Poster Presentation 1B-35

Formation of Glucose 6-phosphate Dehydrogenase from Saccharomyces cerevisiae W303-181 Grown by Batch Fermentation Process

Luiz Carlos Martins das Neves, Adalberto Pessoa-Jr, and Michele Vitolo*

University of São Paulo

School of Pharmaceutical Sciences

Department of Biochemical and Pharmaceutical Technology

Av. Prof. Lineu Prestes, 580, B-16, 05508-900

São Paulo, SP, Brazil

Phone: 55-11-30913862

Fax: 55-11-38156386

E-mail: michenzi@usp.br

In a 5-L fermentor (NBS-MF 105), S.cerevisiae W303-181 (1.0 g/L) was inoculated into a liquid medium containing glucose (10 or 20 g/L), yeast nitrogen base (YNB) (3.7 or 7.4 g/L), L-histidine (0.02 g/L), L-tryptophan (0.02 g/L), uracil (0.02 g/L) and adenine (0.02 g/L). The culture was carried out batchwise by 12h at 30^oC, pH (4.6 or 5.7), aeration of 0, 0.8, 1.7 or 2.2 vvm and agitation of 400 rpm. At each hour, 5 mL of the fermenting broth was taken and the cells harvested by centrifugation (4000g: 20min). The cells were disrupted through stirring with 0.5mm-glass beads by 12min (dry cell matter/mass glass beads ratio of 1:300), the debris removed by centrifugation (4000g; 20 min) and the glucose 6-phosphate dehydrogenase (G6PDH) activity measured in the supernatant. One G6PDH Unit (U) corresponded to 1 mmol of NADP reduced/min under the assay conditions. The highest G6PDH productivity (10.5 U/L.h) and specific activity ( 320 U/mg of protein) occurred at aeration of 2.2 vvm, pH 5.7, 10 g/L of glucose and 3.7 g/L of YNB. The G6PDH specific activity attained was comparable with those of commercial preparations, which are between 50 and 600 U/mg of protein.