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

Abstract

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.

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

“…Wakiyama et al [36] recovered approximately 6 U/mL following the 5 day induction of a recombinant P. pastoris culture. Damaso et al [37] achieved 360 U/mL after 96 h induction, using an optimised medium with 1% (v/v) methanol. Ruanglek et al [38] implemented a fed batch strategy for heterologous xylanase production in P. pastoris, achieving 3676 U/mL over a 165 h period, which consisted of a 96 h induction phase preceded by a 69 h growth phase.…”

“…The banding pattern generated by the recombinant xylanase in Fig. 6a may have been, in part, mediated by inefficient signal sequence cleavage at the N-terminus [37]. Proline residues have been reported to influence the efficacy of both Kex2 and Ste13 proteases, responsible for cleaving the ␣-factor signal upstream of the heterologous gene in the pPic9K vector [14].…”

Production of Xylanase of Bacillus coagulans and its bleaching potential

“…The expression of xylanase from thermophilic microorganisms in fungal and yeast hosts has also received major attention. Pichia pastoris, S. cerevisiae and K. lactis were used as hosts for the heterologous production of xylanase (Damaso et al 2003;Gorgenes et al 2005). Parameters responsible for the improved production of heterologous protein in yeast were studied and it was reported that amino acid supplementation in defined media favours higher production in S. cerevisiae (Gorgenes et al 2005).…”

Activity of cellulase from Thermoactinomycetes and Bacillus spp. isolated from Brassica waste compost

“…The DNS was prepared as described by Bhat & Wood (1988). Carboxymethylcellulose (CMC) was used as the substrate (Damaso et al, 2003). Glucose standard was used to plot the standard curve.…”