Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris,and a simple purification method |
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| Authors: | Akeboshi Hiromi Kashiwagi Yutaka Aoki Hiroyoshi Tonozuka Takashi Nishikawa Atsushi Sakano Yoshiyuki |
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| Institution: | Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. |
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| Abstract: | 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 alpha-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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