constitutive expression of thermobifida fusca thermostable acetylxylan esterase gene in pichia pastoris组成型表达的thermobifida fusca耐热性的acetylxylan pastoris酯酶基因在毕赤酵母.pdfVIP

Int. J. Mol. Sci. 2010, 11, 5143-5151; doi:10.3390/ijm OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 /journal/ijms 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; E-Mails chyang@.tw (C.-H.Y.); ghchen2@.tw (G.-H.C); yfchen@.tw (Y.-F.C.); zychan1268@ (C.-Y.C); wei.lin46@ (W.-L.C) 2 Department of Obstetrics Gynecology, Chang Bing Show Chwan Memorial Hospital, No. 6 Lugong Rd. Lukang Zhen, Changhua County, Taiwan; E-Mail: konyi@ † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: ychuang@.tw. 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