Enhancing Soluble Expression of Phospholipase B for Efficient Catalytic Synthesis of L-Alpha-Glycerylphosphorylcholine

Phospholipase B (PLB) harbors three distinct activities with broad substrate specificities and application fields. Its hydrolyzing of sn-1 sn-2 acyl ester bonds enables it to catalyze the production L-alpha-glycerylphosphorylcholine (L-α-GPC) from phosphatidylcholine (PC) without speed-limiting migration. This work was intended obtain high-level active PLB apply establish an efficient system for L-α-GPC synthesis. Pseudomonas fluorescens co-expressed five different molecular chaperones, including trigger factor (Tf), GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), GroELS DnaKJE, or Tf fused maltose binding protein (MBP) in Escherichia coli BL21(DE3) improve expression. DnaKJE-assisted expression exhibited highest catalytic activity. Further optimization conditions identified optimal induction OD600 0.8, IPTG concentration 0.3 mmol/L, time 9 h, temperature 25 °C. The activity reached a maximum 524.64 ± 3.28 U/mg under conditions. Subsequently, PLB-catalyzed synthesis, series organic-aqueous mixed systems surfactant-supplemented aqueous were designed constructed. Furthermore, factors temperature, reaction pH, metal ions, further systematically identified. Finally, high yield 90.50 2.21% obtained Span 60-supplemented at 40 °C pH 6.0 0.1 mmol/L Mg2+. proposed cost-effective environmentally friendly offer candidate strategy industrial L-α-GPC.