High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

Abstract

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the α-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg⁻¹ protein which was more than a recombinant P. pastoris GS115 (552 U mg⁻¹ protein) or KM71H (539 U mg⁻¹ protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg⁻¹ protein by P. pastoris GS115, 1176 U mg⁻¹ protein by P. pastoris KM71H and 1522 U mg⁻¹ protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 °C) than the wild-type PLC from B. cereus. Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co²⁺ and Mn²⁺ etc., also influenced the activity of the recombinant PLCs.