Two new species of Sordariomycetes (Chaetomiaceae and Nectriaceae) from China.

Rich and diverse fungal species occur in different habitats on the earth. Many new taxa are being reported and described in increasing numbers with the advent of molecular phylogenetics. However, there are still a number of unknown fungi that have not yet been discovered and described. During a survey of fungal diversity in different habitats in China, we identified and proposed two new species, based on the morphology and multi-gene phylogenetic analyses. Herein, we report the descriptions, illustrations and molecular phylogeny of the two new species, Bisifusariumkeratinophilumsp. nov. and Ovatosporasinensissp. nov.

The development of a heterologous gene expression system in thermophilic fungus Thermoascus aurantiacus.

Thermoascus aurantiacus is a thermophilic fungus that belongs to the ascomycetous class and has attracted increasing interest for its ability to produce thermostable cellulolytic enzymes and growth at elevated temperatures. However, studies on this organism have been limited because of the lack of a genetic manipulation system. Here, we developed a polyethylene glycol (PEG)-mediated transformation system for T. aurantiacus based on an orotidine-5'-monophosphate decarboxylase (pyrG)-deficient mutant, with this method achieving a transformation efficiency of 33 ± 3 transformants per microgram of DNA. Intracellular or secretory expression of heterologous proteins, including green fluorescent protein, ß-galactosidase and α-amylase, in T. aurantiacus was successful under the inducible endogenous cellobiohydrolase and endoglucanase gene promoter or the constitutive heterologous pyruvate decarboxylase and enolase gene promoter from Trichoderma reesei. To the best of our knowledge, this is the first report on PEG-mediated transformation of T. aurantiacus, which sets the foundation for strain improvement for biotechnological applications and functional genomic studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02963-w.

Increased Malbranchea pulchella ß-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs.

ß-Glucosidases are a limiting factor in the conversion of cellulose to glucose for the subsequent ethanol production. Here, ß-glucosidase production by Malbranchea pulchella was optimized using Composite Central Designs and Response Surface Methodologies from a medium designed. The coefficient of determination (R2 ) was 0.9960, F-value was very high, and the lack of fit was found to be non-significant. This indicates a statistic valid and predictive result. M. pulchella enzymatic extract was successfully tested as an enzymatic cocktail in a mixture design using sugarcane bagasse, soybean hull and barley bagasse. We proved that the optimization of the ß-glucosidase production and the application in hydrolysis using unexpansive biomass and agricultural wastes can be accomplished by means of statistical methodologies. The strategy presented here can be useful for the improvement of enzyme production and the hydrolysis process, arising as an alternative for bioeconomy.

Valorization of Lignocellulosic Wastes to Produce Phytase and Cellulolytic Enzymes from a Thermophilic Fungus, Thermoascus aurantiacus SL16W, under Semi-Solid State Fermentation.

Agricultural wastes are lignocellulosic biomasses that contain high mineral and nutrient contents. This waste can be used as a raw material in industrial enzyme production by microbial fermentation. Phytase is an important enzyme used in animal feed to enhance the amount of phosphorus available for the growth and overall health improvement of monogastric animals. Fungi offer high potential as an effective source in the production of various extracellular enzymes. In this study, the production of lignocellulolytic enzymes (endoglucanase and xylanase) and phytase by a thermophilic fungus, namely Thermoascus aurantiacus strain SL16W, was evaluated using sixteen different Thai agricultural forms of waste under conditions of high temperature (45 °C). Semi-solid state fermentation was used in the production experiments. The results of this study reveal that the highest phytase activity (58.6 U/g substrate) was found in rice bran, whereas the highest degrees of activity of endoglucanase and xylanase were observed in wheat bran and red tea leaves at 19 and 162 U/g substrate, respectively. Consequently, the optimal conditions for phytase production of this fungus using rice bran were investigated. The results indicate that the highest phytase yield (58.6 to 84.1 U/g substrate) was observed in rice bran containing 0.5% ammonium sulfate as a nitrogen source with 10 discs of inoculum size at a cultivation period of 9 days at 45 °C and moisture content of 95%. Notably, the phytase yield increased by 1.71-fold, while endoglucanase and xylanase were also increased by 1.69- and 1.12-fold, respectively. Furthermore, the crude enzyme obtained from the optimal condition was extracted. The crude enzyme extract was then separately added to red tea leaves, rice straw, corncobs, palm residue, and peanut husks. Subsequently, total reducing sugar and phosphorus contents were determined. The results indicate that the highest level of reducing sugar (122.6 mg/L) and phosphorus content (452.6 mg/L) (p < 0.05) were obtained in palm residue at 36 and 48 h, respectively, after the addition of the crude enzyme extract. This study has provided valuable information on a potentially eco-friendly way to valorize agricultural waste into value-added products as industrial enzymes.

Crystal Structure of a Cu,Zn Superoxide Dismutase From the Thermophilic Fungus Chaetomium thermophilum.

BACKGROUND: Thermophilic fungi have recently emerged as a promising source of thermostable enzymes. Superoxide dismutases are key antioxidant metalloenzymes with promising therapeutic effects in various diseases, both acute and chronic. However, structural heterogeneity and low thermostability limit their therapeutic efficacy. OBJECTIVE: Although several studies from hypethermophilic superoxide dismutases (SODs) have been reported, information about Cu,Zn-SODs from thermophilic fungi is scarce. Chaetomium thermophilum is a thermophilic fungus that could provide proteins with thermophilic properties. METHODS: The enzyme was expressed in Pichia pastoris cells and crystallized using the vapor-diffusion method. X-ray data were collected, and the structure was determined and refined to 1.56 Å resolution. Structural analysis and comparisons were carried out. RESULTS: The presence of 8 molecules (A through H) in the asymmetric unit resulted in four different interfaces. Molecules A and F form the typical homodimer which is also found in other Cu,Zn- SODs. Zinc was present in all subunits of the structure while copper was found in only four subunits with reduced occupancy (C, D, E and F). CONCLUSION: The ability of the enzyme to form oligomers and the elevated Thr:Ser ratio may be contributing factors to its thermal stability. Two hydrophobic residues that participate in interface formation and are not present in other CuZn-SODs may play a role in the formation of new interfaces and the oligomerization process. The CtSOD crystal structure reported here is the first Cu,Zn-SOD structure from a thermophilic fungus.

Crystal Structure of a GH3 ß-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum.

Beta-glucosidases (ß-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 ß-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other ß-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family ß-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in ß-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Characterization and improvement of substrate-binding affinity of D-aspartate oxidase of the thermophilic fungus Thermomyces dupontii.

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic D-amino acids and a neutral D-amino acid, D-Gln, with the highest activity toward D-Glu. The Km and kcat values for D-Glu were 2.16 mM and 217 s-1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and D-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

Heterologous expression, purification and biochemical characterization of a new xylanase from Myceliophthora heterothallica F.2.1.4.

Myceliophthora heterothallica is a thermophilic fungus potentially relevant for the production of enzymes involved in the degradation of plant biomass. A xylanase encoding gene of this species was identified by means of RT-PCR using primers designed based on a xylanase coding sequence (GH11) of the fungus M. thermophila. The obtained gene was ligated to the vector pET28a(+) and the construct was transformed into Escherichia coli cells. The recombinant xylanase (r-ec-XylMh) was heterologously expressed, and the highest activity was observed at 55 °C and pH 6. The enzyme stability was greater than 70% between pH 4.5 and 9.5 and the inclusion of glycerol (50%) resulted in a significant increase in thermostability. Under these conditions, the enzyme retained more than 50% residual activity when incubated at 65 °C for 1 h, and approximately 30% activity when incubated at 70 °C for the same period. The tested cations did not increase xylanolytic activity, and the enzyme indicated significant tolerance to several phenolic compounds after 24 h, as well as high specificity for xylan, with no activity for other substrates such as CMC (carboxymethylcellulose), Avicel, pNPX (p-nitrophenyl-ß-D-xylopyranoside) and pNPA (p-nitrophenyl-α-L-arabinofuranoside), and is thus, of potential relevance in pulp bleaching.

Purification, Cloning, Functional Expression, Structure, and Characterization of a Thermostable ß-Mannanase from Talaromyces trachyspermus B168 and Its Efficiency in Production of Mannooligosaccharides from Coffee Wastes.

Highly thermostable ß-mannanase, belonging to glycoside hydrolase family 5 subfamily 7, was purified from the culture supernatant of Talaromyces trachyspermus B168 and the cDNA of its transcript was cloned. The recombinant enzyme showed maximal activity at pH 4.5 and 85 °C. It retained more than 90 % of its activity below 60 °C. Obtaining the crystal structure of the enzyme helped us to understand the mechanism of its thermostability. An antiparallel ß-sheet, salt-bridges, hydrophobic packing, proline residues in the loops, and loop shortening are considered to be related to the thermostability of the enzyme. The enzyme hydrolyzed mannans such as locust bean gum, carob galactomannan, guar gum, konjac glucomannan, and ivory nut mannan. It hydrolyzed 50.7 % of the total mannans from coffee waste, producing mannooligosaccharides. The enzyme has the highest optimum temperature among the known fungal ß-mannanases and has potential for use in industrial applications.

Crystal structure and biological implications of a glycoside hydrolase family 55 ß-1,3-glucanase from Chaetomium thermophilum.

Crystal structures of a ß-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum were determined at 1.20 and 1.42Å resolution in the free and glucose-bound form, respectively. This is the third structure of a family 55 glycoside hydrolase (GH55) member and the second from a fungus. Based on comparative structural studies and site-directed mutagenesis, Glu654 is proposed as the catalytic acid residue. The substrate binding cleft exhibits restricted access on one side, rendering the enzyme as an exo-ß-1,3-glucanase as confirmed also by thin layer chromatography experiments. A lack of stacking interactions was found at the substrate binding cleft, suggesting that interactions at positions -1, +1 and +2 are sufficient to orientate the substrate. A binding pocket was identified that could explain binding of branched laminarin and accumulation of laminaritriose.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable ß-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

Mycothermus thermophilus (Syn. Scytalidium thermophilum): Repertoire of a diverse array of efficient cellulases and hemicellulases in the secretome revealed.

Mycothermus thermophilus (Syn. Scytalidium thermophilum/Humicola insolens), a thermophilic fungus, is being reported to produce appreciable titers of cellulases and hemicellulases during shake flask culturing on cellulose/wheat-bran/rice straw based production medium. The sequential and differential expression profile of endoglucanases, ß-glucosidases, cellobiohydrolases and xylanases using zymography was studied. Mass spectrometry analysis of secretome (Q-TOF LC/MS) revealed a total of 240 proteins with 92 CAZymes of which 62 glycosyl hydrolases belonging to 30 different families were present. Cellobiohydrolase I (17.42%), ß glucosidase (8.69%), endoglucanase (6.2%), xylanase (4.16%) and AA9 (3.95%) were the major proteins in the secretome. In addition, carbohydrate esterases, polysaccharide lyases, auxiliary activity and a variety of carbohydrate binding modules (CBM) were identified using genomic database of the culture indicating to an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. The cellulases from the strain hydrolyzed alkali treated rice straw and bagasse into fermentable sugars efficiently.

Heterologous production of an acidic thermostable lipase with broad-range pH activity from thermophilic fungus Neosartorya fischeri P1.

Thermophilic Neosartorya fischeri P1 is an excellent lipase producer and harbors seven lipase genes. All genes were found to be functional after heterologous expression in Escherichia coli. One of them, LIP09, showed high-level expression in Pichia pastoris with the yield of 2.0 g/L in a 3.7-L fermentor. Deduced amino acid sequence of LIP09 consists of a putative signal peptide (residues 1-19) and a mature polypeptide (residues 20-562). Compared with other fungal counterparts, purified recombinant LIP09 has some superior properties. It exhibited maximum activity at 60°C and pH 5.0, had broad pH adaptability (>60% activity at pH 3.5-8.0) and stability (retaining >90% activity after incubation at pH 3.0-7.0 for 1 h at 40°C), and was highly thermostable (retaining >96% activity after incubation at 50°C for 30 min). The r-LIP09 had a preference for the medium-chain length p-nitrophenyl esters (C12) rather than short and long-chain length substrates. The high-level expression and excellent properties make LIP09 a potential enzyme candidate in food and feed industries.

Structural and functional studies of the glycoside hydrolase family 3 ß-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. ß-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the ß-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the ß-glucosidase from H. jecorina (HjCel3A) with the ß-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 ß-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Expression, purification and crystallization of a family 55 ß-1,3-glucanase from Chaetomium thermophilum.

A ß-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum was overexpressed in Pichia pastoris, purified and crystallized in the presence of 1.8 M sodium/potassium phosphate pH 6.8 as a precipitant. Data to 2.0 Å resolution were collected in-house at 293 K from a single crystal. The crystal was found to belong to space group P2(1), with unit-cell parameters a = 64.1, b = 85.8, c = 68.5 Å, ß = 93.1° and one molecule in the asymmetric unit.

Immobilization of a thermostable, fungal recombinant chitinase on biocompatible chitosan beads and the properties of the immobilized enzyme.

A recombinant, thermostable fungal chitinase from the thermophilic fungus, Thermomyces lanuginosus, was immobilized on glutaraldehyde cross-linked chitosan beads, and the properties of the immobilized chitinase were studied. The enzyme was found to be almost completely immobilized in 6 H under shaking condition at 30 °C. The immobilized enzyme exhibited much wider pH optimum and was more stable at alkaline pH values as compared with the soluble enzyme. Both the forms of the enzyme were optimally active at 60 °C and stable at 50 °C for 3 H, and after 3 H, the activity of the soluble enzyme declined sharply, whereas the immobilized chitinase was stable up to 6 H without any significant loss in the activity. KM and Vmax values of the immobilized enzyme were 1.18 mM and 445.7 µmol/Min/mg of protein, respectively. The immobilized enzyme was stable at least for 1 month at 4 °C without any significant loss in the activity.

A novel method for the immobilization of a thermostable fungal chitinase and the properties of the immobilized enzyme.

The recombinant thermostable fungal chitinase of Thermomyces lanuginosus was immobilized on the phenyl Sepharose matrix, and the properties of the immobilized chitinase were studied. The immobilized enzyme was optimally active at pH 6.0 and 50 °C and showed improved activity in the acidic range of pH values when compared with the soluble enzyme. The recombinant thermostable immobilized enzyme showed remarkable thermostability at 50 °C by retaining about 45% of the activity for more than 6 H. The KM and Vmax values were 1.3 mM and 4.5 mol/min/mg of protein, respectively. Both the free and immobilized forms of the enzymes were inhibited significantly by Ag(+) but behaved similarly to various other metal ions, detergents, and additives. The immobilized enzyme was stable for at least 1 month at 4 °C.