Highly efficient production of a Thermomyces lanuginosus IOC-4145 ␤-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter. P. pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 2 3 factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermentor growth and industrial applications.

The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively.

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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.

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.