Deciphering the recombinant thermostable phosphatidylcholine-specific phospholipase C activity from Bacillus thuringiensis: Biochemical and interfacial properties

A novel alkaline thermostable phosphatidylcholine-specific phospholipase C (PC-PLCBt) was expressed in E. coli system. Recombinant PC-PLCBt (rPC-PLCBt) activity and thermostability were shown to be significantly depen-dent on the Zn2+. The maximum rPC-PLCBt catalytic activity was found to be 1372 U mg(-1) in the presence of 0.1 mM Zn2+ and at 60 degrees C using an Egg PC as substrate. The interfacial kinetic data show that nPC-PLCBt and rPC-PLCBt display similar substrate specificity on various phospholipid monolayers. The maximal rPC-PLCBt activities were recorded, at decreasing order, on 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diacyl-sn-phosphoglycerol (PG), and 1,2-diacyl-sn-phosphoserine (PS) monolayers at interfacial surface pressures of 15, 25, 20, and 25 mN m(-1), respectively. Such important penetrating power could be exploited for pharmacological purposes. The highest activities were recorded on the DLPC monolayer and shown to be 121.61 and 40.13 mmol cm(-2) min(-1) M-1 for native and recombinant PC-PLCBt, respectively. Interestingly, compared to all known Bacillus PLCs, both PC-PLCBt forms showed an exclusive capacity to hydrolyze the PG film with a more pronounced rate of hydrolysis for the native form with a specific activity of 58.29 mmol cm(-2) min(-1) M-1. Therefore, the high enzyme level production of about 14 mg L-1, the thermostability as well as the broad phospholipid specificity of PC-PLCBt represent great potential in the crude oil refining industry.