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1.
World J Microbiol Biotechnol ; 39(1): 21, 2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36422714

RESUMO

Given the important pharmacological activity of ginsenoside Rd but its low content in plants, the production of Rd by enzymatic transformation is of interest. In this study, a ß-xylosidase gene Ta-XylQS from Thermoascus aurantiacus was cloned and overexpressed in Komagataella phaffii. Purified recombinant Ta-XylQS specifically hydrolyzes substrates with xylosyl residues at the optimal pH of 3.5 and temperature of 60 °C. This study established a process for producing Rd by transforming ginsenoside Rb3 in the saponins of Panax notoginseng leaves via recombinant Ta-XylQS. After 60 h, 3 g L- 1 of Rb3 was transformed into 1.46 g L- 1 of Rd, and the maximum yield of Rd reached 4.31 g kg- 1 of Panax notoginseng leaves. This study is the first report of the biotransformation of ginsenoside Rb3 to Rd via a ß-xylosidase, and the established process could potentially be adopted for the commercial production of Rd from Rb3.


Assuntos
Panax notoginseng , Thermoascus , Biotransformação , Folhas de Planta
2.
J Biotechnol ; 347: 1-8, 2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35151712

RESUMO

Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei, L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a supplement to stimulate the growth of probiotic strains.


Assuntos
Probióticos , Saccharum , Thermoascus , Celulose , Endo-1,4-beta-Xilanases/genética , Glucuronatos , Hidrólise , Oligossacarídeos , Xilanos
3.
Biomolecules ; 11(12)2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34944533

RESUMO

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.


Assuntos
Diospyros/química , Inibidores Enzimáticos/farmacologia , Sucos de Frutas e Vegetais/microbiologia , Oxigenases de Função Mista/antagonistas & inibidores , Thermoascus/enzimologia , Hidrolases de Éster Carboxílico/efeitos adversos , Diospyros/microbiologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fermentação , Sucos de Frutas e Vegetais/análise , Proteínas Fúngicas/antagonistas & inibidores , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hidrolases/antagonistas & inibidores , Polietilenoglicóis/efeitos adversos , Taninos/farmacologia , Thermoascus/efeitos dos fármacos
4.
An Acad Bras Cienc ; 93(1): e20191349, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33787686

RESUMO

Microbial ß-glucosidases can be used in several industrial processes, including production of biofuels, functional foods, juices, and beverages. In the present work, production of ß-glucosidase by solid state cultivation of the fungus Thermoascus crustaceus in a low-cost cultivation medium (comprising agroindustrial residues) was evaluated. The highest production of ß-glucosidase, about 415.1 U/g substrate (or 41.51 U/mL), was obtained by cultivating the fungus in wheat bran with 70% humidity, during 96 h at 40°C. The enzymatic activity was optimum at pH 4.5 and 65°C. ß-Glucosidase maintained its catalytic activity when incubated at a pH range of 4.0-8.0 and temperature of 30-55°C. The enzyme was strongly inhibited by glucose; even when the substrate and glucose concentrations were equal, the inhibition was not reversed, suggesting a non-competitive inhibition. In the presence of up to 10% ethanol, ß-glucosidase maintained its catalytic activity. In addition to ß-glucosidase, the enzymatic extract showed activity of 36 U/g for endoglucanase, 256.2 U/g for xylanase, and 18.2 U/g for ß-xylosidase. The results allow to conclude that the fungus T. crustaceus has considerable potential for production of ß-glucosidase and xylanase when cultivated in agroindustrial residues, thereby reducing the cost of these biocatalysts.


Assuntos
Celulase , Thermoascus , Eurotiales , Fermentação , Concentração de Íons de Hidrogênio , Thermoascus/metabolismo , beta-Glucosidase
5.
Biotechnol Lett ; 42(10): 1897-1905, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32557119

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.


Assuntos
Proteínas Fúngicas , Oxigenases de Função Mista , Thermoascus/enzimologia , Biomassa , Celulases/química , Celulases/isolamento & purificação , Celulases/metabolismo , Celulose/análise , Celulose/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Hidrólise , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo
6.
Braz J Microbiol ; 51(1): 107-123, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31776864

RESUMO

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer ß-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a ß-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.


Assuntos
Fermentação , Saccharomyces cerevisiae/genética , Thermoascus/enzimologia , beta-Glucosidase/biossíntese , Biocombustíveis , Biomassa , Engenharia Genética , Microbiologia Industrial , Lignina/metabolismo , Thermoascus/genética , beta-Glucosidase/genética
7.
Chemistry ; 26(2): 454-463, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31603264

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose which have large practical application in the process of refining biomass. The catalytic mechanism of LPMOs still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t1/2 =6-8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-CuI reacts with sub-equimolar amounts of H2 O2 . UV/Vis absorption spectroscopy, electron paramagnetic resonance, resonance Raman and stopped-flow spectroscopy suggest that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). Additionally, activity assays in the presence of sub-equimolar amounts of H2 O2 showed an increase in the LPMO oxidation of phosphoric acid swollen cellulose. Accordingly, this suggests that the long-lived copper-dependent intermediate could be part of the catalytic mechanism for LPMOs. The observed intermediate offers a new perspective into the oxidative reaction mechanism of TaLPMO9A and hence for the biomass oxidation and the reactivity of copper in biological systems.


Assuntos
Cobre/química , Oxigenases de Função Mista/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/química , Cinética , Oxigenases de Função Mista/química , Oxirredução , Thermoascus/enzimologia
8.
Appl Microbiol Biotechnol ; 103(14): 5739-5750, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31152202

RESUMO

Auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) show significant synergism with cellulase in cellulose degradation. In recent years, there have been many reports on AA9 LPMOs; however, the identification of efficient and thermostable AA9 LPMOs with broad potential for industrial applications remains necessary. In this study, a new AA9 LPMO from Talaromyces cellulolyticus, named TcAA9A, was identified. An analysis of the oxidation products of phosphoric acid-swollen cellulose categorized TcAA9A as a type 3 AA9 LPMO, and TcAA9A exhibited a better synergistic effect with cellulase from Trichoderma reesei than what is seen with TaAA9A, a well-studied AA9 LPMO from Thermoascus aurantiacus. Two AA9 LPMOs were successfully expressed in T. reesei, and the transformants were named TrTcAA9A and TrTaAA9A. The activities and thermostabilities of the AA9 LPMOs in TrTcAA9A were higher than those of the AA9 LPMOs in TrTaAA9A or the parent. The enzyme solution of TrTcAA9A was more efficient than that of the parent or TrTaAA9A for the degradation of Avicel and delignified corncob residue. Thus, TcAA9A showed a better performance than TaAA9A in T. reesei cellulase cocktails. This study may offer an innovative solution for improving enzyme cocktail activity for lignocellulosic degradation.


Assuntos
Proteínas Fúngicas/metabolismo , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Thermoascus/enzimologia , Celulase/metabolismo , Celulose/metabolismo , Estabilidade Enzimática , Oxirredução , Temperatura , Trichoderma/metabolismo
9.
Sheng Wu Gong Cheng Xue Bao ; 34(12): 1996-2006, 2018 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-30584710

RESUMO

Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene, Tcxyn10a, was cloned from Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in Pichia pastoris GS115. The recombinant protein was then purified and characterized. The pH and temperature optima of TcXyn10A were determined to be 5.0 and 65-70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0-11.0) or after 1-h treatment at 60 °C. The specific activities of TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose and lichenan were (1 480±26) U/mg, (2 055±28) U/mg, (7.4±0.2) U/mg and (10.9±0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional TcXyn10A has a single catalytic domain, in which the substrate xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.


Assuntos
Celulase , Thermoascus , Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Pichia , Especificidade por Substrato
10.
Carbohydr Res ; 469: 31-37, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30216845

RESUMO

Fermentation conditions for ß-1,3-1,4-glucanase (TaGlu34) production in submerged culture by a thermophilic fungus, Thermoascus aurantiacus CAU830 were optimized. The highest enzyme activity of 3741 U/mL was obtained, and the crude enzyme was purified to homogeneity with a purification fold of 7.3 and a recovery yield of 11.6%. The molecular mass of the purified enzyme was estimated to be approximately 34 kDa on SDS-PAGE. TaGlu34 was most active at pH 6.0 and 75 °C, respectively. It showed excellent thermostability with thermal denaturing half-lives of 209, 130 and 69 min at 50, 60 and 70 °C, respectively. TaGlu34 exhibited strict substrate specificity towards barley ß-glucan (13,527 U/mg), oat ß-glucan (12,502 U/mg) and lichenan (9225 U/mg), but displayed no activity on other tested polysaccharides including laminarin, xylan, pullulan, CMC and starch. TaGlu34 hydrolyzed barley ß-glucan and lichenan to yield both mainly disaccharide and trisaccharide, suggesting that it should be an endo type ß-1,3-1,4-glucanase. Furthermore, TaGlu34 efficiently degraded the ß-glucan component in oat bran to produce mainly oligosaccharides with degrees of polymerization (DP) 3-5, with the highest conversion ratio of 47.1%. The high yield and excellent enzymatic properties of TaGlu34 may make it a good candidate in industries.


Assuntos
Avena/química , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Temperatura , Thermoascus/enzimologia , Estabilidade Enzimática , Glucanos/química , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Oligossacarídeos/química , Especificidade por Substrato
11.
Mol Biotechnol ; 60(10): 736-748, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30076532

RESUMO

Thermostable cellulases offer several advantages like higher rates of substrate hydrolysis, lowered risk of contamination, and increased flexibility with respect to process design. In the present study, a thermostable native endoglucanase nEG (EC 3.2.1.4) was purified and characterized from T. aurantiacus RCKK. Further, it was cloned in P. pastoris X-33 and processed for over expression. Expression of recombinant endoglucanase (rEG) of molecular size ~ 33 kDa was confirmed by SDS-PAGE and western blotting followed by in gel activity determination by zymogram analysis. Similar to nEG, the purified rEG was characterized to harbor high thermostability while retaining 50% of its initial activity even after 6- and 10-h incubation at 80 and 70 °C, respectively, and exhibited considerable stability in pH range 3.0-7.0. CD spectroscopy revealed more than 20% ß-sheets in protein structure consistently when incubated upto 85 °C as a speculated reason for protein high thermostability. Interestingly, both nEG and rEG were found tolerant up to 10% of the presence of 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. Values of the catalytic constants Km and Vmax for rEG were recorded as 2.5 mg/ml and 303.4 µmol/mg/min, respectively. Thermostability, pH stability, and resistance to the presence of ionic liquid signify the potential applicability of present enzyme in cellulose hydrolysis and enzymatic deinking of recycled paper pulp.


Assuntos
Celulase/genética , Celulase/metabolismo , Pichia/crescimento & desenvolvimento , Thermoascus/enzimologia , Técnicas de Cultura Celular por Lotes , Celulase/química , Clonagem Molecular , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peso Molecular , Pichia/genética , Engenharia de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , Thermoascus/química , Thermoascus/genética
12.
Biomol NMR Assign ; 12(2): 357-361, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30117034

RESUMO

The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.


Assuntos
Apoenzimas/química , Apoenzimas/metabolismo , Celulose/metabolismo , Oxigenases de Função Mista/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxigenases de Função Mista/química , Thermoascus/enzimologia
13.
Protein Sci ; 27(9): 1636-1650, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29971843

RESUMO

The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pKa of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H2 O2 boost LPMO activity, whereas excess H2 O2 leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H2 O2 and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.


Assuntos
Histidina/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Aspergillus oryzae/metabolismo , Biocatálise , Histidina/química , Metilação , Oxigenases de Função Mista/química , Oxirredução , Pichia/metabolismo , Polissacarídeos/química , Processamento de Proteína Pós-Traducional , Thermoascus/enzimologia
14.
Int J Biol Macromol ; 109: 1270-1279, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175163

RESUMO

The thermostable fungus, Thermoascus aurantiacus M-2, which produces a novel acidophilic and thermostable xylanase was isolated and identified based on its morphology and comparison of the internal transcribed spacer rDNA gene sequence. The culture conditions and components of medium were optimized for T. aurantiacus M-2 to produce xylanase. T. aurantiacus M-2 produced xylanase at a maximum level of 39.07 U/mL after 8-d fermentation at 45 °C in the optimized medium. The purified xylanase produced by T. aurantiacus M-2 has a relative molecular mass of approximately 31.0 kD. The characteristics of purified xylanase were investigated. The purified T. aurantiacus xylanase exhibited maximum activity at 75 °C and pH 5.0, and it was stable after treatment at a pH range from 2.0 to 10.0 or a temperature range from 30 °C to 80 °C for 2-h. Mn2+ and Ag+ enhanced xylanase activity to 120.0% and 119.6%, respectively, while Mn2+ had the highest inhibition ratio, with a residual activity of 20.7%. This study provided a foundation for scaled-up production and application of xylanase.


Assuntos
Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Thermoascus/enzimologia , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Ativação Enzimática , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Nitrogênio/metabolismo , Fenótipo , Especificidade por Substrato , Temperatura , Thermoascus/genética , Thermoascus/crescimento & desenvolvimento
15.
Rev. iberoam. micol ; 34(4): 225-228, oct.-dic. 2017. ilus
Artigo em Inglês | IBECS | ID: ibc-168717

RESUMO

Background. Fungal peritonitis is a relatively uncommon infection in peritoneal dialysis patients. However, it can be associated with significant morbimortality. In recent reports, Candida species and other filamentous fungi have been reported as being aetiological agents. Thermoascus species are ubiquitous, thermophilic fungi, with an anamorph in the Paecilomyces genus. Here we present the first report of fungal peritonitis by Thermoascus crustaceus from Chile. Case report. We present the case of an 83-year-old female patient, with a history of cholecystectomy, hernia repair, severe arterial hypertension, hip and knee osteoarthritis and several episodes of peritoneal dialysis with a cloudy exudate. Bacterial cultures were negative. In addition, a history of two months with intermittent fever peaks mainly in the evening was reported. Blood culture bottles inoculated with peritoneal fluid revealed the presence of fungal growth. Morphological and molecular studies allowed us to identify the aetiological agent as Thermoascus crustaceus. An antifungal susceptibility test was performed using the M38-A2 method, developed by the Clinical and Laboratory Standards Institute (CLSI). The MIC values to amphotericin B, itraconazole, voriconazole and echinochandins were 0.5, 0.25, 0.25 and 0.125μg/ml, respectively. Antifungal treatment with amphotericin B was prescribed, with good patient progress. Conclusions. Fungal peritonitis is a very rare entity. Moreover, the spectrum of fungal pathogens continues to expand, a reason for which morphological and molecular studies are necessary for a rapid diagnosis (AU)


Antecedentes. La peritonitis fúngica es una infección bastante infrecuente en pacientes en diálisis peritoneal. Sin embargo, puede estar relacionada con una morbimortalidad considerable. En informes recientes, se han registrado las especies de Candida y algunos hongos filamentosos como agentes etiológicos. Thermoascus es un género de hongos ubicuos, termófilos, que tienen su anamorfo en el género Paecilomyces. A continuación presentamos el primer caso de peritonitis fúngica por Thermoascus crustaceous de Chile. Caso clínico. Presentamos el caso de una mujer de 83 años con antecedentes de colecistectomía, hernia, hipertensión arterial grave, artrosis de cadera y rodilla, y varios episodios de diálisis peritoneal con una efusión turbia. Los cultivos bacterianos fueron negativos. Además, la paciente había presentado durante los dos últimos meses picos febriles intermitentes, principalmente por la noche. Los hemocultivos inoculados con líquido peritoneal revelaron el crecimiento de un micelio. Los estudios morfológicos y moleculares permitieron identificar el agente etiológico como Thermoascus crustaceous. Para el estudio de sensibilidad antifúngica se utilizó el método M38-A2, desarrollado por el Clinical and Laboratory Standards Institute. Las CMI obtenidas de anfotericina B, itraconazol, voriconazol y equinocandinas fueron 0,5; 0,25; 0,25, y 0,125 μg/ml, respectivamente. El tratamiento antifúngico con anfotericina B fue el indicado, con una buena evolución de la paciente. Conclusiones. La peritonitis fúngica es una entidad muy poco frecuente. Además, el espectro de hongos patógenos continúa en expansión, razón por la cual los estudios morfológicos y moleculares son necesarios para el diagnóstico rápido (AU)


Assuntos
Humanos , Feminino , Idoso de 80 Anos ou mais , Peritonite/microbiologia , Diálise Peritoneal/efeitos adversos , Thermoascus/isolamento & purificação , Insuficiência Renal Crônica/terapia , Micoses/microbiologia
16.
Rev Iberoam Micol ; 34(4): 225-228, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28625762

RESUMO

BACKGROUND: Fungal peritonitis is a relatively uncommon infection in peritoneal dialysis patients. However, it can be associated with significant morbimortality. In recent reports, Candida species and other filamentous fungi have been reported as being aetiological agents. Thermoascus species are ubiquitous, thermophilic fungi, with an anamorph in the Paecilomyces genus. Here we present the first report of fungal peritonitis by Thermoascus crustaceus from Chile. CASE REPORT: We present the case of an 83-year-old female patient, with a history of cholecystectomy, hernia repair, severe arterial hypertension, hip and knee osteoarthritis and several episodes of peritoneal dialysis with a cloudy exudate. Bacterial cultures were negative. In addition, a history of two months with intermittent fever peaks mainly in the evening was reported. Blood culture bottles inoculated with peritoneal fluid revealed the presence of fungal growth. Morphological and molecular studies allowed us to identify the aetiological agent as Thermoascus crustaceus. An antifungal susceptibility test was performed using the M38-A2 method, developed by the Clinical and Laboratory Standards Institute (CLSI). The MIC values to amphotericin B, itraconazole, voriconazole and echinochandins were 0.5, 0.25, 0.25 and 0.125µg/ml, respectively. Antifungal treatment with amphotericin B was prescribed, with good patient progress. CONCLUSIONS: Fungal peritonitis is a very rare entity. Moreover, the spectrum of fungal pathogens continues to expand, a reason for which morphological and molecular studies are necessary for a rapid diagnosis.


Assuntos
Infecções Relacionadas a Cateter/microbiologia , Micoses/microbiologia , Diálise Peritoneal , Peritonite/microbiologia , Thermoascus/isolamento & purificação , Idoso de 80 Anos ou mais , Anfotericina B/uso terapêutico , Antifúngicos/uso terapêutico , Líquido Ascítico/microbiologia , Chile , DNA Fúngico/genética , Farmacorresistência Fúngica , Feminino , Humanos , Técnicas de Tipagem Micológica , Micoses/tratamento farmacológico , Micoses/etiologia , Diálise Peritoneal/instrumentação , Peritonite/tratamento farmacológico , Peritonite/etiologia , Filogenia , Thermoascus/classificação , Thermoascus/genética
17.
Inorg Chem ; 56(3): 1023-1026, 2017 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-28060494

RESUMO

We report here two copper complexes as first functional models for lytic polysaccharide monooxygenases, mononuclear copper-containing enzymes involved in recalcitrant polysaccharide breakdown. These complexes feature structural and spectroscopic properties similar to those of the enzyme. In addition, they catalyze oxidative cleavage of the model substrate p-nitrophenyl-ß-d-glucopyranoside. More importantly, a particularly stable copper(II) hydroperoxide intermediate is detected in the reaction conditions.


Assuntos
Cobre/química , Oxigenases de Função Mista/química , Compostos Organometálicos/química , Polissacarídeos/química , Biocatálise , Cobre/metabolismo , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/metabolismo , Polissacarídeos/metabolismo , Teoria Quântica , Thermoascus/enzimologia
18.
Appl Biochem Biotechnol ; 181(2): 784-800, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27900666

RESUMO

Thermostable cellulases have wide variety of applications and distinctive advantages, but their low titer becomes the hurdle in their commercialization. In the present work, an assessment of optimum levels of significant factors (temperature, moisture ratio, inoculum size, and ammonium sulfate) and the effect of their interactions on production of thermostable CMCase, FPase, and ß-glucosidase by Thermoascus aurantiacus RCKK under solid-state fermentation (SSF) was carried out using central composite design (CCD) of response surface methodology (RSM). The study revealed 33, 13, and 8 % improvement in FPase, CMCase, and ß-glucosidase production, respectively. Moreover, crude cellulase from T. aurantiacus RCKK efficiently hydrolyzed office waste paper, algal pulp (Gracillaria verulosa), and biologically treated wheat straw at 60 °C with sugar release of about 830 mg/ml, 285 mg/g, and 260 mg/g of the substrate, respectively. The thermostable enzyme from T. aurantiacus RCKK holds potential to be used in biofuel industry.


Assuntos
Celulase/biossíntese , Celulase/química , Resíduos Industriais/prevenção & controle , Papel , Thermoascus/enzimologia , Triticum/química , Biodegradação Ambiental , Estabilidade Enzimática , Eucariotos/química , Temperatura Alta , Caules de Planta/química , Eliminação de Resíduos/métodos , Especificidade da Espécie , Thermoascus/classificação
19.
Appl Microbiol Biotechnol ; 101(1): 173-183, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27510979

RESUMO

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.


Assuntos
Fungos/enzimologia , Glucose Desidrogenase/isolamento & purificação , Glucose Desidrogenase/metabolismo , Temperatura Alta , Talaromyces/enzimologia , Thermoascus/enzimologia , Clonagem Molecular , Coenzimas/análise , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/análise , Fungos/genética , Expressão Gênica , Glucose Desidrogenase/química , Glucose Desidrogenase/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise Espectral , Talaromyces/genética , Thermoascus/genética
20.
Int J Biol Macromol ; 93(Pt A): 20-26, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27554938

RESUMO

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°C and 75°C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.


Assuntos
Endo-1,4-beta-Xilanases/química , Proteínas Fúngicas/química , Thermoascus/enzimologia , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína
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