High-Level Expression of Aspergillus ficuum Acetyl Xylan Esterase Gene in Pichia pastoris,

Pichia pastoris에서 Aspergillus ficuum 유래 Acetyl Xylan Esterase 유전자의 과발현

  • 임재명 (부경대학교 생물공학과) ;
  • 김성구 (부경대학교 생물공학과) ;
  • 박승문 (전북대학교 생물과학부) ;
  • 남수완 (동의대학교 생명공학과)
  • Published : 2002.12.01

Abstract

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ$\alpha$C-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOXI promoter and connected downstream of mating factor u-1 signal sequence. The plasmid linearized by Sacl was integrated into the 5'AOXI region of the chromosomal DNA of P. pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/1 and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/1 of AXEase protein was produced in the extracellular medium.

Keywords

acetyl xylan esterase;fed-batch fermentation;Pichia pastoris;secretion

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