Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate.
Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.
Fernandes
S
S
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden.
Johansson
G
G
Hatti-Kaul
R
R
D001671:Biochemistry / Q000379:methods*; D002087:Butyrates / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D005227:Fatty Acids / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013050:Spectrometry, Fluorescence
D002087:Butyrates; D005227:Fatty Acids; D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C013216:potassium phosphate; D002265:Carboxylic Ester Hydrolases; C019952:cutinase