Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god). The gene was placed under the control of the gpdA promoter of A. nidulans. For more efficient secretion the alpha-amylase signal peptide from A. oryzae was inserted in front of god. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 gl-1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively. With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures.

Aims: To obtain an optimal combination of agitation speed and aeration rate for maximization of speci®c glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between k L a vis-a Á-vis oxygen transfer condition and speci®c glucose oxidase production. Methods and Results: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coef®cient (k L a) 1 were determined at corresponding combinations of impeller speed and aeration rate. The maximal speci®c extracellular glucose oxidase production (3á17 U mg ±1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6á83 mg l ±1 at the impeller speed of 420 rev min ±1 and at the rate of aeration of 0á25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between k L a and speci®c GOD production could not be established. Conclusions: At the agitation speed of 420 rev min ±1 and at 0á25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. Signi®cance and Impact of the Study: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between k L a and speci®c production of GOD, k L a should not be used as one of the scale-up parameters.

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