Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system

Abstract

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (2 4) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500-8000 g/mol), PEG concentration (12.5-17.5% w/w), phosphate (10-15% w/w) concentration, and pH (6-8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box-Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 C and lost hemagglutinating activity at 80 C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.

Effect of Temperature on the Phase Equilibria of Poly(vinylpyrrolidone) 10000 + Trilithium Citrate + Water Aqueous Two-Phase System and Partitioning of Curcumin at T = 298.15, 308.15, and 318.15 K

“…Various inorganic salts such as nitrates, , phosphates, and sulfates , are used in ATPSs for the extraction and purification of biological compounds; however, these salts can cause environmental pollution issues if they are used on large industrial scales. These salts can be replaced by nontoxic ones to provide large-scale systems without such environmental issues and contaminations for the extraction and purification of proteins and biological compounds.…”

Production, characterization and techno-economic evaluation of Aspergillus fusant l-asparaginase

“…Selected significant factors were employed at their optimum levels for ATPS purification as described by Nascimento et al ( 2020 ). Further purification of the ATPS-extracted protein was performed by molecular exclusion chromatography on Sephadex G-100 of diameter 100 cm by 1.5 cm, using 50 mM phosphate buffer (pH 7.0) and 100 mM KCl as mobile phase at a flow rate of 0.2 mLmin −1 .…”

Emerging delivery systems based on aqueous two-phase systems: A review

“…Additionally, state transitions between single and two phases can occur by altering conditions 42 . Generally, a higher pH 43 , lower temperature 39 , larger molecular weight of the ATPS components 44 , 45 , and greater density difference 46 and hydrophobicity gap between the two solutes forming the ATPS 31 cause the biphasic curve to move to the origin, that is, the biphasic region becomes larger ( Fig. 2 ).…”