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Among different microbes, fungi are proficient candidates for the extracellular synthesis of iron nanoparticles. For biogenic synthesis of iron nanoparticles, a thermophilic mould Myceliophthora thermophila BJTLRMDU7 was used in this study. Mycogenic magnetic nanoparticles were used for phosphatase immobilization and therapeutic applications such as antimicrobial and antimalarial activity. Firstly, the phosphatase was immobilized on biogenic iron nanoparticles with an efficiency of >56 %. Immobilized enzyme was optimally active at 60 °C and pH 5. Immobilized phosphatase was recycled using external magnetic field up to 4th cycle retaining >50 % activity. The immobilized phosphatase efficiently released inorganic phosphate from different flours such as wheat, maize and gram at 37 °C and 60 °C. There was continuous increase in the release of inorganic phosphorus from all samples with incubation time at 37 °C and slight reduction at 60 °C. These nanoparticles showed the effective antimicrobial activity against Bacillus subtilis, Escherichia coli and Myceliophthora thermophila. Further, the synthesized iron nanoparticles showed antimalarial potential against Plasmodium falciparum. Biogenic nanoparticles did not exhibit hemolytic activity and cytotoxicity. Therefore, biogenic iron nanoparticles could be used as a suitable matrix for immobilization of enzymes and safe therapeutics., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which are categorized in the CAZy database under auxiliary activities families AA9-11, 13, 14-17. Secreted by various microorganisms, they play a crucial role in carbon recycling, particularly in fungal saprotrophs. LPMOs oxidize polysaccharides through monooxygenase/peroxygenase activities and exhibit peroxidase and oxidase activities, with variations among different families. AA16, a newly identified LPMO family, is noteworthy due to limited studies on its members, thus rendering the characterization of AA16 enzymes vital for addressing controversies around their functions. This study focused on heterologous expression and biochemical study of an AA16 LPMO from Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila), namely MtLPMO16A. Substrate specificity evaluation of MtLPMO16A showed oxidative cleavage of hemicellulosic substrates and no activity on cellulose, accompanied by a strong oxidase activity. A comparative analysis with an LPMO from AA9 family explored correlations between these families, while MtLPMO16A was shown to boost the activity of some AA9 family LPMOs. The results offer new insights into the AA16 family's action mode and microbial hemicellulose decomposition mechanisms in nature., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Antonis Karantonis reports financial support was provided by Hellenic Foundation for Research and Innovation. Evangelos Topakas reports financial support was provided by European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chemoselective arylation of 5-aminopyrazoles was performed through oxidative formation of orthoquinones from catechols catalyzed by Myceliophthora thermophila laccase (Novozym 51003), and subsequently nucleophilic attack of 5-aminopyrazole to the catechol intermediates. The C-4 arylated products were obtained under extremely mild conditions without the need for amine protection or halogenation of the substrates. From this method, 10 derivatives with moderate to good efficiency (42-94%) were prepared., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Shahedi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl-glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 2 3 -(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the -1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi-functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary β-domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Genome-scale metabolic models (GEMs) serve as effective tools for understanding cellular phenotypes and predicting engineering targets in the development of industrial strain. Enzyme-constrained genome-scale metabolic models (ecGEMs) have emerged as a valuable advancement, providing more accurate predictions and unveiling new engineering targets compared to models lacking enzyme constraints. In 2022, a stoichiometric GEM, iDL1450, was reconstructed for the industrially significant fungus Myceliophthora thermophila. To enhance the GEM's performance, an ecGEM was developed for M. thermophila in this study., Results: Initially, the model iDL1450 underwent refinement and updates, resulting in a new version named iYW1475. These updates included adjustments to biomass components, correction of gene-protein-reaction (GPR) rules, and a consensus on metabolites. Subsequently, the first ecGEM for M. thermophila was constructed using machine learning-based k cat data predicted by TurNuP within the ECMpy framework. During the construction, three versions of ecGEMs were developed based on three distinct k cat collection methods, namely AutoPACMEN, DLKcat and TurNuP. After comparison, the ecGEM constructed using TurNuP-predicted k cat values performed better in several aspects and was selected as the definitive version of ecGEM for M. thermophila (ecMTM). Comparing ecMTM to iYW1475, the solution space was reduced and the growth simulation results more closely resembled realistic cellular phenotypes. Metabolic adjustment simulated by ecMTM revealed a trade-off between biomass yield and enzyme usage efficiency at varying glucose uptake rates. Notably, hierarchical utilization of five carbon sources derived from plant biomass hydrolysis was accurately captured and explained by ecMTM. Furthermore, based on enzyme cost considerations, ecMTM successfully predicted reported targets for metabolic engineering modification and introduced some new potential targets for chemicals produced in M. thermophila., Conclusions: In this study, the incorporation of enzyme constraint to iYW1475 not only improved prediction accuracy but also broadened the model's applicability. This research demonstrates the effectiveness of integrating of machine learning-based k cat data in the construction of ecGEMs especially in situations where there is limited measured enzyme kinetic parameters for a specific organism., (© 2024. The Author(s).)

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H 2 O 2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H 2 O 2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H 2 O 2 . Importantly, the fusion of CBM resulted in more efficient consumption of H 2 O 2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H 2 O 2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (T o = 301 °C, T max = 337 °C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

β-Galactosidases are key enzymes in the food industry. Apart from the hydrolysis of the saccharide bond of lactose, they also catalyze transgalactosylation reactions, producing galactooligosaccharides (GOS) with prebiotic activity. Here we report the heterologous production in Pichia pastoris of a novel β-galactosidase from the fungus Thermothielavioides terrestris. The enzyme (TtbGal1) was purified and characterized, showing optimal activity at 60 °C and pH 4. TtbGal1 is thermostable, retaining almost full activity for 24 h at 50 °C. It was applied to the production of GOS from defined lactose solutions and acid whey, a liquid waste from the Greek yoghurt industry, reaching yields of 19.4 % and 14.8 %, respectively. HILIC-ESI-QTOF-MS analysis revealed the production of GOS with up to 4 saccharide monomers. The results demonstrate efficient GOS production catalyzed by TtbGal1, valorizing acid whey, a waste with a heavy polluting load from the dairy industry., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris . Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: Mfe UPO from Myceliophthora fergusii and Mhi UPO from Myceliophthora hinnulea . To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha . In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the postharvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, which subsequently form pigments. The PPO family includes tyrosinases and catechol oxidases, which, in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data in order to better understand the function of a previously characterized PPO from Thermothelomyces thermophila ( Tt PPO). The crystal structure of a Tt PPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data for structure-guided mutants prove the implication of "gate" residue L306, residue H B1 +1 (G292), and H B2 +1 (Y296) in Tt PPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and show that residue H B1 +1 is unlikely to determine monophenolase activity, as was suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. For ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties., (Copyright © 2021 American Society for Microbiology.)

The modification of gum Arabic with ferulic acid oxidation products was performed in aqueous medium, at 30 °C and pH 7.5, in the presence of Myceliophthora thermophila laccase as biocatalyst. First, this study aimed to investigate the structures of the oxidation products of ferulic acid that could possibly be covalently grafted onto gum Arabic. HPLC analyses revealed that this reaction produced several oxidation products, whose structures were investigated using LC-MS/MS analyses (liquid chromatography-mass spectrometry with mass fragmentation analyses) and NMR experiments. The chemical structure of one intermediate reaction product was fully elucidated as the 2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxy-3-methoxyphenyl) methylidene] cyclobutane-1, 3-dione, called by the authors cyclobutadiferulone. Secondly, this study aimed to locate the grafting of the oxidation products onto gum Arabic by performing several NMR experiments. This study did not determine how much and specifically which oxidation products were grafted but some of them were undeniably present onto modified gum Arabic, close to the glucuronic acid C5 carbon or close to the galactose C6 carbon., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: This work aimed to estimate the growth of Myceliophthora thermophila M.7·7 in solid-state cultivation (SSC) through quantification of N-acetyl-d-glucosamine (NAG) and enzyme activity., Methods and Results: The fungus was cultivated in sugarcane bagasse and wheat bran. A consistent statistical analysis was done to assess the reliability of experimental data. Logistic model equation was fitted to experimental data and growth parameters were estimated. The results showed strong influence of the sample size on NAG and a minimum recommended sample size was identified. Scanning electron microscopy (SEM) was used to identify the strategy of substrate colonization. Wheat bran was attacked firstly, while sugarcane bagasse was consumed after wheat bran depletion. The biomass growth was poorly estimated by secretion kinetics of α-amylase, endoglucanase, protease and xylanase, but enzyme kinetics were important for understanding substrate colonization., Conclusions: In conclusion, the NAG concentration was strongly affected by the sample size and sampling procedure. The strategy of fungal colonization on the substrates was well characterized through SEM analysis. The colonization strategy has direct influence on the kinetic parameters of the logistic model. Myceliophthora thermophila has a well-defined dynamic of enzyme secretion to degrade the substrate, although the kinetics of enzyme secretion has shown not adequate to characterize the kinetics of fungal growth., Significance and Impact of the Study: The paper provides reliable growth kinetic parameters in the SSC of the cellulase producer fungus M. thermophila M.7·7, as well as a robust analysis on three indirect methods (NAG, enzymes and SEM) for estimation of fungal development., (© 2020 The Society for Applied Microbiology.)

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails., Competing Interests: Conflicts of interest The authors declare the following conflicts of interest: Nanna Røjel, Trine H. Sørensen, Kim Borch, and Brett McBrayer work for Novozymes A/S, a major manufacturer of industrial enzymes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis., Methods: The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74 cat ) and Myceliophthora thermophila (MtXeg74A) were carried out., Results: MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between C α atoms of aromatic residues involved in xyloglucan binding in -3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10-12 Å. For PoGH74 cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft., Conclusions: These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity., General Significance: Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharide substrates oxidatively. First discovered because of their action on recalcitrant crystalline substrates (chitin and cellulose), a number of LPMOs are now reported to act on soluble substrates, including oligosaccharides. However, crystallographic complexes with oligosaccharides have been reported for only a single LPMO so far, an enzyme from the basidiomycete fungus Lentinus similis ( Ls AA9_A). Here we present a more detailed comparative study of Ls AA9_A and an LPMO from the ascomycete fungus Collariella virescens ( Cv AA9_A) with which it shares 41.5% sequence identity. Ls AA9_A is considerably more thermostable than Cv AA9_A, and the structural basis for the difference has been investigated. We have compared the patterns of oligosaccharide cleavage and the patterns of binding in several new crystal structures explaining the basis for the product preferences of the two enzymes. Obtaining structural information about complexes of LPMOs with carbohydrates has proven to be very difficult in general judging from the structures reported in the literature thus far, and this can be attributed only partly to the low affinity for small substrates. We have thus evaluated the use of differential scanning fluorimetry as a guide to obtaining complex structures. Furthermore, an analysis of crystal packing of LPMOs and glycoside hydrolases corroborates the hypothesis that active site occlusion is a very significant problem for LPMO-substrate interaction analysis by crystallography, due to their relatively flat and extended substrate binding sites.

A novel phospholipase C gene (TtPLC) from Thielavia terrestris CAU709 was cloned and efficiently expressed in Pichia pastoris. The deduced protein sequence of TtPLC shared the highest identity of 33% with the characterized phospholipase C from Arabidopsis thaliana. The highest phospholipase C yield of 98, 970 U mL -1 , with a protein concentration of 4.9 mg mL -1 was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant enzyme (TtPLC) was purified to homogeneity with a recovery yield of 59.1% and a specific activity of 22, 910 U mg -1 . TtPLC was most active at pH 6.5 and 55 °C, respectively. It was stable within the pH range of 4.5-8.0 and up to 45 °C. The enzyme exhibited excellent stability in different surfactants and organic solvents, including Tween 20 (147.6%), Tween 40 (180.6%), Tween 60 (205.4%), cyclohexane (160.0%), n-octane (178.2%), n-heptane (180.7%), acetone (187.5%) etc. The application of TtPLC in crude soybean oil degumming process significantly reduced the residual phosphorus content from 135.4 mg kg -1 to 7.9 mg kg -1 under the optimized conditions, which satisfied the requirement of environmental friendly physical refining process for oil refining industry. Therefore, TtPLC should be a good candidate in oil refining industry., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest. Author statement. Man Xiang, Ling Wang: Conceptualization, Methodology. Man Xiang, Qiaojuan Yan, Data curation, Writing- Original draft preparation. Man Xiang, Ling Wang: Investigation. Shaoqing Yang, Zhengqiang Jiang, Supervision. Shaoqing Yang: Writing- Reviewing and Editing., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present work, a kinetic analysis is made concerning the reaction of an electroactive immobilized enzyme with a free substrate, based on a Michaelis-Menten scheme. The proposed kinetic equations are investigated numerically for conditions describing large amplitude fast Fourier transform alternating current voltammetry (FTacV), under different reaction states (transient or steady state for the reaction intermediate as well as quasi or complete reversibility of the electrochemical step). The dependence of two chief observables that occur from the analysis of the results of the method, that is, the maximum of the harmonics and the potential shift of the corresponding dominant peaks, on substrate concentration is presented. The FTacV method is applied experimentally for an immobilized laccase-like multicopper oxidase from Thermothelomyces thermophila, TtLMCO1, and its reaction with epinephrine. From the experimental findings it is shown that the intrinsic characteristics of the system do not lead to the extraction of the desired kinetic data although indications on the relation between the kinetic constants is revealed. Finally, the response of the third harmonic for the first additions of epinephrine at subnanomolarity range can be exploited for the detection of epinephrine at rather low concentrations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The laccase has the ability to oxidize substituted phenols and the water is the sole byproduct, thus it has been employed to remove and/or modify the lignin in lignocellulosic material. A putative laccase gene, LacSM, from Sordaria macrospora k-hell was screened by a genome mining approach. Then, it was cloned and highly expressed in Escherichia coli. The molecular weight of recombinant LacSM was ~ 67 kDa. The optimal pH values for the LacSM oxidation of guaiacol, syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were 6, 7, 5, and 5, respectively. The optimal activity of laccase was observed at 60, 55, 55, and 50 °C for four respective substrates. LacSM remained stable at pH 5-8 and thermostable at 60 °C with guaiacol as the substrate. 1 mM K + , Na + , or Mn 2+ ions slightly stimulated laccase activity. In addition, LacSM was moderately tolerant to the Cl - ion and showed an ability to remove and/or modify lignin. Thus, LacSM was a potential candidate for industrial applications, such as lignin degradation of lignocellulosic biomass.

3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies, we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical-SAM enzymes. We predict that our work will help in using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An investigation was carried out using sugarcane bagasse as the agricultural residue to study the optimization of xylanase production by solid-state fermentation. Maximum xylanase production (20.35 U/g substrate) was achieved by Bacillus substilis subsp. subtilis JJBS250 using 'one variable at a time approach' at pH 7.0, 40 °C after 48 h. After statistical optimization by response surface methodology (RSM) there was 4.82-fold improvement in xylanase production (98.16 U/g substrate). Further optimization of untreated and sodium carbonate pretreated sugarcane bagasse enzymatic hydrolysis was carried out using both bacterial (Bacillus substilis subsp. subtilis JJBS250) and fungal (Myceliophthora thermophila BJTLRMDU3) xylanases that showed high amount of reducing sugar liberation from untreated sugarcane bagasse (124.24 mg/g substrate) as compared to pretreated (76.23 mg/g substrate) biomass. Furthermore, biophysical characterization of untreated and sodium carbonate pretreated sugarcane bagasse using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM), revealed the structural changes in the pretreated biomass.

A recombinant strain producing a complex of extracellular enzymes including chitinase from Myceliophtora thermophila was created based on the fungus Penicillium verruculosum. The activity of the enzyme preparations obtained from the cultural fluid of the producer strain was 0.55, 0.53, and 0.66 U/mg protein with chitin and chitosans with the molecular weight of 200 and 1000 kDa, respectively. The temperature optimum for the recombinant chitinase was 52-65°C; the pH optimum was 4.5-6.2, which corresponded to the published data for this class of the enzymes. The content of heterologous chitinase in the obtained enzyme preparations was 47% of total protein content in the cultural fluid. Enzyme preparations produced by the recombinant P. verruculosum XT403 strain and containing heterologous chitinase were able to degrade the mycelium of micromycetes, including phytopathogenic ones, and were very efficient in the bioconversion of microbiological industry waste.

A new β-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: k cat of (7.9 ± 0.2) × 10 5 s -1 , and k cat /K m of (9.5 ± 0.12) × 10 7 M -1 ∙s -1 . An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with K I s in the range of 54-95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.

Changes in the tertiary conformation of adsorbed biomolecules can induce detectable shifts (Δθ r ) in the surface plasmon resonance (SPR) angle. Here it is shown how to calculate the corresponding shifts in the adsorbate's center of mass (Δ z avg ) along the sensing surface normal from the measured Δθ r . The novel developed model was used for determining the mean distance between the cytochrome (CYT) and flavodehydrogenase (DH) domains of the enzyme cellobiose dehydrogenase (CDH) isolated from the fungi Neurospora crassa , Corynascus thermophilus , and Myriococcum thermophilum as a function of pH, [Ca 2+ ], and substrate concentration. SPR confirmed the results from earlier electrochemical and SAXS studies stating that the closed conformation, where the two domains are in close vicinity, is stabilized by a lower pH and an increased [Ca 2+ ]. Interestingly, an increasing substrate concentration in the absence of any electron acceptors stabilizes the open conformation as the electrostatic repulsion due to the reaped electrons pushes the DH and CYT domains apart. The accuracy of distance determination was limited mostly by the random fluctuations between replicate measurements, and it was possible to detect movements <1 nm of the domains with respect to each other. The results agreed with calculations using already established models treating conformational changes as contraction or expansion of the thickness of the adsorbate layer ( t protein ). Although the models yielded equivalent results, in this case, the Δ z avg -based method also works in situations, where the adsorbate's mass is not evenly distributed within the layer.

Objective: To identify main protease genes for the proteolytic degradation of cellulases in M. thermophila and generate a lower-proteases fungal host that can be used for further metabolic engineering to increase cellulase production and heterologous protein expression., Results: Systematic transcriptomic analysis were conducted on the expression of proteases genes in M. thermophila genome and five highly expressed genes encoding extracellular proteases were selected for mutation analyses. A series of single- and multi-gene mutants of these five selected genes was constructed using the CRISPR-Cas9 technique. Compared with WT, the ΔMtalp1 and the quintuple mutant showed significantly lower protease activity (decreased 52.7% and 58.4%, respectively) and at least double enhanced cellulase production., Conclusions: The results indicated that Mtalp1 is a critical protease gene in cellulase degradation in M. thermophila and disruption of protease genes showed significantly decreased protease activity and obviously enhanced cellulase production in the fermentation broth of ΔMtalp1 and the quintuple mutant.

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H. insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4 mM Ca 2+ , and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4-6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C 12 E 8 ). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C 12 E 8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C 12 E 8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C 12 E 8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C 12 E 8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/β-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A codon-optimized cutinase gene (TtCutopt) from Thielavia terrestris was over-expressed in Pichia pastoris. An extracellular activity reached 10,200 U/mL using high cell density fermentation. The optimal pH and temperature of TtCutopt were 7.0 and 50 °C, respectively. It displayed high stability over a wide range of pH from 3.0 to 11.0 and up to 85 °C. Among tested p-nitrophenyl esters and triglycerides, TtCutopt showed the highest activity towards p-nitrophenyl butyrate and tributyrin, with specificity activity of 2322.4 U/mg and 1152.5 U/mg, respectively. It was extremely stable in organic solvents and surfactants. TtCutopt efficiently catalyzed the synthesis of butyl butyrate, hexyl butyrate, butyl hexanoate and hexyl hexanoate with esterification efficiency of >95%. Furthermore, it catalyzed the degradation of >90% of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate to release their corresponding monoalkyl phthalates within 24 h. Thus, high yield, high stability, and esterification efficiency of TtCutopt make it an attractive candidate for ester biosynthesis and biodegradation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Degradation of polysaccharides is central to numerous biological and industrial processes. Starch-active polysaccharide monooxygenases (AA13 PMOs) oxidatively degrade starch and can potentially be used with industrial amylases to convert starch into a fermentable carbohydrate. The oxidative activities of the starch-active PMOs from the fungi Neurospora crassa and Myceliophthora thermophila , Nc AA13 and Mt AA13, respectively, on three different starch substrates are reported here. Using high-performance anion-exchange chromatography coupled with pulsed amperometry detection, we observed that both enzymes have significantly higher oxidative activity on amylose than on amylopectin and cornstarch. Analysis of the product distribution revealed that Nc AA13 and Mt AA13 more frequently oxidize glycosidic linkages separated by multiples of a helical turn consisting of six glucose units on the same amylose helix. Docking studies identified important residues that are involved in amylose binding and suggest that the shallow groove that spans the active-site surface of AA13 PMOs favors the binding of helical amylose substrates over nonhelical substrates. Truncations of Nc AA13 that removed its native carbohydrate-binding module resulted in diminished binding to amylose, but truncated Nc AA13 still favored amylose oxidation over other starch substrates. These findings establish that AA13 PMOs preferentially bind and oxidize the helical starch substrate amylose. Moreover, the product distributions of these two enzymes suggest a unique interaction with starch substrates., (© 2019 Vu et al.)

D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from Cryptococcus neoformans, Chaetomium thermophilum, Arabidopsis thaliana, Thermothelomyces thermophila, and Mus musculus were overexpressed in Escherichia coli, respectively. A novel MIOX from Thermothelomyces thermophila (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the Escherichia coli genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (β-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens . These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia., Competing Interests: Partial funding for this project was obtained from Dole Food Company to support a graduate assistantship. This funding does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Fungi have been used in modern scientific research due to their high potential for different enzymes production based on genomic features. The great proportion of soil mycoflora represented by saprobic fungi plays an important role in decomposition, thus contribute to the global carbon cycle. Sordaria fimicola strains (n= 61) collected from different environments were evaluated for catalase enzyme activity at first stage. Among all 61 isolates of S. fimicola, five strains viz. S1, S2, N7, N6 and SF13 were found to be most efficient in catalase enzyme activity. The complete catalase gene including exons and introns was amplified and sequenced from the most efficient strains of S. fimicola and then submitted in the NCBI data base under accession numbers KM282183, KM282184, KM282186, KM282185 and KM282182 for strains S1, S2, N7, N6 and SF13 respectively. The significant differences in the genes sequences and theoretically translated proteins were observed for all five strains of S. fimicola. As regards catalase enzyme activity, S. fimicola strains were found comparable to the Aspergillus niger strains, therefore being a saprophytic fungus with short life cycle S. fimicola can become a fungus of choice to produce catalase enzyme at large scale.

Glucuronoyl esterases (CE15 family) enable targeted cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), particularly those linking lignin and glucuronoyl residues in xylan. A substantial challenge in characterization and kinetic analysis of CE15 enzymes has been the lack of proper substrates. Here, we present an assay using an insoluble LCC-rich lignin fraction from birch; lignin-rich pellet (LRP). The assay employs quantification of enzyme reaction products by LC-MS. The kinetics of four fungal CE15 enzymes, PsGE, CuGE, TtGE, and AfuGE originating from lignocellulose-degrading fungi Punctularia strigosozonata, Cerrena unicolor, Thielavia terrestris, and Armillaria fuscipes respectively were characterized and compared using this new assay. All four enzymes had activity on LRP and showed a clear preference for the insoluble substrate compared with smaller soluble LCC mimicking esters. End-product profiles were near identical for the four enzymes but differences in kinetic parameters were observed. TtGE possesses an alternative active site compared with the three other enzymes as it has the position of the catalytic glutamic acid occupied by a serine. TtGE performed poorly compared with the other enzymes. We speculate that glucuronoyl LCCs are not the preferred substrate of TtGE. Removal of an N-terminal CBM on CuGE affected the catalytic efficiently of the enzyme by reducing K cat by more than 30%. Reaction products were detected from all four CE15s on a similar substrate from spruce indicating a more generic GE activity not limited to the hardwood. The assay with natural substrate represents a novel tool to study the natural function and kinetics of CE15s.

The thermophilic fungus Thielavia terrestris when cultured on cellulose produces a cocktail of thermal hydrolases with potential application in saccharification of lignocellulosic biomass and other biotechnological areas. Glucuronoyl esterases are considered to play a unique role as accessory enzymes in lignocellulosic material biodegradation by cleaving the covalent ester linkage between 4-O-methyl-D-glucuronic acid (MeGlcA) and lignin in lignin-carbohydrate complexes (LCCs). Two glucuronoyl esterases from T. terrestris named TtGE1 and TtGE2 were expressed in Pichia pastoris. Both esterases displayed features of thermophilic enzymes, with the optimal temperature at 45 °C and 55 °C. TtGE1 and TtGE2 exhibited activity towards methyl (4-nitrophenyl β-D-glucopyranosid) uronate (Me-GlcA-pNP) but no catalytic activity to benzyl-D-glucuronate (BnzGlcA), indicating the difference in substrate specificity from previously studied fungal GEs. A substantial increase in the release of monomeric sugars and glucuronic acid from autohydrolysis of corn bran was observed by the supplementing TtGEs into commercial xylanase; the results clearly demonstrated that the TtGEs played a significant role in this degradation process. This research on TtGEs enriches our knowledge of this novel class of fungal GEs. These newly characterized TtGEs could be used as promising accessory enzymes to improve the hydrolysis efficiency of commercial enzymes in saccharification of lignocellulosic materials due to their thermophilic characteristics.

Laccase-like multicopper oxidases (LMCOs) are a heterogeneous group of oxidases, acting mainly on phenolic compounds and which are widespread among many microorganisms, including Basidiomycetes and Ascomycetes. Here, we report the cloning, heterologous expression, purification and characterization of a novel LMCO from the thermophilic fungus Thermothelomyces thermophila. The 1953 bp lmco gene sequence comprises of 3 exons interrupted by 2 introns and according to the LccED database the translated sequence belongs to superfamily 6 of multicopper oxidases. After removal of the introns, the gene was transformed into Pichia pastoris, under the control of the alcohol oxidase (AOX1) promoter. The heterologous enzyme was purified with an apparent molecular weight of 80 kDa. TtLMCO1 displayed optimum activity at pH 4 and 50 °C and appeared thermostable up to 50 °C. A variety of phenolic compounds were oxidized by TtLMCO1, including standard laccase substrates such as ABTS and 2,6 dimethoxyphenol. The UV/Vis spectrum of purified TtLMCO1 indicates that it belongs to yellow laccase-like oxidases. The enzyme was used for the bioconversion of 2',3,4-trihydroxychalcone to 3',4'-dihydroxy-aurone, a bioactive aurone recently shown to possess inhibitory activity against several isoforms of the histone deacetylase complex (HDAC). Overall, the thermophilic yellow LMCO TtLMCO1 presents a number of superior properties with potential use in industrial biocatalysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and is an important part of the natural carbon cycle. Although induction of cellulases has been described for some saprobic filamentous fungi, the regulation of cellulase transcription is complex and many aspects are still poorly understood. Here, we identified and characterized the novel cellulase regulation factor NcCLR-4 in Neurospora crassa and its ortholog MtCLR-4 in Myceliophthora thermophila. Deletion of CLR-4 resulted in similarly defective cellulolytic enzyme production and activities. Transcriptome analyses of ΔNcclr-4/ΔMtclr-4 revealed the down-regulation of genes encoding (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1) and key genes in the cAMP signaling pathway such as adenylate cyclase Nccr-1. Intracellular cAMP levels were markedly lower in ΔNcclr-4/ΔMtclr-4 than in wild-type during cellulose utilization. In electrophoretic mobility shift (EMSA) and DNase I footprinting assays, NcCLR-4/MtCLR-4 can directly bound to the promoters of Nccr-1/Mtcr-1 (encoding adenylyl cyclase). EMSAs also demonstrated that NcCLR-4/MtCLR-4 could directly bound to clr-1 (encoding a key cellulase regulator), Mtclr-2 and Mtxyr-1 (encoding biomass deconstruction regulators). These findings about the novel cellulase expression regulators NcCLR-4 and MtCLR-4 enrich our understanding of how cellulose degradation is regulated and provide new targets for engineering fungi to deconstruct plant biomass in biorefineries., (© 2018 John Wiley & Sons Ltd.)

Lipidic cubic phase systems (LCPs) are excellent carriers for immobilized enzymes due to their biocompatibility and well-defined nanoporous structure. Lipidic cubic phases act as a convenient matrix to incorporate enzymes and hold them in the vicinity of electrode surfaces in their fully active forms. Corynascus thermophilus cellobiose dehydrogenase (CtCDH) was trapped in a monoolein cubic phase, which increased not only its stability, but also its catalytic performance with both enhanced mediated and direct electron transfer with electrodes. For studies of mediated electron transfer, three mediators with different formal potentials (E°') were employed: horse-heart cytochrome c (cyt c), electron acceptor active with the cytochrome domain of CtCDH, and 2,6-dichlorophenolindophenol (DCPIP) as well as hexaammineruthenium(II) chloride [Ru(NH 3 )Cl 2 ] both electron acceptors with the dehydrogenase domain. Ru(NH 3 )Cl 2 , having the most negative E°' of -0.138V vs. Ag|AgCl at pH7.5, gave a catalytic current for lactose oxidation of 32.10μAcm -2 in MOPS buffer at pH7.5. The process carried out in the same solution but under direct electron conditions transfer resulted in a catalytic current of 9.22μAcm -2 . Electrodes covered with CtCDH in a LCP film retained their catalytic activity after 28days showing a slightly increased current density after 6days., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There are many diseases linked to oxidative stress, including cancer. Importantly, endogenous antioxidants are insufficient to protect against this process. Peptides derived from food proteins produced by hydrolysis have been investigated as exogenous antioxidants. The present study aimed to identify novel peptides with antioxidant potential produced from egg and milk proteins hydrolysis with two new fungal proteases isolated from Eupenicillium javanicum and Myceliophthora thermophila . The degree of hydrolysis at several time points was calculated and correlated to DPPH scavenging and metal chelating assays, all hydrolysates presented antioxidant activity. Casein hydrolyzed by the M. thermophila protease showed the best antioxidant activity. The identified sequences showed that the proportions of amino acids that influence antioxidant activity support the antioxidant assay. Our data reveal the conditions necessary for the successful generation of antioxidant peptides using two novel fungal proteases. This opens a potential new avenue for the design and manufacture of antioxidant molecules.

Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0 °C and 80.9 °C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70 °C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In present study, laccase from Myceliophthora thermophila, was immobilized on epoxy-functionalized silica via one-pot three component reaction as a novel and efficient method for immobilization. The results revealed immobilization of 50 mg of M. thermophila laccase on 1 g of the supports in presence of cyclohexyl isocyanide after 12 h of incubation. The immobilized enzyme exhibited notable activity (50 U/g) with improved stability toward pH, temperature and organic solvents. Laccase derivative was used for decolorization of five textile dyes (acid orange 156, acid red 52, coomassie brilliant blue, methyl violet, malachite green) with or without the redox mediators such as 1-hydroxybenzotriazole (HBT), catechol, syringaldehyde and N-hydroxyphthalimide (HPT). The results showed that laccase/mediator systems were effective biocatalysts for the treatment of textile dyes., (Copyright © 2018. Published by Elsevier B.V.)

Polyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome of Thermothelomyces thermophila and expressed in Pichia pastoris The recombinant enzyme ( Tt PPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein. Tt PPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model of Tt PPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant of Tt PPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type. IMPORTANCE A novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis., (Copyright © 2018 Nikolaivits et al.)

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1&#95;3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function-in particular towards large substrates., Competing Interests: Novozymes A/S is producing MtL laccase on a commercial basis. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Thermophilic fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive option for use as cell factory to produce chemicals directly from renewable polysaccharides, such as starch, rather than monomer glucose. To date, there has been no transcriptomic analysis of this thermophilic fungus on starch. This study determined the transcriptomic profile of M. thermophila responding to soluble starch and a 342-gene set was identified as a "starch regulon", including the major amylolytic enzyme (Mycth&#95;72393). Its overexpression led to increased amylase activities on starch by 35%. Furthermore, overexpressing the key amylolytic enzyme regulator AmyR in M. thermophila significantly increased amylase activity by 30%. Deletion of amyR by the CRISPR/Cas9 system led to the relief of carbon catabolite repression and 3-fold increased lignocellulase activities on cellulose. This study will accelerate rational fungal strain engineering for biochemical production from biomass substrates such as raw corn starch and even crop straw., (Copyright © 2018 Elsevier Ltd. All rights reserved.)