Aspergillus oryzae alpha-amylase partition in potassium phosphate-polyethylene

M.C. PORFIRI; G. PICÓ; D. ROMANINI; B, FARRUGGUA

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2011 vol. 49 p. 7 - 13

0141-8130

The aim of this work is to study the partitioning of alpha-amylase from Aspergillus oryzae in polyethylene  glycol-potassium phosphate systems formed by polymers of different molecular masses with different  total concentrations, several NaCl concentrations and different volume ratio between the phases and at  different temperatures. The enzyme was partitioned towards the top phase in the 2000-molecular-mass  polyethylene glycol systems and towards the bottom phase in the other systems analyzed with higher  molecular mass. The protein–medium interaction parameter (A) was determined; it increased in the same  way as PEG molecular mass. The enthalpic and entropic changes found, in general, were negative and  were shown to be associated by an entropic–enthalpic compensation effect suggesting that the ordered  water structure in the chain of polyetyleneglycol plays a role in protein partition. The recovery in each  of the phases was calculated in order to choose the best systems to be applied to enzyme isolation either  from a polymer-rich or a polymer-poor phase.  Enzymatic activity, circular dichroism and fluorescence were studied for the protein alone and in the   presence of the different phases of the aqueous two-phase systems (ATPSs) in order to understand how they affect the enzymatic structure and the role of the protein-polymer interaction in the partitioning  process. Secondary structure is not affected, in general, by the presence of the phases that do affect the  enzymatic activity; therefore, there should be a change in the tertiary structure in the enzyme active site.  These changes are more important for PEG 8000 than for PEG 2000 systems according to the results of the  quenching of the intrinsic fluorescence. In a bio-separation process, the A. oryzae alpha-amylase could  be isolated with ATPSs PEG 2000/Pi or PEG 8000/Pi with a high recovery, in the top or bottom phases,  respectively.Aspergillus oryzae in polyethylene  glycol-potassium phosphate systems formed by polymers of different molecular masses with different  total concentrations, several NaCl concentrations and different volume ratio between the phases and at  different temperatures. The enzyme was partitioned towards the top phase in the 2000-molecular-mass  polyethylene glycol systems and towards the bottom phase in the other systems analyzed with higher  molecular mass. The protein–medium interaction parameter (A) was determined; it increased in the same  way as PEG molecular mass. The enthalpic and entropic changes found, in general, were negative and  were shown to be associated by an entropic–enthalpic compensation effect suggesting that the ordered  water structure in the chain of polyetyleneglycol plays a role in protein partition. The recovery in each  of the phases was calculated in order to choose the best systems to be applied to enzyme isolation either  from a polymer-rich or a polymer-poor phase.  Enzymatic activity, circular dichroism and fluorescence were studied for the protein alone and in the   presence of the different phases of the aqueous two-phase systems (ATPSs) in order to understand how they affect the enzymatic structure and the role of the protein-polymer interaction in the partitioning  process. Secondary structure is not affected, in general, by the presence of the phases that do affect the  enzymatic activity; therefore, there should be a change in the tertiary structure in the enzyme active site.  These changes are more important for PEG 8000 than for PEG 2000 systems according to the results of the  quenching of the intrinsic fluorescence. In a bio-separation process, the A. oryzae alpha-amylase could  be isolated with ATPSs PEG 2000/Pi or PEG 8000/Pi with a high recovery, in the top or bottom phases,  respectively.