RESUMO
The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels.
Assuntos
Acetilesterase/genética , Acetilesterase/metabolismo , Talaromyces/enzimologia , Talaromyces/genética , Acetilesterase/química , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Alinhamento de Sequência , TemperaturaRESUMO
Fungi's ability to convert organic materials into bioactive products offers environmentally friendly solutions for diverse industries. In the nanotechnology field, fungi metabolites have been explored for green nanoparticle synthesis. Silver nanoparticle (AgNP) research has grown rapidly over recent years mainly due to the enhanced optical, antimicrobial and anticancer properties of AgNPs, which make them extremely useful in the biomedicine and biotechnology field. However, the biological synthesis mechanism is still not fully established. Therefore, this study aimed to evaluate the combined effect of time, temperature and pH variation in AgNP synthesis using three different fungi phyla (Ascomycota, Basidiomycota and Zygomycota) represented by six different fungi species: Cladophialophora bantiana (C. bantiana), Penicillium antarcticum (P. antarcticum), Trametes versicolor (T. versicolor), Trichoderma martiale (T. martiale), Umbelopsis isabellina (U. isabellina) and Bjerkandera adusta (B. adusta). Ultraviolet-visible (UV-Vis) spectrophotometry and transmission electron microscopy (TEM) results demonstrated the synthesis of AgNPs of different sizes (3 to 17 nm) and dispersity percentages (25 to 95%, within the same size range) using fungi extracts by changing physicochemical reaction parameters. It was observed that higher temperatures (90 °C) associated with basic pH (9 and 12) favoured the synthesis of monodisperse small AgNPs. Previous studies demonstrated enhanced antibacterial and anticancer properties correlated with smaller nanoparticle sizes. Therefore, the biologically synthesised AgNPs shown in this study have potential as sustainable substitutes for chemically made antibacterial and anticancer products. It was also shown that not all fungi species (B. adusta) secrete metabolites capable of reducing silver nitrate (AgNO3) precursors into AgNPs, demonstrating the importance of fungal screening studies.
RESUMO
Fungal biotechnology research has rapidly increased as a result of the growing awareness of sustainable development and the pressing need to explore eco-friendly options. In the nanotechnology field, silver nanoparticles (AgNPs) are currently being studied for application in cancer therapy, tumour detection, drug delivery, and elsewhere. Therefore, synthesising nanoparticles (NPs) with low toxicity has become essential in the biomedical area. The fungus Chaetomium thermophilum (C. thermophilum) was here investigated-to the best of our knowledge, for the first time-for application in the production of AgNPs. Transmission electronic microscopy (TEM) images demonstrated a spherical AgNP shape, with an average size of 8.93 nm. Energy-dispersive X-ray spectrometry (EDX) confirmed the presence of elemental silver. A neutral red uptake (NRU) test evaluated the cytotoxicity of the AgNPs at different inhibitory concentrations (ICs). A half-maximal concentration (IC50 = 119.69 µg/mL) was used to predict a half-maximal lethal dose (LD50 = 624.31 mg/kg), indicating a Global Harmonized System of Classification and Labelling of Chemicals (GHS) acute toxicity estimate (ATE) classification category of 4. The fungus extract showed a non-toxic profile at the IC tested. Additionally, the interaction between the AgNPs and the Balb/c 3T3 NIH cells at an ultrastructural level resulted in preserved cells structures at non-toxic concentrations (IC20 = 91.77 µg/mL), demonstrating their potential as sustainable substitutes for physical and chemically made AgNPs. Nonetheless, at the IC50, the cytoplasm of the cells was damaged and mitochondrial morphological alteration was evident. This fact highlights the fact that dose-dependent phenomena are involved, as well as emphasising the importance of investigating NPs' effects on mitochondria, as disruption to this organelle can impact health.
RESUMO
The genes encoding xylitol dehydrogenase (Texdh) and L: -arabitol dehydrogenase (Telad) are involved in the fungal pentose pathway and were isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli, and the products purified to homogeneity. TeXDH showed activity toward xylitol and D: -sorbitol. TeLAD was active with L: -arabitol, xylitol, and D: -sorbitol. Phylogenetic analysis showed TeLAD has evolved from D: -sorbitol dehydrogenase as a result of environmental adaptation. Substrate specificity studies indicate that TeXDH is likely to have evolved from the more broadly acting TeLAD. Texdh and Telad expression was inducible by the same carbon sources responsible for induction of genes involved in biomass degradation, suggesting for the first time a coordinated regulatory control mechanism for expression of genes encoding extracellular hydrolases and intracellular metabolic genes in the pentose utilization pathways of T. emersonii. These data also suggest that TeXDH and TeLAD may be valuable in the production of xylitol, L: -arabitol, and ethanol from renewable resources rich in pentose sugars.
Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Desidrogenase do Álcool de Açúcar/genética , Talaromyces/enzimologia , Talaromyces/genética , Sequência de Aminoácidos , Clonagem Molecular , D-Xilulose Redutase/química , D-Xilulose Redutase/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Humanos , Espaço Intracelular/enzimologia , Cinética , Dados de Sequência Molecular , Monossacarídeos/metabolismo , Filogenia , Análise de Sequência de DNA , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Talaromyces/citologiaRESUMO
Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k(cat)/K(m)=2.1 x 10(6) M(-1) s(-1)) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 degrees C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an alpha/beta hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii.
Assuntos
Aminopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , Talaromyces/genéticaRESUMO
Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.
Assuntos
Celulase/química , Fungos/enzimologia , Ligação Competitiva , Catálise , Celobiose/farmacologia , Celulase/antagonistas & inibidores , Celulase/isolamento & purificação , Celulose 1,4-beta-Celobiosidase , Estabilidade Enzimática , Glicosídeos/química , Cinética , Especificidade por SubstratoRESUMO
A coupling of the plasmon on the surface of metal nanoparticles with an incident photon enhances a broad range of useful optical phenomena, such as resonant light scattering (RLS), surface plasmon resonance (SPR) or Raman scattering. Due to these unique optical properties plasmonic nanostructures of different sizes and shapes have gained increasing popularity in areas such as cancer diagnosis, photothermal therapy as well as the imaging of living cells, detection of pathogens, biomolecules, metal ions, and the catalysis of various reactions in wet chemistry. This article reviews the current trends in the synthesis of plasmonic nanoparticles, particularly gold (AuNPs) and silver (AgNPs), using fungi as well as the proposed mechanisms for their mycofabrication. We provide an overview of the theoretical concepts of plasmonic nanoparticles which are sensitive electromagnetic responses that determine these nanoparticles applications.
Assuntos
Fungos/metabolismo , Ouro/metabolismo , Nanopartículas Metálicas/química , Prata/metabolismo , Coloides/química , Coloides/metabolismo , Ouro/química , Prata/químicaRESUMO
A new family of pi-basic chiral selectors has been developed and employed in the separation of enantiomers by liquid chromatography. These chiral selectors, derived from (S)-proline and designed from mechanistic considerations, show high levels of discrimination between the enantiomers of N-(3,5-dinitrobenzoyl)amino acid esters and amides. A considerable amount of chromatographic data has been assembled, all of it consistent with the proposed chiral recognition mechanism. Moreover, this mechanism is supported by induced chemical shift differences and intermolecular NOE data previously obtained in solution with an equimolar mixture of (S)-1 and (S)-2. A crystalline 1:1 complex of (S)-1 and (S)-2 has been obtained and analyzed by X-ray crystallography. The structure of this complex in the solid state illustrates the essential features of the mechanism proposed to account for chiral recognition between chiral stationary phase (CSP) 3 and the enantiomers of 2 and related analytes. In addition, the orientation of the two components in the solid state is in close agreement with the structure of the more stable diastereomeric complex deduced from solution-state NMR evidence relating to the same system.
RESUMO
Chiral stationary phases (CSPs) for liquid chromatography derived from N-(acyl)proline-3,5-dimethylanilides separate the enantiomers of N-(3,5-dinitrobenzoyl)-alpha-amino esters and amides with high levels of selectivity. These CSPs have been used to assemble a large body of chromatographic data which indirectly supports the validity of the mechanistic rationale originally used in the design of these CSPs. We herein report (1)H and (13)C chemical shift data obtained when the (S)-enantiomer of chiral solvating agent (CSA) 3, a soluble analogue of the selector used in CSP (S)-1, acts on each of the enantiomers of the dimethylamide of N-(3,5-dinitrobenzoyl)leucine, 2. The changes in chemical shift in the mixture of (S)-2 and (S)-3 support the existence of those interactions thought to be essential to chiral recognition in this system. In addition, significant intermolecular NOESY enhancements are observed in this mixture. These NOE data are consistent with the structure expected for the more stable diastereomeric adsorbate formed between (S)-2 and the (S)-proline-derived CSP 1. No intermolecular NOEs are observed for corresponding mixtures of the chiral solvating agent (S)-3 and (R)-2, the enantiomer least retained on (S)-CSP 1.
RESUMO
This work reports the first investigation of Remersonia thermophila hemicellulosic hydrolytic enzyme production, with subsequent purification of an extracellular endo-ß-1,4-xylanase (RtXyl) and its application in bread making. The research describes RtXyl purification from sorghum-induced submerged liquid cultures of this moderately thermophilic, aerobic, ascomycete fungus. The purified enzyme is a single subunit protein with a molecular mass of 42 kDa and exhibits glycosyl hydrolase family-10-like activity over a broad pH and temperature range. Optimal activity was measured at pH 6.0 and 65 °C respectively, which is suitable for bread making applications. Substrate specificity studies revealed that RtXyl is purely xylanolytic with no side-activities against other plant polysaccharides. The RtXyl catalytic efficiency (K cat/K m) was highest with oats spelt xylan (810.90 mg mL(-1) s(-1)), wheat arabinoxylan (809.52 mg mL(-1) s(-1)) and beechwood xylan (417.40 mg mL(-1) s(-1)) with less efficiency towards insoluble oats spelt xylan (236.40 mg mL(-1) s(-1)). Hydrolysis products analysed by thin layer chromatography yielded a range of xylosaccharides, predominantly xylotriose and xylobiose. RtXyl application in a basic wheat bread recipe at low dosages (0.297 XU/g) showed its suitability to increase loaf volume by 8.0 % compared with the control bread. RtXyl increased loaf softness by 19.6 % while reducing bread staling by 20.4 % up to 4 days of storage.
Assuntos
Ascomicetos/enzimologia , Pão , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Catálise , Estabilidade Enzimática , Hidrólise , Polissacarídeos/metabolismo , Triticum/química , Xilanos/metabolismoRESUMO
In this paper, we report new sequence data for secreted thermostable fungal enzymes from the un-sequenced xylanolytic filamentous fungus Talaromyces emersonii and reveal novel insights on the potential role of enzymes relevant as wheat dough improvers. The presence of known and de novo enzyme sequences were confirmed through NanoLC-ESI-MS/MS and resultant peptide sequences were identified using SWISS PROT databases. The de novo protein sequences were assigned identity based on homology to known fungal proteins. Other proteins were assigned function based on the limited T. emersonii genome coverage. This approach allowed the identification of enzymes with relevance as wheat dough improvers. Rheological examination of wheat dough and wheat flour components treated with the thermostable fungal enzyme cocktail revealed structural alterations that can be extrapolated to the baking process. Thermoactive amylolytic, xylanolytic, glucanolytic, proteolytic and lipolytic enzyme activities were observed. Previously characterized T. emersonii enzymes present included; ß-glucosidase, xylan-1,4-ß-xyloxidase, acetylxylan esterase, acid trehalase, avenacinase, cellobiohydrolase and endo-glucanase. De novo sequence analysis confirmed peptides as being; α-glucosidase, endo-1,4-ß-xylanase, endo-arabinase, endo-glucanase, exo-ß-1,3-glucanase, glucanase/cellulase, endopeptidase and lipase/acylhydrolase. Rheology tests using wheat dough and fractioned wheat flour components in conjunction with T. emersonii enzymes show the role of these novel biocatalysts in altering properties of wheat substrates. Enzyme treated wheat flour fractions showed the effects of particular enzymes on appropriate substrates. This proteomic approach combined with rheological characterization is the first such report to the authors' knowledge.
Assuntos
Pão , Tecnologia de Alimentos , Talaromyces/enzimologia , Triticum , Sequência de Aminoácidos , Biotecnologia , Pão/análise , Elasticidade , Estabilidade Enzimática , Microbiologia de Alimentos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutens/química , Dados de Sequência Molecular , Reologia , Amido/química , Talaromyces/genética , Triticum/química , Viscosidade , Xilanos/químicaRESUMO
We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the beta-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75 degrees C. The best mutant was the triple mutant whose unfolding temperature was improved by 9 degrees C leading to efficient microcrystalline cellulose hydrolysis at 80 degrees C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.
Assuntos
Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Mutagênese Sítio-Dirigida/métodos , Saccharomyces cerevisiae/genética , Talaromyces/enzimologia , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase/química , Expressão Gênica , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Estabilidade Proteica , TemperaturaRESUMO
In this study, novel extracellular thermozymes were produced by the thermophilic fungus Talaromyces emersonii (IMI 392299) on low-cost carbon inducers. This paper reports the cocktail characterization, substrate hydrolysis studies, and their application in baking. Relevant enzymes were optimally active at pH 4.5-5.0 and 70 degrees C. Model studies confirmed production of significant levels of yeast monosaccharide sugars during cereal flour hydrolysis. The "thermozyme cocktails" are thermostable secreted T. emersonii enzyme blends. In baking trials, these thermozyme cocktails showed significant improvements in bread quality with respect to hardness, staling, and loaf volume (p < 0.5). Thermozyme cocktail B- treated loaf volume was 23.2% greater than the control and 49.5% softer. Staling analysis showed that bread treated with cocktail B was 41.7% softer than the control. This is the first report of T. emersonii thermozymes positively influencing bread quality.
Assuntos
Manipulação de Alimentos , Proteínas Fúngicas/química , Talaromyces/enzimologia , Triticum/química , Pão/análise , Estabilidade Enzimática , Farinha/análise , Temperatura Alta , Concentração de Íons de Hidrogênio , HidróliseRESUMO
Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXR K271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.
Assuntos
Aldeído Redutase , Coenzimas/metabolismo , Proteínas Fúngicas , Especificidade por Substrato/genética , Talaromyces/enzimologia , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Coenzimas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , NADP/metabolismo , Alinhamento de Sequência , Xilose/metabolismoRESUMO
The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1' such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k(cat)/K(m)=2 x 10(5) m(-1) s(-1)). Maximum hydrolysis occurs at pH 3.4 and 50 degrees C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K(i)=1.5 nM), as well as a portion of the propeptide sequence, PT1 (K(i)=32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.
Assuntos
Endopeptidases/metabolismo , Endopeptidases/fisiologia , Regulação Fúngica da Expressão Gênica , Glutamina/química , Talaromyces/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Endopeptidases/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Pepstatinas/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
Recent sequencing of a number of fungal genomes has revealed the presence of multiple putative beta-glucosidases. Here, we report the cloning of two beta-glucosidase genes (bg1 and aven1), which have very different biological functions and represent two of a number of beta-glucosidases from Talaromyces emersonii. The bg1 gene, encoding a putative intracellular beta-glucosidase, shows significant similarity to other fungal glucosidases from glycosyl hydrolase family 1, known to be involved in cellulose degradation. Solka floc, methyl-xylose, gentiobiose, beech wood xylan, and lactose induced expression of bg1, whereas glucose repressed expression. A second beta-glucosidase gene isolated from T. emersonii, aven1, encodes a putative avenacinase, an enzyme that deglucosylates the anti-fungal saponin, avenacin, rendering it less toxic to the fungus. This gene displays high homology with other fungal saponin-hydrolysing enzymes and beta-glucosidases within GH3. A putative secretory signal peptide of 21 amino acids was identified at the N-terminus of the predicted aven1 protein sequence suggesting that this enzyme is extracellular. Furthermore, T. emersonii cultivated on oat plant biomass was shown to deglucosylate avenacin. The presence of the avenacinase transcript was confirmed by RT-PCR on RNA extracted from mycelia grown in the presence of avenacin. The expression pattern of aven1 on various carbon sources was distinctly different from that of bg1. Only methyl-xylose and gentiobiose induced transcription of aven1. Gentiobiose induces synthesis of a number of cellulase genes by T. emersonii and it may be a possible candidate for the natural cellulase inducer observed in Penicillium purpurogenum. This work represents the first report of an avenacinase gene from a thermophilic, saprophytic fungal source, and suggests that this gene is not exclusive to plant pathogens.
Assuntos
Clonagem Molecular , Temperatura Alta , Talaromyces/enzimologia , beta-Glucosidase/classificação , beta-Glucosidase/metabolismo , Sequência de Aminoácidos , Meios de Cultura , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Saponinas/metabolismo , Análise de Sequência de DNA , Talaromyces/genética , Talaromyces/crescimento & desenvolvimento , Talaromyces/metabolismo , beta-Glucosidase/química , beta-Glucosidase/genéticaRESUMO
Xylose reductase is involved in the fi rst step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purifi ed to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of fi ve carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXR K271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.
RESUMO
Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.
Assuntos
Malato Desidrogenase/genética , Malato Desidrogenase/isolamento & purificação , Mitocôndrias/enzimologia , Talaromyces/enzimologia , Talaromyces/genética , Sequência de Aminoácidos , Northern Blotting , Cromatografia , Clonagem Molecular , Escherichia coli , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Dados de Sequência Molecular , RNA/análise , RNA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Talaromyces/citologia , TemperaturaRESUMO
The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of approximately 60 A x 40 A x 50 A and an overall beta-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.