Construction of an Efficient Expression System for Aspergillus Isopullulanase in Pichia pastoris, and a Simple Purification Method
AKEBOSHI, Hiromi | KASHIWAGI, Yutaka | AOKI, Hiroyoshi | TONOZUKA, Takashi | NISHIKAWA, Atsushi | SAKANO, Yoshiyuki
Aspergillus niger ATCC 9642 isopullulanase (IPU) was heterologously expressed by Pichia pastoris GS115 under three different signal sequences of Saccharomyces cerevisiae acid phosphatase, S. cerevisiae α-factor prepro peptide, and A. niger isopullulanase. One-step purification using lectin Con A affinity chromatography yielded recombinant IPU (IPU-PP) with high purity. IPU-PP had a higher carbohydrate content than native IPU and IPU-AO expressed in A. oryzae M-2-3. IPU-PP hydrolyzed various substrates containing the structure of panose, which indicated a strict subsite recognition of the panose motif.
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