Purification of recombinant cutinase by extraction in an aqueous two‐phase system facilitated by a fatty acid substrate

Abstract

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.

Recovery of Recombinant Cutinase Using Detergent Foam

“…Activation is most probably a result of the complex formation between cutinase (a lipase) and the detergent. Observation of the same kind was reported earlier as an effect of low molecular weight poly(ethylene glycol)s during cutinase extraction in an aqueous two‐phase system (25). To provide a fair recovery measure, the percent recovery of the enzyme into the foam phase was thus calculated with respect to the calculated total sum of the activity recovered from foam phase and the unbound activity.…”

“…S. cerevisiae strains MM01‐pUR7320–9 and MM01‐pUR806–6 bearing the recombinant wild‐type cutinase (cut‐wt) and cutinase‐(Trp‐Pro) 4 (cut‐(WP) 4 ), respectively, were provided by Unilever Research Laboratories, Vlaardingen, The Netherlands. The strains were grown as described earlier (25). A 25 mL portion of selective medium made of 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI) and 2% glucose in a 50 mL conical flask was inoculated with colonies from a 48 h culture grown on the plates with the same medium supplemented with 1.5% (w/v) agar (Difco).…”

“…Determination of Cutinase Activity. Cutinase activity was determined according to the procedure described earlier (25). An assay mix was prepared by adding 0.2 mL of 50 mM p ‐nitrophenylbutyrate (ICN Biomedicals, Costa Mesa, CA) to 10 mL buffer (pH 8.0) containing 50 mM taurodeoxycholic acid (TDOC, sodium salt; Sigma, St Louis, USA), 10 mM Tris and 10 mM NaCl.…”

Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier

“…4000)-based ATPS. The bioaffinity between an enzyme and substrate was also used for the separation of cutinase with a fatty acid substrate in an ATPS [21].…”

Extração de adenovírus em sistemas micelares de duas fases aquosas

“…O princípio do método de separação em um sistema de duas fases é a distribuição seletiva das substâncias entre elas (FERNANDES, S et al, 2001). Essa distribuição é governada por uma gama de parâmetros relacionados com as propriedades das fases do sistema e das substâncias e a interação entre as duas (ALBERTSSON, P. A et al, 1987;HASMANN et al, 2007).…”