Abstract
A fast and efficient method based on a polyethylene glycol (PEG) 600/(NH4)2SO4 aqueous two-phase system for extracting lectin from Zihua snap-bean (Phaseolus vulgaris) seeds was established. According to a Box–Behnken design (BBD), involving four factors at three levels each subjected to analysis of variance (ANOVA) and response surface analysis, the protein recovery and the purification factor of lectin in the top phase were used as the response values of the variance analysis to acquire the multivariate quadratic regression model. SDS–PAGE electrophoresis and the hemagglutination test were used to detect the distribution of lectin in the aqueous two-phase system (ATPS). The obtained data indicated that lectin was preferentially partitioned into the PEG-rich phase, and the ATPS, composed of 15% (NH4)2SO4 (w/w), 18% PEG 600 (w/w), 0.4 g/5 g NaCl and 1 mL crude extract, showed good selectivity for lectin when the pH value was 7.5. Under the optimal conditions, most of the lectin was assigned to the top phase in the ATPS, and the hemagglutination activity of the purified lectin in the top phase was 3.08 times that of the crude extract. Consequently, the PEG 600/(NH4)2SO4 aqueous two-phase system was an effective method for separating and enriching lectin directly from the crude extract of Zihua snap-bean seeds.
Highlights
Due to the advantage of being able to bind to monosaccharides, polysaccharides and glycoproteins [1], plant lectin has played a huge role and value in medicine, materials science and agronomy [2,3]
In order to investigate the effect of different variables on the distribution of lectins in aqueous two-phase system (ATPS), each parameter—including concentration of ammonium sulfate, concentration of polyethylene glycol (PEG) 600, NaCl content and pH—was changed while others were fixed, to form the ATPS
Based on the protein recovery in the top phase (Y), the hemagglutinating activity of lectin’s partition coefficient (K) and the purification factor of lectins in the top phase (PF), the ATPS rendering the most-effective partitioning was chosen for further study
Summary
Due to the advantage of being able to bind to monosaccharides, polysaccharides and glycoproteins [1], plant lectin has played a huge role and value in medicine, materials science and agronomy [2,3]. Glycoproteins, with at least one carbohydrate or derivate binding site without catalytic function (nonenzymatic) or immunological characteristics [1]. Lectins are highly sought-after proteins due to their wide potential in many cellular and molecular recognition processes, as well as in pharmacology, biochemistry, medicine and clinical analysis [4,5]. Plant lectins are classified into seven families according to their evolutionary and structural characteristics [7], and among these families, lectins of the Leguminosae family are, far, the most-studied group. Nearly a thousand species of plant lectins have been found, among which leguminous plant lectins are the most abundant, accounting for more than 600 species, of which more than 70 species have been isolated and purified [8]
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