Partial purification of glucose-6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems

Brazilian Journal of Microbiology

Marcela Zanella Ribeiro²  Daniel Pereira Silva²  Michele Vitolo²  Inês Conceição Roberto¹  Adalberto Pessoa-jr² 

[1] ,University of São Paulo School of Pharmaceutical Sciences Biochemical-Pharmaceutical Technology Dept.São Paulo SP ,Brazil

关键词: aqueous two-phase systems;    glucose-6-phosphate dehydrogenase;    liquid-liquid extraction;    Saccharomyces cerevisiae;    sistema de duas fases aquosas;    glicose-6-fosfato desidrogenase;    extração líquido-líquido;    Saccharomyces cerevisiae;   

DOI  :  10.1590/S1517-83822007000100016

来源: SciELO

【 摘 要 】

Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme used in biochemical and medical studies and in several analytical methods that have industrial and commercial application. This work evaluated the extraction of G6PDH in aqueous two-phase system (ATPS) of poly(ethyleneglycol) (PEG)/phosphate buffer, using as enzyme source a medium prepared through commercial baker's yeast disruption. Firstly, the effects of PEG molar mass on the enzyme partition and of homogenization and rest on the system equilibrium were investigated. Afterwards, several ATPS were prepared using statistical analysis (2² factorial design). The results, including kinetic and thermodynamic parameters for the G6PDH activity, showed partial purification of this enzyme in ATPS composed of 17.5% (w/w) PEG400 and 15.0% (w/w) phosphate. A high enzymatic recovery value (97.7%), a high partition coefficient (351), and an acceptable purification factor (2.28 times higher than in cell homogenate) were attained from the top phase. So, it was possible to attain an effective enzyme pre-purification by separating some contaminants with a simple method such as liquid-liquid extraction in aqueous two-phase systems (ATPS).

【 授权许可】

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