Fungal Glucoamylase Research Articles

Biochemical Characterization of a Novel Thermostable 1,4-α-Glucoamylase from Aspergillus brasiliensis Strain Isolated in the Brazilian Atlantic Forest.

Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60°C. GlaAb was stable at 50°C for 7h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21mgmL-1, Vmax of 155 U mg-1, Kcat 179s-1, and Kcat/Km 81.06mgmL-1s-1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process.

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.

Novel glucoamylase-resistant gluco-oligosaccharides with adjacent α-1, 6 branches at the non-reducing end discovered in Japanese rice wine, sake

Sake, a traditional Japanese rice wine, contains various oligosaccharides (Sake oligosaccharides; SAOs) derived from rice starch. We previously found that SAOs reach a high degree of polymerization (DP). In this study, we developed a hydrophilic interaction liquid chromatography-time-of-flight/mass spectrometry (HILIC-TOF/MS) based analytical method to separate isomeric SAOs. Isomers of SAOs with DP = 6, 7, and 8, which were named DP6-1, DP7-1, DP8-1 and DP8-2, respectively, were purified from sake and their structures were determined by two-dimensional NMR spectroscopy. These were novel oligosaccharides containing two α-1, 6 bonded branches on an α-1, 4-linked glucose main chain. Interestingly, adjacent double α-1, 6 branches that have not been identified in starch, were found in DP6-1, DP7-1, and DP8-1, suggesting the presence of the branching pattern in starch. DP6-1 was poorly digested by fungal glucoamylase, and this may be attributed to its adjacent double branches at the non-reducing end.

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.

INVESTIGATION OF THE EXPEDIENCE OF MODIFICATION OF THE CARBOHYDRATE COMPOSITION OF RICE FLOUR IN THE TECHNOLOGY OF GLUTEN-FREE BREAD

The research considers a possibility of using modification of the carbohydrate composition of rice flour in the technology of bread of the special destination for celiachia. There has been conducted an analysis of the condition of the carbohydrate-amylase complex of rice flour. Revealed regularities have demonstrated that this flour has an unessential amylolytic activity that negatively influences the course of microbiological processes in gluten-free dough and quality parameters of ready products. The aim of the research was to develop arrangements as to accumulation of sugars in dough at the expanse of own reserves of rice bread for providing the necessary intensity of rice dough fermentation by using enzymes of the amylolytic effect. It is a precondition for improving structural-mechanical and organoleptic properties of gluten-free rice bread. At the same time accumulation of products of incomplete hydrolysis of flour starch in the process of its fermentative modification – dextrins will favor deceleration of ready products staling. There has been established the influence of α-amylase of the fungal origin and glucoamylase on accumulation of sugars at hydrolysis of rice flour starch. It has been studied, that dosage of α-amylase in amount 0,005 % and glucoamylase – 0,03 % of a flour mass results in creating sugars in amount 5,5–6 %. There has been proved the effectiveness of using α-amylase by accumulation of dextrins, in which composition the essential raise of the content of low-molecular ones – archo- and maltodextrins has been determined. Based on the conducted studies, it has been determined, that hydrolysis products of rice flour starch are created as a result of its fermentative modification by α-amylase and glycoamylase that is testified by the increase of gas-creation and acid-accumulation. There has been observed the positive influence of using modification of the carbohydrate composition of flour in the technology of rice bread on the specific volume, porosity structure and flexible-elastic properties of crumb of ready products. It has been established, that realization of hydrolysis of flour starch at producing rice bread favors prolongation of its fresh storage term as a result of raising the amount of dextrins, created under the effect of α-amylase.

Structural insight into industrially relevant glucoamylases: flexible positions of starch-binding domains.

Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.

Optimization of gluco-amylase production from Aspergillus spp. for its use in saccharification of liquefied corn starch.

Fungal gluco-amylase is required for the production of sugars from starchy substrates. Commercially available fungal gluco-amylase is quite costly which makes the process uneconomical. This study was undertaken to standardize physico-chemical parameters for optimum production of gluco-amylases from Aspergillus spp. Two fungal cultures, i.e., Aspergillus niger and Aspergillus terreus, were compared for gluco-amylase activity both under stationary and shake flask conditions. Among two fungal cultures, maximum gluco-amylase activity was shown by A. niger (243.09U/ml) under stationary conditions as compared to A. terreus (126.34U/ml). Gluco-amylase activity of A. niger increases by 42.48% from 243.09 to 346.35U/ml after optimization using response surface methodology, whereby a substrate concentration of 7%, yeast extract 0.25%, temperature 32.5°C and pH 5.5 were found to be optimum for gluco-amylase production. Crude enzyme was compared with commercial enzyme and it was found that when 500U of Glucoamylase ex. Rhizopus were inoculated into starch-supplemented minimal media (SSMM) liquefied using 2g of fungal diastase, it increases the reducing sugar concentration from 2.19to 21.15mg/ml and a saccharification efficiency of 77.7% was achieved, whereas 1.5ml of crude enzyme (extracted from A. niger) was able to produce 14.46mg/ml of reducing sugars with a saccharification efficiency of 53.2%.

Bioprospection and characterization of the amylolytic activity by filamentous fungi from Brazilian Atlantic Forest

Abstract Filamentous fungi are widely diverse and ubiquitous organisms. Such biodiversity is barely known, making room for a great potential still to be discovered, especially in tropical environments - which are favorable to growth and species variety. Filamentous fungi are extensively applied to the production of industrial enzymes, such as the amylases. This class of enzymes acts in the hydrolysis of starch to glucose or maltooligosaccharides. In this work twenty-five filamentous fungi were isolated from samples of decomposing material collected in the Brazilian Atlantic Forest. The two best amylase producers were identified as Aspergillus brasiliensis and Rhizopus oryzae. Both are mesophilic, they grow well in organic nitrogen-rich media produce great amounts of glucoamylases. The enzymes of A. brasiliensis and R. oryzae are different, possibly because of their phylogenetical distance. The best amylase production of A. brasiliensis occurred during 120 hours with initial pH of 7.5; it had a better activity in the pH range of 3.5-5.0 and at 60-75°C. Both fungal glucoamylase had wide pH stability (3-8) and were activated by Mn2+. R. oryzae best production occurred in 96 hours and at pH 6.5. Its amylases had a greater activity in the pH range of 4.0-5.5 and temperature at 50-65ºC. The most significant difference between the enzymes produced by both fungi is the resistance to thermal denaturation: A. brasiliensis glucoamylase had a T50 of 60 minutes at 70ºC. The R. oryzae glucoamylase only had a residual activity when incubated at 50°C with a 12 min T50.

Purification and characterization of a novel cold adapted fungal glucoamylase

BackgroundAmylases are used in various industrial processes and a key requirement for the efficiency of these processes is the use of enzymes with high catalytic activity at ambient temperature. Unfortunately, most amylases isolated from bacteria and filamentous fungi have optimal activity above 45 °C and low pH. For example, the most commonly used industrial glucoamylases, a type of amylase that degrades starch to glucose, are produced by Aspergillus strains displaying optimal activities at 45–60 °C. Thus, isolating new amylases with optimal activity at ambient temperature is essential for improving industrial processes. In this report, a glucoamylase secreted by the cold-adapted yeast Tetracladium sp. was isolated and biochemically characterized.ResultsThe effects of physicochemical parameters on enzyme activity were analyzed, and pH and temperature were found to be key factors modulating the glucoamylase activity. The optimal conditions for enzyme activity were 30 °C and pH 6.0, and the Km and kcat using soluble starch as substrate were 4.5 g/L and 45 min−1, respectively. Possible amylase or glucoamylase encoding genes were identified, and their transcript levels using glucose or soluble starch as the sole carbon source were analyzed. Transcription levels were highest in medium supplemented with soluble starch for the potential glucoamylase encoding gene. Comparison of the structural model of the identified Tetracladium sp. glucoamylase with the solved structure of the Hypocrea jecorina glucoamylase revealed unique structural features that may explain the thermal lability of the glucoamylase from Tetracladium sp.ConclusionThe glucoamylase secreted by Tetracladium sp. is a novel cold-adapted enzyme and its properties should render this enzyme suitable for use in industrial processes that require cold-active amylases, such as biofuel production.

Starch Biorefinery Enzymes.

Nature uses enzymes to build and convert biomass; mankind uses the same enzymes and produces them on a large scale to make optimum use of biomass in biorefineries. Bacterial α-amylases and fungal glucoamylases have been the workhorses of starch biorefineries for many decades. Pullulanases were introduced in the 1980s. Proteases, cellulases, hemicellulases, and phytases have been on the market for a few years as process aids, improving yields, performance, and costs. Detailed studies of the complex chemical structures of biomass and of the physicochemical limitations of industrial biorefineries have led enzyme developers to produce novel tailor-made solutions for improving yield and profitability in the industry. This chapter reviews the development of enzyme applications in the major starch biorefining processes.

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.

Small intestine mucosal α-glucosidase: A missing feature of in vitro starch digestibility

The quality of starch digestion related to the rate and extent of glucose release is associated with high glycemia-related health issues such as diabetes and other metabolic syndromes. In humans, two types of digestive enzymes, α-amylase and mucosal α-glucosidase, are involved in breaking down starch molecules to absorbable glucose. To evaluate starch digestion in vitro, which is an affordable and efficient way to examine the nutritional quality of starchy foods during the process of product development, it is common to use a combination of porcine α-amylase and fungal glucoamylase to determine the digestibility. Fungal glucoamylase converts α-amylase hydrolyzates to glucose and directly hydrolyzes starch molecules to glucose. Although its functions look similar to human small intestinal α-glucosidase, its hydrolytic mechanism and influence in physiologic responses are quite different. In this article, we review the structure and properties of the small intestine α-glucosidases, including their protein structure, hydrolytic activity, digestion capability, distribution in the small intestine, and the dietary influence in their activity and synthesis.

Reverse micellar extraction of fungal glucoamylase produced in solid-state fermentation culture.

Partial purification of glucoamylase from solid-state fermentation culture was, firstly, investigated by reverse micellar extraction (RME). To avoid back extraction problems, the glucoamylase was kept in the original aqueous phase, while the other undesired proteins/ enzymes were moved to the reverse micellar organic phase. The individual and interaction effects of main factors (i.e., pH and NaCl concentration in the aqueous phase, and concentration of sodium bis-2-ethyl-hexyl-sulfosuccinate (AOT) in the organic phase) were studied using response surface methodology. The optimum conditions for the maximum recovery of the enzyme were pH 2.75, 100 mM NaCl, and 200 mM AOT. Furthermore, the optimum organic to aqueous volume ratio (Vorg/Vaq) and appropriate number of sequential extraction stages were 2 and 3, respectively. Finally, 60% of the undesired enzymes including proteases and xylanases were removed from the aqueous phase, while 140% of glucoamylase activity was recovered in the aqueous phase and the purification factor of glucoamylase was found to be 3.0-fold.

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.

Suppression of Fusarium graminearium growth by differently structured starch types

AbstractWe investigated the growth behavior and amylolytic enzymes of Fusarium graminearum cultivated on different types of native starch granules including barley (A‐type crystalline polymorph), potato and Curcuma zedoaria (B‐type crystalline polymorph), cassava (C‐type crystalline polymorph), and high AM maize (A + Vh‐type crystalline polymorphs). F. graminearum grew poorly on B‐type starches and the accumulation of biomass was similar to that obtained for fungi cultivated under carbohydrate starvation conditions. In comparison, three‐ to fivefold higher accumulation of fungal biomass was observed for growth on the A‐, C‐ and A + Vh‐type starches. Fungal glucoamylase and α‐amylase activity increased over time in the presence of native starch granules. Interestingly, resistant B‐type starches induced the highest amylolytic activity indicating that F. graminearum interacts with B‐type granules although only limited degradation occur. Starch degradation products maltose and malto‐oligosacharides was found to increase glucoamylase and α‐amylase activity, whereas glucose acted as a catabolite repressor.

Activated Charcoal—A Potential Material in Glucoamylase Recovery

The potential of activated charcoal in the purification of fungal glucoamylase was investigated. Various concentrations of activated charcoal (1–4% w/v) were used to concentrate crude glucoamylase from Rhizopus oligosporus at different temperature values (30–50°C). Effects of pH (3.0–6.0) and contact time (0–60 min) on enzyme purification were also monitored. Activated charcoal (3% w/v) gave a 16-fold purification in a single-step purification at 50°C for 20 min and pH 5.5. The result of SDS-PAGE analysis of purified glucoamylase showed two major protein bands with corresponding molecular weight of 36 kDa and 50 kDa. The method is inexpensive, rapid, and simple which could facilitate downstream processing of industrial enzyme.

English

A new amylase gene APGA1 was cloned from Aureobasidium pullulans NRRL 12974 and expressed in Pichia pastoris . This is the first report on cloning and expression of amylolytic gene from the industrially important microorganism A. pullulans . The purified recombinant protein with MW of 66 kDa and specific activity of 298.02 Umg -1 protein was verified as a glucoamylase by its hydrolytic mode. This recombinant glucoamylase with optimal pH of 4.5, and temperature of 60°C, showed good hydrolytic activity against raw potato starch. At 60°C, 83.1% of raw potato starch slurry (150 gl -1 ) was hydrolysed into glucose by 0.1 U mg -1 starch purified recombinant glucoamylase in less than 2.5 h. This is the highest raw starch hydrolysis efficiency report about recombinant fungal glucoamylase. This useful property indicated that this glucoamylase may find important applications in the starch saccharification industry and in bioethanol production. Key words: Glucoamylase, Aureobasidium pullulans, expression, raw starch degradation.

Glucoamylases: structural and biotechnological aspects

Glucoamylases, one of the main types of enzymes involved in starch hydrolysis, are exo-acting enzymes that release consecutive glucose units from the non-reducing ends of starch molecules. Glucoamylases are microbial enzymes, present in bacteria, archaea, and fungi but not in plants and animals. Structurally, they are classified in family 15 of glycoside hydrolases and characterised by the invariable presence of a catalytic domain with (α/α)₆-fold, often bound to a non-catalytic domain of diverse origin and function. Fungal glucoamylases are biotechnologically very important as they are used industrially in large amounts and have been extensively studied during the past 30 years. Prokaryotic glucoamylases are of biotechnological relevance for being generally thermophilic enzymes, active at elevated temperatures.

The novel proapoptotic activity of nonnatural enantiomer of Lentiginosine

D-(-)-Lentiginosine [(-)-4], the nonnatural enantiomer of the iminosugar indolizidine alkaloid L-(+)-lentiginosine, acts as apoptosis inducer on tumor cells of different origin, in contrast to its natural enantiomer. Although D-(-)-4 exhibited a proapoptotic activity towards tumor cells at level lower than the chemotherapeutic agent, SN38, it was less proapoptotic towards normal cells and less cytotoxic. Apoptosis induced by D-(-)-4 was caspase-dependent, as shown by the increased expression and activity of caspase-3 and -8 in treated cells, and by inhibition following treatment with the pan caspase inhibitor, ZVAD-FMK. This study highlighted how a natural iminosugar alkaloid and its synthetic enantiomer, which were simply known for their inhibition against a fungal glucoamylase, could behave in a complete different way when tested towards cell growth and death of cells of different origin.

Airlift-driven fibrous-bed bioreactor for continuous production of glucoamylase using immobilized recombinant yeast cells

Continuous production of a fungal glucoamylase by immobilized recombinant Saccharomyces cerevisiae strain C468 containing plasmid pGAC9. Yeast cells were immobilized on hydrophilic cotton cloth in an inverse internal loop airlift-driven bioreactor. Free-cell culture in the airlift and stirred tank bioreactors confirmed the plasmid instability of the recombinant yeast. Enhanced glucoamylase productivity and plasmid stability were observed both in the free and immobilized cell cultures in the airlift bioreactor system. The glucoamylase level of the free-cell culture in the airlift bioreactor was ∼20% higher than that in the in stirred tank bioreactor due to high cell density (cell dry weight/volume of bioreactor) and fraction of the plasmid-carrying cells. A potentially high glucoamylase activity of 161 U/L and a corresponding volumetric productivity of 3.5 U/L h were achieved when a cell density of ∼85 g/L (or 12.3 g/g fiber) was attained in the fibrous-bed immobilized cell bioreactor system. The stable glucoamylase production was achieved after five generations, at which time a fraction of ∼62% of the plasmid-carrying cells was realized in the immobilized cell system. Plasmid stability was increased for the immobilized cells during continuous culture at the operating dilution rate. The volumetric and specific productivities and fraction of plasmid-carrying cells in the immobilized cell system were higher than in the free-cell counterpart, however. This was in part due to the high viability (∼80%) in the immobilized cell system and the selective immobilization of the plasmid-carrying cells in the fibrous bed, and perhaps increased plasmid copy number.