Overproduction and high level secretion of glucose oxidase in saccharomyces cerevisiae = Glucose oxidase의 Saccharomyces cerevisiae에서의 대량생산 및 고효율 분비

Abstract

The overproduction and high level secretion of Glucose Oxidase (GOD) from A. niger in S. cerevisiae was carried out by cloning GOD gene. For this purpose, using two different strong promotors (ADH1 promotor, GAL1O promotor) and signal sequences (α-MF signal sequence of S. cerevisiae and α-amylase signal sequence of A. oryzae) and GAL7- and GOD terminator, four expression vectors were constructed. All the expression vectors were transformed in S. cerevisiae 2805 using auxotroph method. By the flask culture, transformants of pGAL expression vector series containing GAL 10 promotor showed much higher GOD productivity than transformants of pADH expression vector series containing ADH1 promotor. Transformants of pGALGO2 containing GAL10 promotor and α-amylase signal sequence has shown the best productivity of GOD (GOD(total): 10.3 unit/mL, GOD(ex): 8.7 unit/mL) at 115 hr. This value was three fold higher than that of pGALGO1 containing GAL 10 promotor and α-MF signal sequence, even if the same promotor was involved. Through the α- amylase signal sequence of A. oryzae, GOD was secreted much more than the case of α-MF signal sequence from S. cerevisiae. These results suggest that signal sequence may play a important roles in not only the secretion but also the overproduction of foreign protein. Secretion rate of GOD in pGALGO1 and pGALGO2 was 89% and 84%, respectively. Because of the overglycosylation in S. cerevisiae the molecular weight of recombinant GOD in S. cerevisiae was much larger (250 kDa) than that of nature GOD in A. niger (170 kDa).