PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resulted in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi

Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose

Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5-8 and 37-45 degreesC when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors

Avicel-adsorbable endoglucanase production by the thermophilic fungus Scytalidium thermophilum type culture Torula thermophila

Scytalidium thermophilum type culture Torula thermophila was isolated from mushroom compost and the total cellulase, endoglucanase, Avicel-adsorbable endoglucanase activities, as well as the fungal biomass generation and cellulose utilisation were analyzed in shake flask cultures with Avicel (microcrystalline cellulose) as the carbon source. Results were compared with an industrial strain of Scyalidium thermophilum type culture Humicola insolens. The pH and temperature optima for endoglucanase activities during enzyme assays were also analyzed for both organisms and determined to be pH 6.0 and 65 degrees C for type culture Torula thermophila, and pH 6.5 and 60 degrees C for type culture Humicola insolens. Analysis of the effect of growth temperature showed that type culture T. thermophila can grow and produce cellulases in the range of 35 to 55 degrees C although 40 to 50 degrees C seemed to favor growth and cellulase production. Although 45 degrees C was found optimal for fungal growth, both the specific endoglucanase and Avicel-adsorbable endoglucanase activities (U/mg protein) as well as the percentage of Avicel-adsorbable endoglucanase activity reached maxima at 50 degrees C and were higher as compared to type culture H. insolens. Results indicate that type culture T. thermophila, with further optimisations, is of potential use in the industrial production of cellulases. (C) 2000 Elsevier Science Inc. All rights reserved

Xylanase and itaconic acid production by Aspergillus terreus NRRL 1960 within a biorefinery concept

WOS:000331648300009Production of two industrially important products, xylanase and itaconic acid (IA), by Aspergillus terreus NRRL 1960 from agricultural residues was investigated within a biorefinery concept. Biological pretreatment was applied to lignocellulosic materials by using A. terreus, which produced xylanase while growing on agricultural residues. For IA production, already grown cells were transferred into a new medium. The first step provided not only the pretreatment of lignocellulosic material in order to be used as feedstock but also production of xylanase. For this purpose, cotton stalk, sunflower stalk and corn cob were used as carbon sources as lignocellulosic material. Among them, the highest xylanase production was obtained on corn cob. By application of two-step fermentation, about 70 IU/mL xylanase and 18 g/L IA production levels were achieved. This study shows the stepwise usage potential of the microorganism as a tool in a biorefinery concept.Middle East Technical University Scientific Research Found ProjectMiddle East Technical University [BAP-08-11-DPT2002K120510]This project was supported by the Middle East Technical University Scientific Research Found Project BAP-08-11-DPT2002K120510. The authors thank Dr. Leo de Graaff for providing the organism