Aqueous two-phase systems: new strategies for separation and purification of lectin from crude extract of Cratylia mollis seeds

• The direct extraction of a lectin from a crude extract using ATPS.
• The lectin showed higher affinity for the salt rich phase.
• The extracted lectin was stable for a temperature up to 60 °C.
• An efficient recovery in just one extraction step was obtained.
• ATPS (PEG/citrate) proved to be a viable alternative for lectin extraction.

Plant lectins are reversible carbohydrate-binding proteins (or glycoproteins) of non-immune origin that can agglutinate cells or precipitate polysaccharides and glycoconjugates. A polymer/salt aqueous two-phase system (ATPS) based on polyethylene glycol (PEG)/sodium citrate was used for the extraction of lectins from a crude extract (CE) of Cratylia mollis seeds in 24 and 22 previously experimental designs. The effects of pH (4, 5, 6 and 7), PEG molar mass (1500, 4000 and 8000 g/mol), PEG concentration (15, 18.5 and 22% w/w) and citrate concentration (10, 15, 20 and 22% w/w) were analysed to establish the optimal extraction conditions. The optimal performance system consisted of 20% (w/w) citrate and 22% (w/w) PEG 8000 g/mol at pH 5.0 using 20% (w/w) crude extract, which allowed for a 125% activity yield of lectins in the bottom phase, with a purification factor of 13.28. A direct comparison of chromatography and ATPS processes for the recovery of lectin Cramoll 1,4 from the CE revealed a reduction in the number of unit operations (from 10 to 4). An electrophoretic study revealed three bands with molar masses of 30, 16 and 14 kDa, consistent with what has been previously observed for Cramoll 1,4. After purification using ATPS, the lectin was stable until 60 °C, with a decrease in hemagglutinating activity (512–128) at 80 °C. These results indicate that liquid–liquid extraction is an adequate tool for separating and purifying lectins from the CE of C. mollis seeds while maintaining their biological activity, which may have great potential for biotechnological applications.