The present work is aimed to hypothesize that fungal endophytes associated with wheat (Triticum aestivum L.) plants can play a variety of roles in biotechnology including plant growth. Out of 67 fungal isolates, five maximum drought-tolerant isolates were used to check their various plant growth-promoting traits, antioxidants, and antifungal activities under secondary screening. Fungal isolate #8TAKS-3a exhibited the maximum drought tolerance capacity and potential to produce auxin, gibberellic acid, ACC deaminase, phosphate, zinc solubilization, ammonia, siderophore, and extracellular enzyme activities followed by #6TAKR-1a isolate. In terms of antioxidant activities, #8TAKS-3a culture also showed maximum DPPH scavenging, total antioxidant, and NO-scavenging activities. However, #6TAKR-1a exhibited maximum total flavonoid content, total phenolic content, and Fe-reducing power and also the highest growth inhibition of Aspergillus niger (ITCC 6152) and Colletotrichum sp. (ITCC 6152). Based on morphological characters and multi-locus phylogenetic analysis of the nuc rDNA internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS), β-tubulin (TUB 2), and RNA polymerase II second largest subunit (RPB2) genes, potent fungal isolate #8TAKS-3a was identified as Talaromyces purpureogenus. Under the in vitro conditions, T. purpureogenus (#8TAKS-3a) was used as a bioinoculant that displayed a significant increase in various physio-biochemical growth parameters under normal and stressed conditions (p < 0.05). Our results indicate that drought stress-tolerant T. purpureogenus can be further used for field testing as a growth promoter.
- MeSH
- antioxidancia MeSH
- endofyty MeSH
- fylogeneze MeSH
- období sucha MeSH
- pšenice MeSH
- semenáček * MeSH
- Talaromyces * genetika MeSH
- Publikační typ
- časopisecké články MeSH
Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 μg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi.
- MeSH
- endofyty chemie genetika růst a vývoj MeSH
- kokultivační techniky MeSH
- listy rostlin mikrobiologie MeSH
- Phanerochaete chemie genetika růst a vývoj metabolismus MeSH
- Tabebuia mikrobiologie MeSH
- Talaromyces chemie genetika růst a vývoj metabolismus MeSH
- terpeny analýza metabolismus farmakologie MeSH
- trypanocidální látky analýza metabolismus farmakologie MeSH
- Trypanosoma cruzi účinky léků růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.
- MeSH
- beta-N-acetylhexosaminidasy chemie metabolismus MeSH
- glykosidy metabolismus MeSH
- glykosylace MeSH
- kinetika MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- oligosacharidy metabolismus MeSH
- substrátová specifita MeSH
- Talaromyces enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- blastomykóza diagnóza patofyziologie terapie MeSH
- chromoblastomykóza diagnóza patofyziologie terapie MeSH
- histoplazmóza * diagnóza patofyziologie terapie MeSH
- kokcidioidomykóza * diagnóza patofyziologie terapie MeSH
- kryptokokóza * diagnóza patofyziologie terapie MeSH
- lidé MeSH
- lobomykóza patofyziologie terapie MeSH
- mukormykóza diagnóza patofyziologie terapie MeSH
- mycetom diagnóza patofyziologie terapie MeSH
- parakokcidioidomykóza diagnóza patofyziologie terapie MeSH
- phaeohyphomykóza patofyziologie terapie MeSH
- rinosporidióza etiologie patofyziologie terapie MeSH
- sporotrichóza * diagnóza patofyziologie terapie MeSH
- Talaromyces MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 - a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.
- MeSH
- Aspergillus enzymologie MeSH
- beta-N-acetylhexosaminidasy chemie genetika izolace a purifikace metabolismus MeSH
- chromatografie iontoměničová MeSH
- disacharidy metabolismus MeSH
- exprese genu MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- Pichia genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- Talaromyces enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
A filamentous fungus displaying high cellulase activity was isolated from a compost heap with triticale (a wheat-rye hybrid) as the main constituent. It was preliminarily identified as a Talaromyces pinophilus species. A 2577 base pair β-glucosidase gene was cloned from complementary DNA and heterologously expressed in Saccharomyces cerevisiae. The recombinant β-glucosidase production profile was assessed and compared to that of the Saccharomycopsis fibuligera β-glucosidase which served as a benchmark. The enzyme was also characterised in terms of pH and temperature tolerance as well as response to inhibitors. Maximal extracellular β-glucosidase activity of 0.56 nkat/mg total protein was measured using p-nitrophenyl-β-D-glucopyranoside as substrate. The recombinant protein displayed a pH optimum of 4.0, and good thermostability as 70% of maximal enzyme activity was retained after 1 h at 60 °C. Activity of the recombinant β-glucosidase was adversely affected by the presence of glucose and ethanol at higher concentrations while xylose had no effect. The expression of the T. pinophilus β-glucosidase did not reach the same titres as for the benchmark; however, in the context of constructing a yeast strain for bioethanol production in a consolidated bioprocess, the enzyme may still show good potential.
- MeSH
- ethanol metabolismus MeSH
- exprese genu * MeSH
- fenotyp MeSH
- fermentace MeSH
- genotyp MeSH
- glukosylceramidasa genetika metabolismus MeSH
- klonování DNA MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Talaromyces enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Klíčová slova
- isavuconazol,
- MeSH
- antifungální látky * farmakologie terapeutické užití MeSH
- dexamethason farmakologie terapeutické užití MeSH
- kongresy jako téma MeSH
- kryptokoková meningitida * farmakoterapie mortalita MeSH
- lidé MeSH
- mykózy * farmakoterapie MeSH
- randomizované kontrolované studie jako téma MeSH
- Talaromyces účinky léků MeSH
- vzácné nemoci MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- zprávy MeSH
BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.
- MeSH
- beta-N-acetylhexosaminidasy chemie metabolismus MeSH
- fylogeneze MeSH
- glykosylace MeSH
- katalytická doména MeSH
- kinetika MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- reprodukovatelnost výsledků MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- substrátová specifita MeSH
- Talaromyces enzymologie MeSH
- výpočetní biologie * MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The β-N-acetylhexosaminidase from Talaromyces flavus has a remarkable synthetic ability, processing even carbohydrates with various functionalities. Its broader use is partially hampered by low-yield production in the native fungus. Here, we present an optimized 3-day production of this enzyme in the eukaryotic host of Pichia pastoris, in ca 10-fold higher volume activity (10 U/ml) and close-to-perfect purity (one chromatographic step needed). Importantly, the recombinant enzyme features the same biochemical and catalytic properties, including the syntheses with derivatized carbohydrate substrates. This is the first example of the overexpression of a fungal β-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.
- MeSH
- beta-N-acetylhexosaminidasy genetika izolace a purifikace MeSH
- exprese genu MeSH
- klonování DNA MeSH
- komplementární DNA genetika MeSH
- Pichia genetika MeSH
- rekombinantní proteiny genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- Talaromyces enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
