Glucoamylase Research Articles

Modification of Ordered Macroporous Silica by a Functional Polymer Layer and Immobilization of Glucoamylase on the Macropore Walls

Modification of Ordered Macroporous Silica by a Functional Polymer Layer and Immobilization of Glucoamylase on the Macropore Walls

Sakarifikasi pati ubi kayu menggunakan amilase <i>Aspergillus niger</i> ITB CC L74

Saccharification of cassava starch using amilase Aspergillus niger ITB CC L74 Bioethanol is a derivative product from cassava that is thoroughly developed nowadays. One of the bioethanol research program of the Chemical Engineering Product Design and Development Research Group, Faculty of Industrial Technology, Institut Teknologi Bandung in the year of 2008 was to increase productivity and the performance of α-amylase and glucoamylase from Aspergillus niger ITB CC L74 for saccharification of cassava starch in bioethanol production. In conjunction with the optimum condition of saccharification process, research on the effect of pH, temperature, Ca2+ concentration, substrate concentration, and enzyme volume percentage have been carried out. The performance of α-amylase was determined by the iodine method while the performance of glucoamylase was done by the Somogyi-Nelson method. Research variable to determine the optimum performance condition of amylase extract were pH (3.15–7.0), temperature (30–89 oC), Ca2+ concentration (0–200 ppm), substrate concentration (0.5–20 %-w/v), and enzyme volume percentage (1.0–50 %-v). The results of this research showed that the optimum performance of amylase complex were pH 4.5, temperature 60 oC, Ca2+ concentration 75 m/L, and substrate concentration 7 %-w/v. The performances of amylase complex increased with the increase in the amount of enzyme percentage, but the increase was limited by the amount of glucose that could inhibit enzyme activity. Keywords: amylase, glucoamylase, Aspergillus niger, saccharification, starch AbstrakBioetanol merupakan produk turunan ubi kayu yang sekarang sedang giat dikembangkan. Salah satu program riset bioetanol Kelompok Keahlian Perancangan dan Pengembangan Produk Teknik Kimia, FTI, ITB tahun 2008 adalah peningkatan produktivitas dan kinerja enzim α-amilase dan glukoamilase Aspergillus niger ITBCC L74 untuk proses sakarifikasi pati ubi kayu pada produksi bioetanol. Sehubungan dengan kondisi optimum proses sakarifikasi, penelitian tentang pengaruh pH, temperatur, konsentrasi Ca2+, konsentrasi substrat, dan persentase volume enzim, telah dilakukan. Analisis kinerja kompleks amilase yang dilakukan meliputi analisis kinerja enzim α-amilase dengan metode iodin dan analisis kinerja enzim glukoamilase dengan metode Somogyi-Nelson. Variabel yang diteliti untuk menentukan kondisi optimum kinerja ekstrak amilase adalah pH (3,5–7,5), temperatur (25–80 oC), konsentrasi Ca2+ (25–200 ppm) konsentrasi substrat (0,5–20 %-b/v) dan persentase volume enzim (1-50 %-v). Hasil penelitian menunjukkan bahwa kinerja kompleks enzim amilase optimum berada pada pH 4,5, temperatur 60 oC, konsentrasi Ca2+ 75 ppm, dan konsentrasi substrat 7 %-b/v. Kinerja kompleks amilase makin baik seiring dengan peningkatan persentase volume enzim, namun peningkatan ini dibatasi oleh kandungan glukosa dalam enzim yang dapat menyebabkan inhibisi terhadap aktivitas enzim.Kata kunci: amilase, glukoamilase, Aspergillus niger, sakarifikasi, pati

Development of a novel bi-enzymatic silver dendritic hierarchical nanostructure cascade catalytic system for efficient conversion of starch into gluconic acid

The natural cascade processes lead to inspire researchers to bring diverse biocatalysts together in an artificial way. In this work, we are going to introduce a brilliant double enzyme microsystem prepared from co–immobilization of glucose oxidase (GOD) and glucoamylase (GA) on silver dendrites hierarchical (Ag–DH) nanostructure through Ugi four-component reaction (Ugi–4CR) in water, as the green solvent at ambient temperature. The success preparation of the unique biocatalyst system was confirmed by FT–IR, UV–Vis, TGA, XRD, TEM, FE–SEM, AFM and elemental analysis. The properties of free and immobilized enzymes were investigated and compared. The immobilized GA and GOD had higher Km (Michaelis constant) and lower Vmax (maximum reaction velocity) than their native forms. The values of activation energy (Ea) for both the immobilized enzymes were smaller than those with native enzymes, implying that the immobilized enzymes are more temperature insensitive. The obtained double enzyme microsystem was employed as a biocatalyst for one pot transformation of starch to gluconic acid as an exclusive cascade reaction under mild conditions and in aqueous medium, and the final product obtained in high yield. Moreover, the artificial biomimetic microsystem exhibited high stability and reusability after eight cycles. These results demonstrated the feasibility of this approach for co-immobilization of enzyme on hierarchical structures may be extended to other biocatalytic cascades, thereby opening a new window for the other artificial biotransformations in chemistry.

Co-Immobilization of pullulanase, glucoamylase and glucose isomerase together with cross-linked enzyme aggregates: Fructose conversion from three enzymes and starch in one step

In this study, in order to obtain fructose syrup from starch, pullulanase (PUL), glucoamylase (GA) and glucose isomerase (GI) were immobilized together with cross-linked enzyme aggregates (CLEAs), and named as 'combi-CLEAs'. The values of Vmaxand Kmfrom the kinetic parameters of combi-CLEAs [(GA + PUL) + GI] were found as 12 U g-1 and 0.1157 mg ml-1 in optimum conditions, respectively. After 50 uses, it was observed that the remaining activity of combi-CLEAs [GA + PUL + GI] was preserved at 46%. The thermal stability of combi-CLEAs [GA + PUL) + GI] was observed to be 44% and 30%, respectively, at 60 and 80 ° C after 24 hours. At the end of 30 days, the storage stabilities of combi-CLEAs [(GA + PUL) + GI] was determined as 56% and 13% at the temperatures of 4 and 25°C, respectively.

Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.

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.

Static and dynamic half-life and lifetime molecular turnover of enzymes

The static half-life of an enzyme is the half-life of a free enzyme not working without substrate and the dynamic half-life is that of an active enzyme working with plenty amount of substrate. These two half-lives were measured and compared for glucoamylase (GA) and β-galactosidase (BG). The dynamic half-life was much longer than the static half-life by one to three orders of magnitude for both enzymes. For BG, the half-life of the enzyme physically entrapped in a membrane reactor was also measured. In this case also, the half-life of BG in the membrane reactor was much longer than the free enzyme without substrate. These results suggest the large difference in stabilities between the free enzyme and the enzyme-substrate complex. This may be related to the natural enzyme metabolism. According to the difference in half-life, the lifetime molecular turnover (LMT), which is the number of product molecules produced by a single molecule of enzyme until it loses its activity completely, was much higher by one to four orders of magnitude for the active enzyme than the free enzyme. The concept of LMT, proposed here, will be important in bioreactor operations with or without immobilization.

Microfluidic Distance Readout Sweet Hydrogel Integrated Paper-Based Analytical Device (μDiSH-PAD) for Visual Quantitative Point-of-Care Testing.

A disposable, equipment-free, versatile point-of-care testing platform, microfluidic distance readout sweet hydrogel integrated paper-based analytical device (μDiSH-PAD), was developed for portable quantitative detection of different types of targets. The platform relies on a target-responsive aptamer cross-linked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and microfluidic paper-based analytic devices (μPADs) for visual distance-based quantitative readout. A "sweet" hydrogel with trapped glucoamylase (GA) was synthesized using an aptamer as a cross-linker. When target is present in the sample, the "sweet" hydrogel collapses and releases enzyme GA into the sample, generating glucose by amylolysis. A hydrophilic channel on the μPADs is modified with glucose oxidase (GOx) and colorless 3,3'-diaminobenzidine (DAB) as the substrate. When glucose travels along the channel by capillary action, it is converted to H2O2 by GOx. In addition, DAB is converted into brown insoluble poly-3,3'-diaminobenzidine [poly(DAB)] by horseradish peroxidase, producing a visible brown bar, whose length is positively correlated to the concentration of targets. The distance-based visual quantitative platform can detect cocaine in urine with high selectivity, sensitivity, and accuracy. Because the target-induced cascade reaction is triggered by aptamer/target recognition, this method is widely suitable for different kinds of targets. With the advantages of low cost, ease of operation, general applicability, and disposability with quantitative readout, the μDiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring, security inspection, personalized healthcare, and clinical diagnostics.

Application of the Electronic Nose on the Online Feedback Control of Methanol Concentration during Glucoamylase Fermentation Optimization by Pichia pastoris

Application of the Electronic Nose on the Online Feedback Control of Methanol Concentration during Glucoamylase Fermentation Optimization by Pichia pastoris

Development and assessment of a novel Arxula adeninivorans androgen screen (A-YAS) assay and its application in analysis of cattle urine

The novel A-YAS assay for the detection of androgenic activity in liquid samples such as urine has been developed and assessed. The assay is based on transgenic Arxula adeninivorans yeast cells as the bio-component. The cells were engineered to co-express the human androgen receptor (hAR) gene and the inducible phytase reporter gene (phyK, derived from Klebsiella sp. ASR1), under the control of an Arxula derived glucoamylase (GAA) promoter, which had been modified by the insertion of hormone-responsive elements (HREs). The Arxula transformation/expression platform Xplor®2 was used to select stable mitotic resistance marker free transformants and the most suitable cells were characterized for performance as a sensor bio-component. The assay is easy-to-use, fast (6–25h) and is currently the most sensitive yeast-based androgen screen with an EC50, limit of detection and of quantification values for 5α-dihydrotestosterone (DHT) of 277.1±53.0, 56.5±4.1 and 76.5±6.7ngL−1, respectively. Furthermore, the assay allows the determination of androgenic and anti-androgenic activity of various compounds such as naturally occurring androgens and estrogens, pharmaceuticals and biocides. The robustness of the A-YAS assay enables it to be used for analysis of complex samples such as urine. The results of the analysis of a number of cattle urine samples achieved by the A-YAS assay correlate well with GC-MS analysis of the same samples.

Introduction of additional thiol groups into glucoamylase in Aspergillus awamori and their effect on the thermal stability and catalytic activity of the enzyme

Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\A14C, 6D-A14C\Y419C\G137A, 10D-V13C\G396C, 11D-V13C\G396C\A14C\Y419C\G137A, and 20D-G137A\A246C\A14C) were constructed using computer simulation and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth alpha-helices and that of the loop located close to a catalytic residue--E400--made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\A14C, V13C\G396C\A14C\Y419C\G137A, and G137A\A246C\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\Y419C\G137A and V13C\G396C proteins led to the destabilization of their structure and the loss of thermal stability.

Co-immobilization of multi-enzyme on control-reduced graphene oxide by non-covalent bonds: an artificial biocatalytic system for the one-pot production of gluconic acid from starch

Here we report a simple and efficient approach to fabricate a new biocatalytic system by co-immobilizing multi-enzyme on chemically reduced graphene oxide (CRGO) via non-covalent bonds. The obtained artificial biocatalyst was characterized by UV/Vis, FTIR, AFM, TEM and SEM. Compared with native and graphene oxide (GO) bounded enzymes, it was found that the glucose oxidase (GOD) or glucoamylase (GA) immobilized on CRGO exhibited significantly higher enzymatic activity, due to the positive effect of the CRGO carrier. This multi-enzyme microsystem was employed as a biocatalyst to accomplish the starch-to-gluconic acid reaction in one pot, and the yield of gluconic acid could reach 82% within 2 hours. It was also proved that the stability of the multi-enzyme biocatalyst immobilized on CRGO was dramatically enhanced compared with the GO microsystem. About 85% of the activity of the artificial biocatalyst could be preserved after four cycles. These results demonstrated the feasibility of the novel strategy to construct bio-microsystems with multi-enzyme on 2D CRGO via non-covalent bonds to accomplish some complex conversions.

Production of fungal glucoamylase for glucose production from food waste.

The feasibility of using pastry waste as resource for glucoamylase (GA) production via solid state fermentation (SSF) was studied. The crude GA extract obtained was used for glucose production from mixed food waste. Our results showed that pastry waste could be used as a sole substrate for GA production. A maximal GA activity of 76.1 ± 6.1 U/mL was obtained at Day 10. The optimal pH and reaction temperature for the crude GA extract for hydrolysis were pH 5.5 and 55 °C, respectively. Under this condition, the half-life of the GA extract was 315.0 minutes with a deactivation constant (kd) 2.20 × 10−3 minutes−1. The application of the crude GA extract for mixed food waste hydrolysis and glucose production was successfully demonstrated. Approximately 53 g glucose was recovered from 100 g of mixed food waste in 1 h under the optimal digestion conditions, highlighting the potential of this approach as an alternative strategy for waste management and sustainable production of glucose applicable as carbon source in many biotechnological processes.

Co-immobilization of glucoamylase and glucose oxidase for electrochemical sequential enzyme electrode for starch biosensor and biofuel cell

A novel electrochemical sequential biosensor was constructed by co-immobilizing glucoamylase (GA) and glucose oxidase (GOD) on the multi-walled carbon nanotubes (MWNTs)-modified glassy carbon electrode (GCE) by chemical crosslinking method, where glutaraldehyde and bovine serum albumin was used as crosslinking and blocking agent, respectively. The proposed biosensor (GA/GOD/MWNTs/GCE) is capable of determining starch without using extra sensors such as Clark-type oxygen sensor or H2O2 sensor. The current linearly decreased with the increasing concentration of starch ranging from 0.005% to 0.7% (w/w) with the limit of detection of 0.003% (w/w) starch. The as-fabricated sequential biosensor can be applicable to the detection of the content of starch in real samples, which are in good accordance with traditional Fehling's titration. Finally, a stable starch/O2 biofuel cell was assembled using the GA/GOD/MWNTs/GCE as bioanode and laccase/MWNTs/GCE as biocathode, which exhibited open circuit voltage of ca. 0.53V and the maximum power density of 8.15μWcm−2 at 0.31V, comparable with the other glucose/O2 based biofuel cells reported recently. Therefore, the proposed biosensor exhibited attractive features such as good stability in weak acidic buffer, good operational stability, wide linear range and capable of determination of starch in real samples as well as optimal bioanode for the biofuel cell.

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.

EFFECT OF ADSORBENT pKa ON SELECTIVE REMOVAL OF GLUCOAMYLASE FROM UNPASTEURIZED SAKE MATERIALS

To selective adsorption of glucoamylase (GA) from the raw materials of the unpasteurized sake, various polymer beads were prepared by immobilization of cationic or anionic functional groups onto cellulose beads (Cellufine). Under conditions which mimic sake (GA 500 µg mL−1, ethanol 20 vol%, pH 4), Aspergillus species GA (As-GA) (pI 3.5) was strongly adsorbed by cationic Cellufines with anion-exchange capacity (AEC) of 0.5–1.3 meq g−1, but Rhizopus sp. GA (pI 6.5) was strongly adsorbed by anionic adsorbents with cation-exchange capacity of 0.3–1.1 meq g−1. The As-GA selectivity of the cationic Cellufines depended on their apparent pKa (pKa,app) but not necessarily on their AEC. The Cellufine-poly(ethyleneimine) (pKa,app 7.9, AEC 0.4 meq g−1) was effective at adsorbing As-GA (>99%) in a sake model solution (GA 250 µg ml−1, ethanol 20 vol%, 0.016 M acetate buffer, pH 4), but it also adsorbed organic acid, such as acetic acid. By contrast, Cellufine-poly(ϵ-lysine) (pKa,app 7.3–7.4, AEC 0.6–1.0 meq g−1) selectively removed the As-GA from the sake-model solution without adsorption of the organic acid; the residual As-GA concentration was lower than 1%, and the recovery of organic acid was more than 99% as total acidity.

Optimization of Cassava Starch Hydrolysis by Malt Extract, α-Amylase and Glucoamylase

Cassava starch hydrolysis was investigated in this study using malt extract, alpha amylase and glucoamylase. The effects of process variables, namely: temperature, pH and time were studied and optimized for hydrolysis of cassava (Manihot esculenta) flour to glucose syrup. Three levels of process variables were used for the study. The three levels of process variables were: temperature (60, 67 and 74 oC), time (1.5, 2.0 and 2.5 h) and pH (4.5, 5 and 5.5). A polynomial regression model was developed using the experimental data. The results showed that production of reducing sugar was strongly affected by the variation of variables on malt extract, alpha amylase and glucoamylase hydrolysis of cassava starch. The fit of the model was expressed by the coefficient of determination R2 which was found to be 0.987 indicating that 98.7 % of the variability in the response can be explained by the model. The value also indicates that only 1.3% of the total variation is not explained by the model. This shows that equation (2) is a suitable model to describe the response of the experiment pertaining to reducing sugar production. The statistical significance of the model was validated by F-test for analysis of variance (p ≤ 0.05). For malt extract, alpha amylase and glucoamylase hydrolysis, the optimum value of temperature, time and pH were found to be 74 oC; pH 5.5 and time 1.5 h. The maximum reducing sugar production at optimum condition was 268 g/l representing 76.57 % conversion or 76.57 dextrose equivalent (DE)

Efficient saccharification for non-treated cassava pulp by supplementation of Clostridium thermocellum cellulosome and Thermoanaerobacter brockii β-glucosidase

Cassava pulp containing 60% starch and 20% cellulose is a promising renewable source for bioethanol. The starch granule was observed to tightly bind cellulose fiber. To achieve an efficient degradation for cassava pulp, saccharification tests without pre-gelatinization treatment were carried out using combination of commercial α-amylase with cellulosome from Clostridium thermocellum S14 and β-glucosidase (rCglT) from Thermoanaerobacter brockii. The saccharification rate for cassava pulp was shown 59% of dry matter. To obtain maximum saccharification rate, glucoamylase (GA) from C. thermocellum S14 was supplemented to the combination. The result showed gradual increase in the saccharification rate to 74% (dry matter). Supplementation of GA to the combination of commercial α-amylase, cellulosome and rCglT is powerful method for efficient saccharification of cassava pulp without pretreatment.

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of "starch" dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7mg/g from 2.0mg/mL enzyme solution at pH4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55°C and pH4.5 compared to free enzyme (50°C and pH5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.

Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins

A procedure for determination of apparent affinity constants KDapp between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10−7 to 9×10−6M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.