Fed-batch production of glucose 6-phosphate dehydrogenase using recombinant Saccharomyces cerevisiae.
The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L L-tryptophan, 0.02 g/L L-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30 degrees C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (L-tryptophan, L-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 micromol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.
das Neves
Luiz Carlos Martins
LC
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580 B.16, 05508-900, São Paulo SP, Brazil.
Pessoa
Adalberto
A
Vitolo
Michele
M
D005947:Glucose / Q000378:metabolism*; D005952:Glucose-6-Phosphatase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005952:Glucose-6-Phosphatase; D005947:Glucose