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 (Q_max) of the enzyme was 10.7 ± 0.29 mg/g and the desorption constant (K_d) 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%.