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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
,
1
,
1
,
1
 and
1,*
1 Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung, 43301, Taiwan 2 Department of Obstetrics & Gynecology, Chang Bing Show Chwan Memorial Hospital, No. 6 Lugong Rd. Lukang Zhen, Changhua County, Taiwan These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 17 November 2010 / Revised: 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 (CC BY 3.0).

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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.

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