KINETICS OF GLUCOSE-OXIDASE EXCRETION BY RECOMBINANT ASPERGILLUS-NIGER

The kinetics of glucose oxidase (GOD) excretion by recombinant Aspergi llus niger NRRL-3 (GOD3-18) were investigated using enzymatic activity measurements as well as gel electrophoresis techniques. The majority of GOD was produced during rapid growth in the first phase of the cult ivation. The high excretion rate during this phase did not prevent the endocellular accumulation of GOD up to 40% of the total soluble cell protein demonstrating that the production rate exceeded the excretion rate of the enzyme into the culture medium. During the second phase of the cultivation, excretion of GOD occurred at a slower rate, although the majority of GOD produced during the first phase was excreted duri ng the second phase of the cultivation. At the end, about 90% of the t otal GOD produced was recovered from the culture medium. Two-dimension al gel electrophoresis provided evidence that endo- and exocellular GO D were indistinguishable, revealing identical posttranslational modifi cations (e.g., signal sequence cleavage, glycosylation pattern). The r esults demonstrate that the initial steps of the secretory pathway are fast and that the excretion of the enzyme into the culture fluid was most likely delayed due to retention by the cell wail. (C) 1996 John W iley & Sons, Inc.