Produção de glycerol quinase recombinante utilizando o sistema Pichia pastoris

The methylotrophic yeast Pichia pastoris has been developed into an efficient expression system for the production of recombinant protein under the tight control of the methanol-induced alcohol oxidase promoter (pAOX1). In this study, a 2.5-liter culture system was developed for the growth of a P. pastoris strain bearing the GUT1 gene from Saccharomyces cerevisiae for the expression of recombinant glycerol kinase (GK). The best culture conditions to produce high levels of secreted GK were investigated by growing the recombinant strain of P. pastoris in shake flasks and a fermenter. Cell growth and enzyme production were found to be optimal after two days of growth. Enzyme production was affected by the nitrogen source, Difco peptone being the most appropriate for this purpose. Three different rates of air flow (1 to 3 L/min) were tested to observe their effect on cell growth and the secretion of GK into a medium containing 1% methanol as the sole carbon source. Increasing the rate of air bubbling in the culture medium enhanced both cell growth and GK activity, reaching a dry biomass of 7.84 mg/mL, cell viability of 98.4% and a maximal GK activity of 1.57 U/mL, at a flow rate of 2.0 L/minute, at 30° C and pH 6.0. Moreover, the enzyme activity in the P. pastoris culture medium was 2.3 times higher under these conditions than in the shake-flask culture, demonstrating the significant influence of aeration on biomass production and GK activity secreted by P. pastoris.(AU) A levedura metilotrofica Pichia pastoris possui umsistema de expressao eficiente para a producao deproteinas recombinantes. A inducao da producao daproteina de interesse e feita com metanol, que e capazde ativar a transcricao do gene de interesse clonadosob controle do promotor do gene AOX1. Um meio decultura de 2.5 litros foi elaborado para o crescimentoda cepa Pichia pastoris construida com o gene GUT1de Saccharomyces cerevisiae para expressar a enzimarecombinante glicerol quinase (GK). As condicoes ideaisde cultura, para alcancar altos niveis de expressao deGK foram investigados em crescimentos realizados emfrascos e fermentador. Crescimento celular e producaode enzima atingiram valores otimos em dois dias decultura. A producao enzimatica foi afetada pela fonte denitrogenio no meio. Peptona da marca Difco foi a fontede nitrogenio mais adequada para a expressao destaenzima. Tres diferentes concentracoes (1-3 L / min) defluxo de ar foram analisados em ensaios de crescimentocelular e secrecao da GK, no meio contendo 1 % demetanol como unica fonte de carbono. O aumentodo fluxo do ar no meio de cultura produziu melhoresresultados para o crescimento celular e atividade daGK, atingindo 7,84 mg / mL de biomassa seca e 98,4%de viabilidade. A maxima atividade de GK foi de 1,57U / mL, com a concentracao de fluxo de ar de 2,0 L/ minuto a 30 ° C e pH 6.0. O aumento da atividadeenzimatica foi 2,3 vezes maior no meio de cultura daPichia pastoris nestas condicoes, revelando a influenciadeste parâmetro na producao de biomassa e atividadeda GK.(AU)