Affinity adsorption of phospholipase A1 with designed ligand binding to catalytic pocket.

An affinity ligand was designed from 1-aminocyclohexane based on the crystal structure of Streptomyces albidoflavus phospholipase A1 (saPLA1) by using Discovery Studio software. The molecular docking results indicated that the designed ligand could interact with the active pocket of saPLA1. Epichlorohydrin, cyanuric chloride and 1-aminocyclohexane were used to synthesize the affinity ligand, which was composed to Sepharose beads. The density of the ligand on Sepharose beads was 22.5 ± 1.1 μmol/g wet gel. Adsorption analysis of the sorbent indicated the maximum adsorption (Qmax) of the enzyme was 10.7 ± 0.29 mg/g and the desorption constant (Kd) was 426.6 ± 29.7 μg/mL. The sorbent could bind the enzyme in the supernatant of disrupted recombinant Escherichia coli through one step of affinity adsorption. After the optimization of the purification process, a single band was obtained at approximately 30 kDa, which was confirmed as saPLA1 by the matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry and activity assay. The purity of the isolated enzyme was about 96.6% with the purify fold at 7.62, and the activity recovery was 52.5%.