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 (2⁴) 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.

Keywords:

• ATPS
• Cratylia mollis seeds
• lectins
• partitioning
• purification factor

Additional information

Funding

We are grateful to the following bodies for the grants awarded: CAPES (Coordination for the Improvement of Level Personnel Superior); FACEPE (Pernambuco Science and Technology Foundation): Researcher's scholarship grant: BFP-0017-5.05/18 CNPq (National Council for Scientific Development and Technological) process: 427153/2016-6 and we also thank the reviewers for their valuable comments and suggestions as these helped us to improve the manuscript.