Thermophilic Fungus Thermoascus Aurantiacus Research Articles

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass will promote the use of this renewable material. Here, we have studied several process parameters (reaction temperature, type of enzyme blend, type of substrate, type of reductant, and in situ supply of hydrogen peroxide) to better understand how saccharification could be optimized, focusing on the role of lytic polysaccharide monooxygenases (LPMOs). Comparison of a simple, LPMO-rich cellulolytic secretome from the thermophilic fungus Thermoascus aurantiacus with the commercial cellulase preparation Cellic CTec2 showed that saccharification of (lignin-poor) sulfite-pulped spruce at 60 °C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 °C. Quantification of LPMO products showed that while LPMO activity contributed to saccharification efficiency, high levels of LPMO activity were not necessarily beneficial. Reactions with steam-exploded birch, rich in redox-active lignin, highlighted a strong impact of the feedstock on enzyme performance. In this case, the reaction with Cellic CTec2 at 50 °C was clearly most efficient. At 60 °C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H2O2-generating glucose oxidase to reactions with Cellic CTec2 at 50 °C led to strongly increased LPMO activity and, only for reactions with the lignin-poor substrate, improved saccharification yields. These results underpin the potential of the T. aurantiacus secretome for hydrolysis of lignin-poor substrates, and the usefulness of glucose oxidase for optimizing their saccharification. They also show that the efficiency of LPMO-containing cellulase preparations is highly dependent on the nature of the reductant and the substrate.

Purification and Structural Characterization of the Auxiliary Activity 9 Native Lytic Polysaccharide Monooxygenase from Thermoascus aurantiacus and Identification of Its C1- and C4-Oxidized Reaction Products

Auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are copper-dependent oxidoreductases that use O2 or H2O2 to perform oxidative cleavage of cellulose in the presence of an electron donor. Combined with cellulases, they can assist in a more efficient cleavage of cellulose. AA9 LPMOs have therefore attracted considerable attention in recent years for use in biotechnological applications. Here, a native AA9 LPMO (nTaAA9A) from the thermophilic fungus Thermoascus aurantiacus was purified and characterized. The enzyme was shown to be active and able to cleave cellulose and xylan to produce C1- and C4-oxidized products. It was also found to retain about 84.3, 63.7, and 35.3% of its activity after incubation for 30 min at 60, 70, and 80 °C, respectively, using quantitative activity determination. The structure was determined to 1.36 Å resolution and compared with that of the recombinant enzyme expressed in Aspergillus oryzae. Structural differences in the glycosylated Asn138 and in solvent-exposed loops were identified.

CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars

BackgroundFilamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood.ResultsA fed-batch system with plant biomass-derived sugars d-xylose, l-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways.ConclusionThis work has identified two additional CAZyme inducers for T. aurantiacus, l-arabinose and cellobiose, along with d-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

BackgroundFungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production.ResultsHere, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week.ConclusionThe genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

Xylose induces cellulase production in Thermoascus aurantiacus

BackgroundLignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains.ResultsInduction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated.ConclusionsXylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

Production of xylanase by a new strain of Thermoascus aurantiacus: obtainment of enzymatic extract with reduced cellulolytic activity for application in pulp and paper industries

Xylanases are useful in several industrial segments, including pulp and paper bleaching, animal feed, and bread-making processes. However, the industrial use of these enzymes is closely related to its production cost and its catalytic properties. The process of solid state fermentation enables the use of agro-industrial residues as substrates for microbial cultivation and enzymes production, reducing costs. In the present study, different cultivation parameters were evaluated for the xylanase production by the thermophilic fungus Thermoascus aurantiacus , by solid state fermentation, using agro-industrial residues as substrates. High production of xylanase (1701.9 U g -1 of dry substrate) was obtained using wheat bran containing 65% of initial moisture, at 120 h of cultivation, and 45°C. The xylanase showed optimal activity at pH 5.0 and 75°C; its stability was maintained at pH 3.0–11.0. The enzyme retained its catalytic potential after 1 h, at 75°C. The enzymatic extract produced under optimized conditions showed reduced activities of endoglucanase and FPase. Our results, including the xylanase production by T. aurantiacus in low-cost cultivation medium, high structural stability of the enzyme, and reduced cellulolytic activity, encourage the application of this enzymatic extract in pulp and paper bleaching processes.

Purification and Properties of Polygalacturonase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI-756 on Solid-State Fermentation

Polygalacturonases are enzymes involved in the degradation of pectic substances, being extensively used in food industries, textile processing, degumming of plant rough fibres, and treatment of pectic wastewaters. Polygalacturonase (PG) production by thermophilic fungus Thermoascus aurantiacus on solid-state fermentation was carried out in culture media containing sugar cane bagasse and orange bagasse in proportions of 30% and 70% (w/w) at 45°C for 4 days. PG obtained was purified by gel filtration and ion-exchange chromatography. The highest activity was found between pH 4.5 and 5.5, and the enzyme preserved more than 80% of its activity at pH values between 5.0 and 6.5. At pH values between 3.0 and 4.5, PG retained about 73% of the original activity, whereas at pH 10.0 it remained around 44%. The optimum temperature was 60–65°C. The enzyme was completely stable when incubated for 1 hour at 50°C. At 55°C and 60°C, the activity decreased 55% and 90%, respectively. The apparent molecular weight was 29.3 kDa, K m of 1.58 mg/mL and V max of 1553.1 μmol/min/mg. The presence of Zn+2, Mn+2, and Hg+2 inhibited 59%, 77%, and 100% of enzyme activity, respectively. The hydrolysis product suggests that polygalacturonase was shown to be an endo/exoenzyme.

Effect of a Thermoascus aurantiacus thermostable enzyme cocktail on wheat bread qualitiy

Thermophilic fungus Thermoascus aurantiacus (CBMAI 756) on solid-state fermentation using corncob as a nutrient source produces an enzyme pool with the potential to be used in bread making. In this paper, the use of this enzyme cocktail as a wheat bread improver was reported. Both products released by flour arabinoxylan degradation and bread quality were investigated. The main product released through enzyme activity after prolonged incubation was xylose indicating the presence of xylanase; however, a small amount of xylobiose and arabinose also confirmed the presence of xylosidase and α-l-arabinofuranosidase, respectively. Enzyme mixture “in vitro” mainly attacked water-unextractable arabinoxylan contributing to beneficial effect in bread making. The use of an optimal enzyme concentration (35U xylanase/100g of flour) increased specific volume (22%), reduced crumb firmness (25%), and reduced amylopectin retrogradation (17%) during bread storage. In conclusion, the enzyme cocktail produced by T. aurantiacus CBMAI 756 can improve wheat bread quality.

Production and characterization of polygalacturonase from thermophilic Thermoascus aurantiacus on submerged fermentation

Microbial pectinase production by submerged fermentation (SMF) has been conducted mainly using mesophilic microorganisms. However, the use of thermophilic fungal strains capable of producing thermostable and thermophilic enzymes offers specific alternatives to biotechnology processes that use enzymes with such characteristics. Pectinases produced by thermophilic fungi may exhibit interesting characteristics, such as high thermostability, in terms of industrial application. Polygalacturonase (PG) production by the thermophilic fungus Thermoascus aurantiacus on SMF was carried out in culture media containing commercial pectin or agro-industrial by-products as carbon source under different fermentation conditions. The highest enzyme production occurred when the liquid waste from orange juice processing was used as substrate, after 5 days of cultivation. PG activity was highest when the fungus was cultivated at 45°C with an initial pH of 5.5. PG showed an optimum temperature of 60°C and optimum pH of 5.0. The enzyme showed greater stability in acid pH (3.0–4.5) and remained stable when incubated at 55°C for 1 h.

Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus

The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.

Purification and characterization of the α-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756

An alpha-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70 degrees C. Enzyme showed stability stable in the pH range of 3.0-9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60 degrees C. In the presence of ions Na(+), Ba(2+), Co(2+), Ni(2+), Mg(2+), Mn(2+), Al(3+), Zn(2+), Ca(2+) this enzyme maintained 90-105% of its maximum activity and was inhibited by Cr(3+), Ag(+), and Hg(2+). The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and alpha-PNPG. The K(m) measured for the alpha-glucosidase was 0.07 microM, with a V(max) of 318.0 micromol/min/mg.

Properties of the purified glycosylated alpha-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756 in submerged fermentation

Properties of the purified glycosylated alpha-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756 in submerged fermentation

Adsorption of major endoglucanase from Thermoascus aurantiacus on cellulosic substrates

A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated Amax and Kad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing.

Production and characteristics comparison of crude β-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes

The purified β-glucosidase of Aureobasidium pullulans ER-16 is one of more thermostable enzyme reported to date. Considering the unfeasibility of using purified enzyme for industrial application, it was interesting to analyze this property for the crude enzyme. Thermophilic fungus Thermoascus aurantiacus CBMAI-756 and mesophilic A. pullulans ER-16 were cultivated in different hemicellulosic materials on solid-state cultivation for β-glucosidase production. Wheat bran was most appropriate for β-glucosidase production by both microorganisms. T. aurantiacus exhibited maximum enzyme production (7.0 U/ml or 70 U/g) at 48–72 h and A. pullulans a maximum (1.3 U/ml or 13 U/g) at 120 h. Maximum activities were at 75 °C with optimum pH at 4.5 and 4.0, for T. aurantiacus and A. pullulans, respectively. A. pullulans's β-glucosidase was more pH stable (4.5–10.0 against 4.5–8.0) and more thermostable (90% after 1 h at 75 °C against 85% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t (1/2) at 80 °C were 50 and 12.5 min for A. pullulans and T. aurantiascus, respectively. These data confirm the high thermostability of crude β-glucosidase from A. pullulans. Both β-glucosidases were strongly inhibited by glucose, but ethanol significantly increased the activity of the enzyme from T. aurantiacus.

Partially purified and characterization of the α-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756 in submerged fermentation

Partially purified and characterization of the α-glucosidase produced by thermophilic fungus Thermoascus aurantiacus CBMAI 756 in submerged fermentation

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t (1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol −1 for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.

Production of thermostable β-mannosidase by a strain of Thermoascus aurantiacus: Isolation, partial purification and characterization of the enzyme

The production of an extracellular thermostable β-mannosidase by the thermophilic fungus Thermoascus aurantiacus Miehe was investigated in shake flask cultures. Among five different carbon sources and inducers tested, locust bean gum at 15 g l −1 induced the highest (4 nkat ml −1) β-mannosidase activity. Although the fungus grew very well on mannose at an initial concentration of 20 g l −1 there was practically no enzyme synthesis. Low level of enzyme synthesis occurred only when mannose had dropped to about 1 g l −1. The β-mannosidase was purified to apparent homogeneity by ethanol precipitation, differential solubility at pH 4.0 and cation ion exchange chromatography. The enzyme had a molecular mass of 99.9 kDa and a p I value of 4.8. The β-mannosidase showed maximum activity at pH 2.5–3.0 and 76 °C. It exhibited highest pH stability at 5.9 after 5 h incubation at 50 °C. It had a half-life of 10 min at 76 °C. The K m and V max values for p-nitrophenyl-β- d-mannopyranoside ( p-NP-β-MP) were 1.1 mM and 61 nkat mg −1, respectively. Low transglycosylation activity was observed when the enzyme was incubated with p-NP-β-MP as glycosyl donor and methyl-α- d-mannopyranoside as acceptor at 50 °C and pH 5.0 and small amounts of β-configured methylmannobioside was formed.

Production and characterization of cellulolytic enzymes from the thermophilic fungus Thermoascus aurantiacus under solid state cultivation of agricultural wastes

Extracellular cellulolytic enzymes were produced under solid state cultivation by the thermophilic fungus Thermoascus aurantiacus and characterized. Elevated levels of endoglucanase and β-glucosidase activities were produced simultaneously by optimization of growth factors. Under optimal growth conditions, 1572 U endoglucanase and 101.6 U β-glucosidase per g of carbon source were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl-β-glycosides of glucose (MUG) and cellobiose (MUG 2) were used to characterize the cellulolytic multienzyme components after separation by isoelectric focusing. The zymogram indicated one endoglucanase and one β-glucosidase with pI values 3.5 and 3.9, respectively. Both enzymes exhibited significant thermostability, with half-lives of 42 and 18 min, respectively, at 80 °C.

Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus.

beta-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps--ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degrees C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, Km values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Performance of an intermittent agitation rotating drum type bioreactor for solid-state fermentation of wheat straw.

A laboratory bioreactor, designed for solid-state fermentation of thermophilic microorganisms, was operated for production of cellulases and hemicellulases by the thermophilic fungus Thermoascus aurantiacus. The suitability of the apparatus for the effective control of important operating variables affecting growth of microbes in solid-state cultivation was determined. Application of the optimum conditions found for the moisture content of the medium, growth temperature and airflow rate produced enzyme yields of 1709 U endoglucanase, 4 U cellobiohydrolase, 79 U beta-glucosidase, 5.5 U FPA, 4490 U xylanase and 45 U beta-xylosidase per g of dry wheat straw. The correlation between microorganism growth and production of enzymes was efficiently described by the Le Duy kinetic model.