An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.
The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic beta- D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of beta- D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial beta-xylosidase induced by xylan was purified using a procedure that included heating at 50 degrees C, ammonium sulfate fractioning (30-75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified beta- D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60 degrees C and 5.0, respectively. beta- D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60 degrees C and has half-lives of 11 and 30 min at 65 degrees C in the absence or presence of calcium, respectively. The purified beta- D-xylosidase hydrolyzed p-nitrophenol-beta- D-xylopyranoside and p-nitrophenol-beta- D-glucopyranoside, exhibiting apparent K(m) and V(max) values of 1.3 mM, 88 micromol min(-1) protein(-1) and 0.5 mM, 20 micromol min(-1) protein(-1), respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true beta- D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most beta-xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum beta-xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.
Many enzymes produced by fungi have relevant biotechnological applications in several industrial areas. The purpose of this study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the São Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose-oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose-oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C-8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var. rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of São Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti-microbial or anti-parasitic activities.
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis. Currently, the treatment approach involves the use of antifungal drugs and requires years of medical therapy, which can induce nephrotoxicity and lead to resistance in yeast strains. Photodynamic inactivation (PDI) is a new therapy capable of killing microorganisms via the combination of a nontoxic dye with visible light to generate toxic reactive oxygen species (ROS). We investigated the phototoxic effect of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP), a cationic porphyrin, on the survival of P. brasiliensis following exposure to light. Phototoxicity was found to depend on both the fluence and concentration of the photosensitizer (PS). Although the biological effects of PDI are known, the molecular mechanisms underlying the resultant damage to cells are poorly defined. Therefore, we evaluated the molecular response to PDI-induced oxidative stress by gene transcription analysis. We selected genes associated with the high-osmolarity glycerol (HOG)-mitogen-activated protein kinase (MAPK) pathway and antioxidant enzymes. The genes analyzed were all overexpressed after PDI treatment, suggesting that the oxidative stress generated in our experimental conditions induces antioxidant activity. In addition to PDI-induced gene expression, there was high cell mortality, suggesting that the antioxidant response was not sufficient to avoid fungal mortality.
This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.
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