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

International Journal of Molecular Sciences

Chao-Hsun Yang¹  Kun-I Lin²  Gen-Hung Chen¹  Yu-Fen Chen¹  Cheng-Yu Chen¹  Wei-Lin Chen¹ 

[1] Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung, 43301, Taiwan; E-Mails;Department of Obstetrics & Gynecology, Chang Bing Show Chwan Memorial Hospital, No. 6 Lugong Rd. Lukang Zhen, Changhua County, Taiwan; E-Mail:

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

DOI  :  10.3390/ijms11125143

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2010 by the authors; licensee MDPI, Basel, Switzerland.