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Applied and Environmental Microbiology, October 2003, p. 6064-6072, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6064-6072.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Departamento de Engenharia Bioquímica, Escola de Química,¹ Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro,² Departamento de Bioquímica, Universidade do Estado do Rio de Janeiro, Rio de Janeiro,⁴ Rio Technology Center-White Martins Gases Industriais LTDA, Duque de Caxias, Brazil³

Received 25 June 2003/ Accepted 30 July 2003

Highly efficient production of a Thermomyces lanuginosus IOC-4145 ß-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 2³ 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.