Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris, and a simple purification method.
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.
Akeboshi
Hiromi
H
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
Kashiwagi
Yutaka
Y
Aoki
Hiroyoshi
H
Tonozuka
Takashi
T
Nishikawa
Atsushi
A
Sakano
Yoshiyuki
Y
D000135:Acid Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D000135:Acid Phosphatase; D006026:Glycoside Hydrolases; C047371:isopullulanase