Glucoamylase Production Research Articles

Novel Fungal Co-Culture Technique for Enhanced Bioconversion of Agro-Industrial Waste to Amylase

Global strategies for management of solid waste occasionally lead to the environmental pollution. Now a days environmental friendly methods to valorize this waste are more demand to reduce the global warming. Solid-state fermentation (SSF) which is a potential waste recycling method to convert these solid wastes into value-added products by microbial population. In the present study, SSF was carried out using four substrates namely castor husk, rice husk, groundnut fodder, sugarcane bagasse and saw dust for the selection of renewable and chief substrate for the industrial production of amylase enzyme. We used two indigenous strains i.e., Aspergillus protuberus and Aspergillus unguis. Maximum production of α-amylase 1.614 U/g of substrate and 0.958 U/g of substrate on 2nd day of incubation in rice husk respectively. Groundnut waste (0.847 U/g of substrate) and castor husk (0.692 U/g of substrate) were also showed highest production of glucoamylase on 1st day and 2nd day of incubation in SSF. Further, prominent increase in the production of α-amylase (12 U/ml) and glucoamylase (3 U/ml) and extra cellular secretion of protein (20 mg/ml) was noticed in co-culture system on 2nd day of incubation in submerged fermentation (SmF).

Optimization of nutrient media for glucoamylase production by Plackett-Burman design using Aspergillus niger

Background: Glucoamylase is an important enzyme produced by various bacterial and fungal species as an intracellular or extra cellular enzyme. It is used to treat gastrointestinal issues, autoimmune diseases, inflammation, psoriasis, allergies, eczema, emphysema and asthma, and thus, supplements are often included in complexes for digestive disorders and also applications in different industries. Methods: Natural ingredients are screened for glucoamylase production by stationary culture of solid-state fermentation technique and Plackett-Burman design microorganism used Aspergillus niger. Eight nutrient medias are designed and from this glucoamylase isolated from the medium by centrifugation at 1500 rpm and assayed by spectrophotometric method. Results: high glucoamylase levels are found in nutrient media VI with optimized physical conditions like percentage inoculum- 2%, pH- 6, incubation temperature- 400C upon incubation for 4 days. Conclusions: High amount of glucoamylase is produced in the media VI due to the presence of high amounts of sucrose and optimum concentration of potato, peptone, wheat bran and yeast extract.

Effect of pulsed electric field on spore germination rate and enzyme activity of Aspergillus niger

Pulsed electric field (PEF) treatment on Aspergillus niger, which is an important source of several enzymes, was conducted to investigate the effects of different PEF parameters on spore germination of A. niger and the production of lipase and glucoamylase. The results showed that the spore germination rate and the relative activities of lipase and glucoamylase could be promoted when the electric field strength was in the range of 5– 15 kV/cm and the treatment time was in the range of 1– 7.5 ms. During the processing, the concentration of spores was maintained within a range of approximately 3.5 × 106 CFU/mL. Maximum increase of spore germination rate, lipase and glucoamylase activities were 217%, 25.6% and 31.3% respectively, which were observed when the spore suspension was subjected to PEF treatment with 10 kV/cm field strength for 5 ms. Lipase activity was inhibited with the increase of field strength and treatment time. Further analysis of the input energy density during PEF showed that the spore germination rate and the relative activities of enzymes were also promoted and then inhibited with increasing input energy density, but the electric field strength was more decisive. Industrial relevancePEF is an emerging non-thermal microbial treatment technology with the characteristics of short treatment time and low energy consumption. Aspergillus niger is an important microbial species in the food industry, as the enzymes it produces play an important role in various fields. This study applied PEF technology to A. niger and significantly promoted the germination of its spores, and the activity of its lipase and glucoamylase, which suggests PEF technology has the potential to be used to shorten the industrial production cycle of A. niger and its enzyme products.

Effect of Gamma Irradiation on Aspergillus tamarii FNCC 6151 to enhance Alpha-Amylase and Glucoamylase Production

Molds can carry out amylase production, but the production of the enzyme is affected by a carbon catabolite repression system, thus reducing amylase production. This study aimed to increase amylase production in Aspergillus tamarii FNCC 6151 with irradiation by gamma rays. Spore suspensions were irradiated at 100, 200, 300, 400 and 500 Gy and the survival of the spores was determined. The spore suspension irradiated at 400 Gy was diluted and grown on medium plates containing starch as a carbon source and 2% glucose as a repressor. After staining with iodine, colonies showing clear zones around them were purified and grown in liquid media for four days and their alpha-amylase and glucoamylase activities were analyzed. The mutant Mut1 showed a clear zone. This mutant produced 1.127 U/mL of alpha-amylase and 0.011 U/mL of glucoamylase. Meanwhile, the parental strain produced 0.68 U/mL of alpha-amylase and 0.079 U/mL of glucoamylase. The random mutagenesis using gamma-ray into A. tamarii FNCC 6151 increased alpha-amylase and glucoamylase production by 1.8- and 1.4-fold respectively.

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

BackgroundOur recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction.ResultsBased on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction.ConclusionThis study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

Production of Glucoamylase from Novel Strain of Alternaria Alternata under Solid State Fermentation.

Glucoamylase has an essential role as biocatalyst in various important industries of Pakistan. It is synthesized by using various fungal and bacterial strains, and different ecocultural conditions are applied under solid substrate fermentation method (SSF) to get the highest yield of glucoamylase. Alternaria alternata is an important fungus that can grow on industrial raw material like wheat bran, dried potato powder, tea leaves, rice husk, and sugar cane peel which are used as substrate. Among all, dried potato powder (10g) proved the best fermentation media for growth of fungal strain as well as maximum glucoamylase producer. Moreover, several chemical and physical states were also explored through solid substrate fermentation technique on glucoamylase yield. The highest glucoamylase production was recorded after 72 hours incubation in incubation chamber with 10g raw substrate, 1ml inoculum spore solution, 30°C temperature, and 5 pH. Further, phosphate buffer (5 pH) as moistening agent, 5% starch concentration and media additive as nitrogen (yeast extract), and carbon source (maltose) were screened for maximum glucoamylase titer (17.3 ± 0.05a°U/ml/min) and the highest specific activity (39.2U/mg). These cultural conditions were most appropriate for growth of A. alternata on solid media and production of highest glucoamylase under solid state fermentation procedure that could be utilized for commercial synthesis of glucoamylase.

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger.

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.

Effects of glucose as carbon catabolite repressor on alpha-amylase and glucoamylase production in Indonesian indigenous fungi

Effects of glucose as carbon catabolite repressor on alpha-amylase and glucoamylase production in Indonesian indigenous fungi

Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

Statistical approach of Ca2+ effect on the amylases production by Saccharomyces diastaticus ATCC 13007 using corn and cassava starch as inducers

Amylase production from starch sources is associated with the nature of this carbon source, particle size and crystalline architecture of natural starch granules. The aim of this work was to evaluated the amylase production by Saccharomyces diastaticus ATCC 13007, using different CaCO3 concentration and corn starch (Zea mays) and cassava starch (Manihot esculenta) as inducers. A 22 Central Composite Rotatable Design with three replications at the center point was used to determine the influence of calcium and starch concentrations on the production process. Besides, the partial characterization of the crude enzymatic extracts obtained was investigated. Ca2+ influenced the α-amylase production by both evaluated carbon sources. The production of glucoamylase was not influenced by the variables under study. Partial characterization of crude extracts indicated that, for corn substrate, the enzyme showed maximum activity at pH 5.0, and no influence of temperature was observed. Regarding cassava starch, the results showed that conditions at central point (pH 6.5 and 50ºC) should be avoided in order to maximize enzyme activity.

Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification

BackgroundStarch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase.ResultsIn this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%.ConclusionsOur study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.

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

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

Optimization of α‐amylase and Glucoamylase Production Using Cull Potatoes as Substrate

AbstractAmylases are required for the conversion of starchy substrate into sugars. Commercially available enzymes are quite costly which makes the process uneconomical. This study is conducted to standardize physico‐chemical parameters such as substrate concentration (1.0–5.0%), temperature (25–40 °C), pH (4.0–7.0), incubation time (18–24) h for α‐amylase and 3–6 days for glucoamylase production) for optimum production of α‐amylase from bacterial isolate (AM1) and glucoamylase from Aspergillus niger by using cull potatoes as a substrate. Various organic and inorganic nitrogen sources (urea, yeast extract and soybean meal, and ammonium sulphate) are tried for maximum enzyme production. Among the four nitrogen sources, yeast extract supported maximum α‐amylase and glucoamylase production. Among the different amylolytic isolates from fermented jalebi dough and AM1culture, maximum α‐amylase activity is shown by AM1 (90.46 U mL−1) as compared to J1 (71.02 U mL−1). After optimization, α‐amylase activity of AM1 increases by 41.73% from 198.20 to 280.92 U mL−1 at 5.0% substrate concentration, 30 °C temperature, 24 h of incubation and pH 5.0. Glucoamylase activity of A. niger increases by 38.78% from 228.72 to 317.44 U mL−1 after optimization at 5% substrate concentration, pH 5, incubation period of 4 days and temperature 30 °C.

Renewable Plant Waste As Substrates for Enzyme Production, Saccharification and Direct Bioethanol Production by Indigenous Yeast Strain Pichia Exigua

Renewable plant wastes constitute environmental nuisance. Their conversion by enzymes into bioethanol can be beneficial. We investigated the use of renewable plant waste as substrate for enzyme production and hydrolysis of the plant waste for ethanol production using an indigenous yeast strain. Five yeast strains; MCC-1, MCC-2, MCC-3, MCC-4 and MCC-5 were evaluated for production of sugars, α-amylase, glucoamylase and bioethanol using soluble starch. Phylogenetic analysis using partial sequence of the ITS gene classified MCC-4 as Pichia exigua. Proximate composition of plant wastes – cassava, wild yam, mango seed, udara seed and breadfruit were determined. Results showed total carbohydrate of (83.9%) for cassava flour. The ability of yeast to utilize these substrates and the effect of culture conditions (inoculum, pH, nitrogen source and substrate concentration) were also determined. Cassava pulp flour was the best substrate producing reducing sugar (1.471 ± 0.056mg/mL), α-amylase (0.573 ± 0.019U/mL), glucoamylase (1.605 ± 0.119U/mL), and ethanol (4.440 ± 0.014g/L). Culture conditions revealed optimum for inoculum concentration as (1mL), pH (4), nitrogen source (soya bean, 3g/L) and substrate concentration of (8%). Pichia exigua (MCC-4) a natural yeast strain isolated from the soil has the potential for both enzyme and ethanol production in a single step process.

Dynamic response of Aspergillus niger to periodical glucose pulse stimuli in chemostat cultures.

In industrial large-scale bioreactors, microorganisms encounter heterogeneous substrate concentration conditions, which can impact growth or product formation. Here we carried out an extended (12 h) experiment of repeated glucose pulsing with a 10-minperiod to simulate fluctuating glucose concentrations with Aspergillus niger producing glucoamylase, and investigated its dynamic response by rapid sampling and quantitative metabolomics. The 10-minperiod represents worst-case conditions, as in industrial bioreactors the average cycling duration is usually in the order of 1 min. We found that cell growth and the glucoamylase productivity were not significantly affected, despite striking metabolomic dynamics. Periodical dynamic responses were found across all central carbon metabolism pathways, with different time scales, and the frequently reported ATP paradox was confirmed for this A. niger strain under the dynamic conditions. A thermodynamics analysis revealed that several reactions of the central carbon metabolism remained in equilibrium even under periodical dynamic conditions. The dynamic response profiles of the intracellular metabolites did not change during the pulse exposure, showing no significant adaptation of the strain to the more than 60 perturbation cycles applied. The apparent high tolerance of the glucoamylase producing A. niger strain for extreme variations in the glucose availability presents valuable information for the design of robust industrial microbial hosts.

Biodiesel production using microbial lipids derived from food waste discarded by catering services

Biodiesel production using microbial oil derived from food waste discarded by the hospilatity sector could provide a sustainable replacement for diesel fuel. Discarded potato peels were used in solid-state fermentations of Aspergillus awamori for the production of glucoamylase (30 U/g) and protease (50 U/g). Hospitality food waste hydrolysis led to 98% (w/w) starch to glucose conversion yields. Crude hydrolyzates were used in shake flask fermentations with the oleaginous yeast Rhodosporidium toruloides Y-27012 leading to 32.9 g/L total dry weight (TDW) with 36.4% (w/w) intracellular lipid content. Fed-bath bioreactor cultures resulted in TDW of 53.9 g/L and lipid concentration of 26.7 g/L. Principal component analysis showed a fatty acid profile similar to soybean oil and solid food waste oil. Microbial oil was transesterified into biodiesel with satisfactory performance considering the European standard EN 14214. This work demonstrated that valorization of food waste for biodiesel production is feasible.

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

BackgroundNicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction.ResultsWe thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production.ConclusionsThis data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

Optimisation of fungal glucoamylase production by Response Surface Methodology and characterisation of the purified enzyme

A statistical approach was made for the production of glucoamylase by Aspergillus niger using wheat bran and green gram as fermentation medium. Box-Behnken design used to analyse the simultaneous impact of the substrate, hydration, inoculum volume, fermentation time and temperature, using a second-order polynomial. The experimental results were in good agreement with the proposed regression model with R2 = 0.9322 (p<0.05). The maximum yield (313 U/gds against the predicted value of 306 U/gds) was obtained using 12 gm substrate, 55% moisture, 1 gm sucrose and 0.05 gm tryptone. The purification and characterisation of the enzyme were also studied. Optimum thermal and pH stability was 60 °C and 5 respectively.

Solid State Fermentation of Wheat Bran for Production of Glucoamylase by Aspergillus niger

Solid State Fermentation of Wheat Bran for Production of Glucoamylase by Aspergillus niger

Production of Glucoamylase, α-amylase and Cellulase by Aspergillus oryzae F-923 Cultivated on Wheat Bran under Solid State Fermentation

Filamentous fungi have been widely utilized in production of enzymes which have many industrial applications. In this study, twenty five local fungal isolates, belonging to Aspergillus sp., Trichoderma sp. and Penicillium sp., were screened under solid state fermentation conditions (SSF) for the production of α-amylase, glucoamylase and cellulase. Asperigillus oryzae F-923, cultivated on wheat bran, was the most promising isolate for production of the target enzymes under this study. Physical parameters of moisture content, pH, temperature and incubation time, optimized were 1:2(w/v), 5.5, 28°C and 72 hr, respectively. The production of enzymes was enhanced when ammonium sulfate was supplemented as a nitrogen source to wheat bran. The production of α-amylase and glucoamylase was also enhanced when 10% (w/w) soluble starch was added as a carbon source to wheat bran. However xylose supplementation at 10% (w/w) was observed to be best for cellulase production. Tap water was found to be efficient for enzymes' extraction from the fermentation medium. Three successive extractions were needed to obtain the produced enzymes from the fermented substrate. Characterization of the produced enzymes revealed that, the optimum temperature for α-amylase and glucoamylase was 60°C, while 50°C was the optimum temperature for cellulase activity. Isopropanol 1:1(v/v) was proved to be more suitable for partial purification of enzymes. Following partial purification of enzymes of glucoamylase, α-amylase and cellulase increased to 10.8, 11.8 and 11.4 folds, respectively.