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• Yang, C
• Lin, K
• Chen, G
• Chen, Y
• Chen, C
• Chen, W
• Huang, Y
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• Lin, K
• Chen, G
• Chen, Y
• Chen, C
• Chen, W
• Huang, Y
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• Lin, K
• Chen, G
• Chen, Y
• Chen, C
• Chen, W
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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

Chao-Hsun Yang ^1,† , Kun-I Lin ^2,† , Gen-Hung Chen ¹ , Yu-Fen Chen ¹ , Cheng-Yu Chen ¹ , Wei-Lin Chen ¹  and Yu-Chun Huang ^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; 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 P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

Keywords: acetylxylan esterase (AXE); Pichia pastoris; Thermobifida fusca; constitutive expression

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