Thesis for Master of Science

Maria Catarina Marques Dias de Almeida
“Purificação de uma Proteína Recombinante por Partição Bifásica Aquosa”
MSc. Chemical and Biological Engineering, UM, 1997
Supervisor: J. A. Teixeira

Abstract:

Aqueous two-phase systems are used in many biotechnological applications, mainly as a purification method for

 biological materials. The different fractionation of biomolecules, organelles, ions, cells and tissue cells in the 

aqueous two-phase systems provides a way to separate the components of a mixture – for instance, an extract or

 a broth. The high percentage of water in each of the phases of the system makes them apropriate to deal with labile 

biomaterials.

The interest for the use of enzymes is increasing in different fields of industry, from detergents to food and 

pharmaceutical products, but their availability depends on the existence of efficient and less expensive purification

 procedures.

Cutinase, an enzyme with lipolytic activity, has a high potential for industrial applications. During the present work, 

the use of aqueous two-phase systems, as a way of avoiding the usual proceedings (based on chromatographic 

methods, expensive and time consuming), has been tested to purify the recombinant cutinase from the fungus 

Fusarium solani pisi. The polymers used were polyethylene glycol and hydroxypropil starch. The partitioning 

behaviour of  cutinase was compared for systems prepared with a purified hydroxypropyl starch (Reppal® PES 100) 

and a crude one (HPS). All the laboratory work was made using lyophilized cutinase.

To describe the cutinase partition in aqueous two-phase systems, partition coefficients for the cutinase (Kcut) 

and for the total protein (Kp) were calculated, as well as the percent yield of cutinase for the top phase of the system.

The influence of

a)                 polyethylene glycol molecular mass

b)                 pH of the system

c)                 tie-line lenght

was investigated in systems composed of PEG/Reppal and PEG/HPS.

The effect of the presence of different salts – sodium chloride, sodium sulphate and ammonium sulphate – on 

cutinase partitioning was also tested in systems composed by PEG 4000/Reppal and PEG 4000/HPS, at different 

pH values.

The results lead to the conclusion that ATPS composed by polyethylene glycol and hydroxypropyl starch are not

 efficient to purify cutinase. The partition coefficients for cutinase are, in the majority of the cases studied, very 

close or below 1. The larger differences between Kcut values are observed for PEG/HPS, varying the systems’ pH.

Partition coefficients for cutinase and for total protein, as well as percent yields of cutinase for the top phase of 

the system were higher for systems that included salts. The maximum value for the partition coefficient for cutinase 

was 3,7, observed for systems PEG 4000/Reppal (at pH 4, 0,5 M (NH4)2SO4 and 12, for systems composed by 

PEG/HPS (at pH 4, 1 M NaCl).

The pI of 7,8 for cutinase was confirmed by constructing a cross partition graphic from the results obtained in

 the experiences with the different salts.

Keywords: separation method, aqueous two-phase systems, recombinant cutinase, polyethylene glycol, 

crude hydroxypropyl starch, partition coefficient