Use of Corncob for Endoxylanase Production by Thermophilic Fungus Thermomyces lanuginosus IOC-4145

Abstract

The production of cellulase-free endoxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated in semisolid fermentation and liquid fermentation. Different process variables were investigated in semisolid fermentation, employing corncob as the carbon source. The best results were with the following conditions: grain size = 4.5 mm, solid:liquid ratio = 1:2, and inoculum size = 20% (v/v). Corncob, xylan, and xylose were the best inducers for endoxylanase production. Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities. The crude enzyme had optimum activity at pH 6.0 and 75 degrees C, displaying a high thermostability. The apparent Km and Vmax were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

“…Escherichia coli strain XL1Blue (Stratagene) was used for propagation of plasmids, and P. pastoris strain GS115 (his4) (Invitrogen) was used for protein expression. Thermomyces lanuginosus was isolated from Brazilian soil and identified by Fundação Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, under the code IOC-4145 (9).…”

“…T. lanuginosus was grown in 1% birchwood xylan (9), and total RNA was extracted with Trizol reagent (Invitrogen). First-strand cDNA synthesis was performed with 2 g of total RNA, 200 ng of random hexamers (Amersham Biosciences), 1ϫ reverse transcription buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl 2 ), 10 mM dithiothreitol, 0.5 mM deoxynucleoside triphosphate mix, 5 U of RNase inhibitor (Amersham Biosciences), and 200 U of Superscript II reverse transcriptase (Invitrogen) in a total volume of 20 l at 42°C for 50 min.…”

“…Purkarthofer et al (28) were the first to report that the T. lanuginosus strain DSM 5826 secreted a xylanase during submerged cultivation in a complex medium. Accordingly, we demonstrated that T. lanuginosus strain IOC4145, isolated from Brazilian soil, secreted cellulase-free xylanase activity in submerged and semisolid fermentation with corncob as the substrate (9). Xylanase activity in the semisolid fermentation culture medium was high, displayed considerable thermostability, and was stable over a broad pH range (4.0 to 11.0).…”

“…Several strains of the thermophilic fungus Thermomyces lanuginosus secrete high levels of xylanases, which are very active and stable at elevated temperatures (9,27,31). Purified and unpurified extracellular xylanase preparations from this fungus have been successfully used for the bleaching of pulps (4,11,20,21).…”

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

“…Thermomyces lanuginosus is a thermophilic fungus commonly found in composting plant material, with an optimum growth temperate of 40-50 • C [8][9][10]. Enzyme production by solid state fermentation (SSF) has been shown to be directly influenced by growth media; however, T. lanuginosus is noted for its inability to produce cellulolytic enzymes [11].…”