Monday, November 9, 2009
P28

Production of an amylolytic fusion protein with a PGK-αG Pichia pastoris strain in fed batch culture

Pablo Savoy1, Lidia M. P. de Moraes2, Faustino Sineriz3, and Maria E. Lucca3. (1) Fermentation Department, PROIMI, Belgrano y Caseros, Tucumán, Argentina, (2) Departamento de Biologia Celular, Universidade de Brasilia, 70910-900, Brasilia, Brasilia, DF, Brazil, (3) Fermentation Department, PROIMI and FBQF, Universidad Nacional de Tucuman, Belgrano y Caseros, Tucumán, Argentina

Production of heterologous proteins with recombinant strains of Pichia pastoris is extensively used because high cell density cultures can be obtained. Usually, the inducible AOX1 promoter is preferred for fed-batch production since methanol can be a disadvantage for some recombinant proteins as amylolytic enzymes. Pichia pastoris  PGK-αG producing a fusion protein (α-amylase from Bacillus subtilis and glucoamylase from Aspergillus awamori ) under the constitutive PGK1 promoter gene was used in this work. Fed-batch cultures with cerelose as sole carbon source was performed in a 1 L fermentor  at 28ºC, 400rpm, 60% oxygen saturation and pH 5.5. The production media used was (in g/L): cerelose 30; yeast extract 3; MgS04  1; Mn S04 0.1; (NH4 )2 HPO4 1 and ZnS04 0.1. When exponential phase was reached, the culture was fed with a concentrated cerelose solution (50g/L) at a constant flow rate of 20ml/L . Final biomass concentration (OD540nm ) in batch culture was 35 and was increased during the fed-batch culture to 98. Enzyme activities were measured in the supernatant culture during batch and fed-batch growth.  Glucoamylase and α-amylase maximum activities obtained were (U/ml) 114 and 55 respectively. Specific activities (U/mg biomass) referred to final biomass concentration, 30 (g dry weight /L) were 3.8  for glucoamylase and 1.6 for α-amylase respectively.
The results obtained allow to consider the fusion protein production using this recombinant PGK-αG strain of Pichia pastoris in a single fed-batch step using commercial glucose avoiding traditional glycerol growth and methanol induction.  Supported by CIUNT 26/D434

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