Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris.
We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.
Yamaguchi
Masaki
M
Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan. yamag@eng.toyama-u.ac.jp
Tahara
Yusuke
Y
Nakano
Atsunori
A
Taniyama
Tadayoshi
T
D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D015374:Biosensing Techniques; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005949:Glucose Oxidase / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D005949:Glucose Oxidase