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Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris
Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung, 43301, Taiwan
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
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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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.
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.
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.