title: Constitutive expression of Thermobifida fusca thermostable Acetylxylan Esterase gene in Pichia pastoris.
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.
authors: Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung, 43301, Taiwan; E-Mails chyang@pu.edu.tw (C.-H.Y.); ghchen2@pu.edu.tw (G.-H.C); yfchen@pu.edu.tw (Y.-F.C.); zychan1268@gmail.com (C.-Y.C); wei.lin46@gmail.com (W.-L.C).
authors: Chao-Hsun
authors: CH
authors: Yang
authors: Kun-I
authors: KI
authors: Lin
authors: Gen-Hung
authors: GH
authors: Chen
authors: Yu-Fen
authors: YF
authors: Chen
authors: Cheng-Yu
authors: CY
authors: Chen
authors: Wei-Lin
authors: WL
authors: Chen
authors: Yu-Chun
authors: YC
authors: Huang
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