, which were produced by using commercial strain Lactobacillus casei and bacteriocin-like inhibitory substances (BLIS) producing strains Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7 and Pediococcus pentosaceus KTU05-8 separately, caused significant reduction of acrylamide in mixed rye bread. All bread loafs of 1000 g contained less by 27% acrylamide concentrations versus loafs of 500 g. Acrylamide formation was affected (r2 = 0.7193) by total reducing sugar content in bread and slightly correlated (r2 = 0.5587) with reducing sugar content in sourdough. The treatment of extruded rye wholemeal with Aspergillus niger glucoamylase as compared to the control sample was found to have a positive effect on the acidification process lowering the acrylamide formation on an average by 59.4% and 40% in 500 g and 1000 g loafs of bread, respectively. This study demonstrates that acrylamide content could be reduced by using LAB excreting lower amylolytic activity in the medium, while the higher proteolytic activity is preferred.

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE222nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE222nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in Ksv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100°C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.

Immobilization of Glucoamylase onto Lewis Metal Ion Chelated Magnetic Affinity Sorbent: Kinetic, Isotherm and Thermodynamic Studies

In this study, magnetic metal-chelate beads, m-poly(ethylene glycol dimethacrylate-vinyl imidazole) [m-poly(EGDMA–VIM)] with an average diameter 150–200 μm was synthesized by copolymerizing ethylene glycol dimethacrylate (EGDMA) with vinyl imidazole (VIM). The spesific surface area of the m-poly(EGDMA–VIM) beads was found 63.1 m2/g. Cu2+ ions were chelated on the m-poly(EGDMA–VIM) beads and used in immobilization of Aspergillus niger glucoamylase in a batch system. The maximum glucoamylase adsorption capacity of the m-poly(EGDMA–VIM)–Cu2+ beads was observed as 120 mg/g at pH 6.5. The optimum pH for free and m-poly(EGDMA–VIM)–Cu2+ immobilized glucoamylase were found 4.0 and 4.5, respectively. The optimum temperature of glucoamylase was not changed after immobilization and determined as 60 o C for free and immobilized enzyme preparations. The glucoamylase adsorption capacity and adsorbed enzyme activity slightly decreased after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications. Storage stability was found to increase with immobilization. The effect of various experimental parameters such as pH, glucoamylase concentration, contact time and temperature in aqueous solution were also investigated. Adsorption isotherm obtained for m-poly(EGDMA–VIM)–Cu2+ was consistent with Langmuir model. Kinetic studies showed that the adsorption process agreed with both the pseudo-second-order kinetic model and the modified Ritchie's-second-order kinetic model. Various thermodynamic parameters, free energy (ΔG 0), enthalpy (ΔH 0) and entropy (ΔS 0), were also calculated and the results showed that the adsorption process strongly depended on temperature of medium.

Vinyl triazole carrying metal‐chelated beads for the reversible immobilization of glucoamylase

AbstractPoly(ethylene glycol dimethacrylate–1‐vinyl‐1,2,4‐triazole) [poly(EGDMA–VTAZ)] beads with an average diameter of 100–200 μm were obtained by the copolymerization of ethylene glycol dimethacrylate (EGDMA) with 1‐vinyl‐1,2,4‐triazole (VTAZ). The copolymer hydrogel bead composition was determined by elemental analysis and was found to contain 5 EGDMA monomer units for each VTAZ monomer unit. The poly(EGDMA–VTAZ) beads were characterized by swelling studies and scanning electron microscopy (SEM). The specific surface area of the poly(EGDMA–VTAZ) beads was found 65.8 m2/g. Cu2+ ions were chelated on the poly(EGDMA–VTAZ) beads. The Cu2+ loading was 82.6 μmol/g of support. Cu2+‐chelated poly(EGDMA–VTAZ) beads with a swelling ratio of 84% were used in the immobilization of Aspergillus niger glucoamylase in a batch system. The maximum glucoamylase adsorption capacity of the poly(EGDMA–VTAZ)–Cu2+ beads was 104 mg/g at pH 6.5. The adsorption isotherm of the poly(EGDMA–VTAZ)–Cu2+ beads fitted well with the Langmuir model. Adsorption kinetics data were tested with pseudo‐first‐ and second‐order models. The kinetic studies showed that the adsorption followed a pseudo‐second‐order reaction model. The Michaelis constant value for the immobilized glucoamylase (1.15 mg/mL) was higher than that for free glucoamylase (1.00 mg/mL). The maximum initial rate of the reaction values were 42.9 U/mg for the free enzyme and 33.3 U/mg for the immobilized enzyme. The optimum temperature for the immobilized preparation of poly(EGDMA–VTAZ)–Cu2+–glucoamylase was 65°C; this was 5°C higher than that of the free enzyme at 60°C. The glucoamylase adsorption capacity and adsorbed enzyme activity slightly decreased after 10 batch successive reactions; this demonstrated the usefulness of the enzyme‐loaded beads in biocatalytic applications. The storage stability was found to increase with immobilization. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Stereoselective enzymatic synthesis of monoglucosyl- myo-inositols with in vivo anti-inflammatory activity

The monoglucosyl-inositols α- d-glucopyranosyl-(1→4)-4 d- myo-inositol 3 and α- d-glucopyranosyl-(1→1)-1 d- myo-inositol 4 were synthesized by a combined enzymatic transglucosylation and hydrolysis strategy, using cyclodextrin glucosyl transferase (CGTase) from Thermoanaerobacter sp., followed by hydrolysis with Aspergillus niger glucoamylase. The glucosides were separated by preparative HPLC and fully characterized by extensive 1D and 2D NMR studies. The structure of the regioisomer 4 was confirmed by X-ray crystallography of its perbenzoylated derivative 4a. Both isomers demonstrated in vivo anti-inflammatory activity at comparative levels to corticosterone on mouse ear oedema induced by 12- O-tetradecanoylphorbol-13-acetate (TPA) and in rat hind paw oedema induced by carrageenan.

ORIGINAL RESEARCH: Analysis of an Aspergillus niger glucoamylase strain pedigree using comparative genome hybridization & real-time quantitative polymerase chain reaction

Abstract Microarray-based comparative genome hybridization (CGH) analysis was used to obtain information regarding chromosomal changes that occurred during successive rounds of mutagenesis in a pedigree of Aspergillus niger glucoamylase production strains. This analysis revealed both deletions and amplifications of discrete DNA segments ranging in size from less than one kb to as much as 400 kb. Most importantly, we observed repeated amplification of a 216 kb region that included the glaA (glucoamylase) gene. CHEF (Contour-clamped Homogeneous Electric Field) gel analyses indicated that the amplified DNA segment was translocated nonreciprocally and several chromosomes were rearranged in successive strains of the lineage. The number of glaA gene copies increased from 2 in the original parent strain to approximately 10 in the final strain of the pedigree. Quantitative real-time PCR analyses confirmed the CGH results, leading us to hypothesize that at least some of the incremental improvements in glucoamylase...

Thermal Unfolding of Mutant Forms C509G and C509S of Starch Binding Domain-Fragment of Aspergillus niger Glucoamylase

Thermal Unfolding of Mutant Forms C509G and C509S of Starch Binding Domain-Fragment of Aspergillus niger Glucoamylase

Directed evolution of Aspergillus niger glucoamylase to increase thermostability.

SummaryUsing directed evolution and site‐directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2‐1. CR2‐1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2‐1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2‐1 GA has a catalytic efficiency (kcat/Km) at 35°C and a specific activity at 50°C similar to that of wild‐type GA. Irreversible inactivation tests indicated that CR2‐1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol−1 compared with that of wild‐type GA. Thus, CR2‐1 is more thermostable (by 5 kJ mol−1 at 80°C) than the most thermostable A. niger GA variant previously described, THS8. In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol−1, respectively, at 80°C.

Small Angle X-ray Studies Reveal That Aspergillus niger Glucoamylase Has a Defined Extended Conformation and Can Form Dimers in Solution

The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.

In-depth analysis of the Aspergillus niger glucoamylase ( glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques

An in-depth characterization of the Aspergillus niger glucoamylase ( glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.

Thermodynamic Effects of Disulfide Bond on Thermal Unfolding of the Starch-Binding Domain ofAspergillus nigerGlucoamylase

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1/2 of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t1/2 of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.