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Int. J. Mol. Sci. 2010, 11(12), 5143-5151; doi:10.3390/ijms11125143
Article

Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris

1,†
, 2,†
, 1
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 and 1,*
Received: 17 November 2010; in revised form: 1 December 2010 / Accepted: 9 December 2010 / Published: 15 December 2010
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Abstract: A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the Ppastoris transformant are similar to those of the AXE from an E. coli transformant.
Keywords: acetylxylan esterase (AXE); Pichia pastoris; Thermobifida fusca; constitutive expression acetylxylan esterase (AXE); Pichia pastoris; Thermobifida fusca; constitutive expression
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Yang, C.-H.; Lin, K.-I.; Chen, G.-H.; Chen, Y.-F.; Chen, C.-Y.; Chen, W.-L.; Huang, Y.-C. Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris. Int. J. Mol. Sci. 2010, 11, 5143-5151.

AMA Style

Yang C-H, Lin K-I, Chen G-H, Chen Y-F, Chen C-Y, Chen W-L, Huang Y-C. Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris. International Journal of Molecular Sciences. 2010; 11(12):5143-5151.

Chicago/Turabian Style

Yang, Chao-Hsun; Lin, Kun-I; Chen, Gen-Hung; Chen, Yu-Fen; Chen, Cheng-Yu; Chen, Wei-Lin; Huang, Yu-Chun. 2010. "Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris." Int. J. Mol. Sci. 11, no. 12: 5143-5151.


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