RESUMO
Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.
Assuntos
Ácido Aspártico Proteases/biossíntese , Colágeno/química , Escherichia coli/metabolismo , Mucorales/enzimologia , Saccharomycetales/metabolismo , Simulação por Computador , Fermentação , Tecnologia de Alimentos , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Íons , Peptídeos/química , Proteínas Recombinantes/biossíntese , TemperaturaRESUMO
The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of ß-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of ß-glucosidase produced by L. corymbifera was 39 U/g dry substrate (or 3.9 U/mL), and that by B. spectabilis was 77 U/g (or 7.7 U/mL). The optimum pH and temperature were 4.5 and 55 °C and 4.0 and 50 °C for the enzyme from L. corymbifera and B. spectabilis, respectively. ß-Glucosidase produced by L. corymbifera was stable at pH 4.0-7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0-6.0. Regarding the thermostability, ß-glucosidase produced by B. spectabilis remained stable for 1 h at 50 °C, and that from L. corymbifera was active for 1 h at 45 °C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (Ea), and half-life t(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.
Assuntos
Byssochlamys/enzimologia , Celulases/química , Proteínas Fúngicas/química , Mucorales/enzimologia , Byssochlamys/crescimento & desenvolvimento , Catálise , Celulases/antagonistas & inibidores , Celulases/isolamento & purificação , Técnicas de Cultura/métodos , Inibidores Enzimáticos/química , Etanol/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/isolamento & purificação , Glucose/química , Concentração de Íons de Hidrogênio , Cinética , Mucorales/crescimento & desenvolvimento , Temperatura , TermodinâmicaRESUMO
The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia ramosa (mesophilic) and Thermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2 U/g of dry substrate (or 41.72 U/mL) and 144.5 U/g of dry substrate (or 14.45 U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed higher catalytic activity at pH 6.0 at 60°C. The amylases produced by L. ramosa and T. aurantiacus were stable between pH 3.5-10.5 and pH 4.5-9.5, respectively. The amylase of L. ramosa was stable at 55°C after 1 hour of incubation, whereas that of T. aurantiacus maintained 60% of its original activity under the same conditions. Both enzymes were active in the presence of ethanol. The enzymes hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L. ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented only saccharifying potential, releasing glucose monomers as the main hydrolysis product.
Assuntos
Amilases/química , Fermentação , Mucorales/enzimologia , Thermoascus/enzimologia , Hidrólise , Microbiologia Industrial , Amido/metabolismoRESUMO
The aim of this study was to explore the use of a new coagulant from Thermomucor indicae-seudaticae N31 for the manufacture of a high-cooked starter-free cheese variety, by evaluating its physicochemical and functional characteristics in comparison to cheeses made with a traditional commercial coagulant. Coalho cheese was successfully produced with the new protease as it exhibited comparable characteristics to the one produced using the commercial enzyme: pH behavior during manufacture; cheese composition; protein and fat recovery; and cheese yield. In addition, during storage, melting was low and not affected by storage time; the increase of TCA 12% soluble nitrogen (% of total nitrogen) was lower than half of that of pH 4.6 soluble nitrogen (% of total nitrogen); concentration of ß-CN significantly decreased, whereas αs1 -CN concentration was not affected by storage time.
Assuntos
Queijo/análise , Endopeptidases/metabolismo , Manipulação de Alimentos/métodos , Mucorales/enzimologia , Coagulantes , Culinária , Humanos , Nitrogênio/análiseRESUMO
Background β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Results A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5-10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes.
Assuntos
beta-Glucosidase/metabolismo , Mucorales/enzimologia , Temperatura , Celulases , Celulases/biossíntese , Agroindústria , Biocatálise , Fermentação , Concentração de Íons de Hidrogênio , Resíduos IndustriaisRESUMO
Background: Enzyme production by solid state bioprocess (SSB) using residues as substrate for microorganisms is an alternative for costs reduction and to avoid their disposal into environment. The aim of this work was to evaluate the physiology of the fungus Lichtheimia ramosa in terms of microbial growth and production of amylases, β-glucosidases, carboxymethylcellulase (CMCase), and xylanases, via SSB, utilizing wastes of the Brazilian savannah fruits bocaiuva (Acrocomia aculeata), guavira (Campomanesia pubescens) and pequi (Caryocar brasiliense) as substrate at different temperatures (25, 30, and 35ºC) during 168 hrs. Results: Samples were taken every 24 hrs, which resulted in 8-points kinetic experiments to determine microbiological and enzymatic contents. The best substrate for β-glucosidase activity was pequi waste after 48 hrs at 30ºC (0.061 U/mL). For amylase activity, bocaiuva presented itself as the best substrate after 96 hrs at 30ºC (0.925 U/mL). CMCase activity was higher in guavira waste after 96 hrs at 35ºC (0.787 U/mL). However, the activity was more expressive for xylanase in substrate composed of bocaiuva residue after 144 hrs at 35ºC (1.802 U/mL). Conclusions: It was concluded that best growth condition for L. ramosa is at 35ºC for all substrates and that xylanase is the enzyme with more potential in SSB, considering the studied Brazilian savannah fruit wastes.
Assuntos
Xilosidases/metabolismo , Celulases/metabolismo , Amilases/metabolismo , Mucorales/enzimologia , Resíduos , Brasil , Reatores Biológicos , Frutas , Mucorales/crescimento & desenvolvimentoRESUMO
Brazil is known for its great potential for production of renewable resources such as agro-industrial residues. These residues can be used as alternative sources of new products. Meanwhile, solid-state fermentation, with its advantages of energy conservation and pollution reduction, has been identified as a process of great potential for the production of bioactive compounds, especially enzymes. In the present work, a 2(3) factorial design was used to evaluate the effects of pH, temperature and moisture on the production of phytase and xylanase by Lichtheimia blakesleeana URM 5604 through the fermentation of citrus pulp. Statistical analyses of the results showed that the only the pH influenced the production of these enzymes, with the best phytase production (264.68 U/g) ocurring at pH 6.0, 34 °C, initial moisture 50%, after 48 hours of culture. The best conditions for xylanase production (397.82 U/g) were fermentation for 120 hours at pH 4.0, 26 °C and initial moisture of 70%. The best parameters for the simultaneous production of phytase (226.92 U/g) and xylanase (215.59 U/g) were determined to be initial moisture of 50%, pH 6.0, 26 °C, and 48 hours of fermentation.
Assuntos
6-Fitase/biossíntese , Mucorales/enzimologia , Xilosidases/biossíntese , Ativação Enzimática/fisiologia , FermentaçãoRESUMO
Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45 degrees C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1:1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.
Assuntos
Microbiologia Industrial , Mucorales/enzimologia , Mucorales/crescimento & desenvolvimento , Poligalacturonase/biossíntese , Brasil , Meios de Cultura , Fermentação , Temperatura Alta , Resíduos Industriais , Mucorales/classificação , Mucorales/isolamento & purificação , Pectinas/metabolismo , Microbiologia do Solo , Especificidade por SubstratoRESUMO
The biotransformation of 1R-(-)-camphorquinone, achieved by growing cells of four fungi species isolated from soil (Mucor plumbeus, Lecanicillium muscarium, Thamnostylum sp. and Syncephalastrum racemosum), was investigated in optimized culture media for each species. Fungi were grown aerobically under shaking and their activities with respect to camphorquinone were monitored for 20 days by gas chromatography coupled to mass spectrometry (GCMS). Camphorquinone was found to be stable in control flasks throughout the experiment. The most interesting results were found for M. plumbeus, which was only able to perform monoreduction of camphorquinone when cultivated on a glucose-peptone-yeast extract medium. Large-scale experiments were set up and the camphorquinone biotransformation products formed by M. plumbeus were purified by column chromatography and identified by (1)H and (13)C nuclear magnetic resonance (NMR). Theoretical calculations were employed as a complementary technique to unambiguously identify the biotransformation products. These findings suggest that M. plumbeus could be of great use for the selective reduction of camphorquinone and related compounds.
Assuntos
Proteínas Fúngicas/metabolismo , Mucorales/enzimologia , Oxirredutases/metabolismo , Terpenos/metabolismo , Aerobiose , Biotransformação , Meios de Cultura/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectroscopia de Ressonância Magnética/métodos , Mucorales/isolamento & purificação , Mucorales/metabolismo , Microbiologia do Solo , Especificidade por SubstratoRESUMO
Ornithine decarboxylase (ODC) is the first enzyme in polyamine biosynthesis in numerous living organisms, from bacteria to mammalian cells. Its control is under negative feedback regulation by the end products of the pathway. In dimorphic fungi, ODC activity and therefore polyamine concentrations are related to the morphogenetic process. From the fission yeast Schizosaccharomyces pombe to human, polyamines induce antizyme synthesis which in turn inactivates ODC. This is hydrolyzed by the 26S proteasome without ubiquitination. The regulatory mechanism of antizyme on polyamines is conserved, although to date no antizyme homology has been identified in some fungal species. The components that are responsible for regulating polyamine levels in cells and the current knowledge of ODC regulation in dimorphic fungi are presented in this review. ODC degradation is of particular interest because inhibitors of this pathway may lead to the discovery of novel antifungal drugs.