High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization.
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 alpha-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 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.
Seo
Kook-Hwa
KH
Faculty of Applied Chemical Engineering, BioProcess Technology Laboratory, Chonnam National University, YongBong-dong 300, GwangJu 500-757, Republic of Korea.
Rhee
Jong Il
JI
D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D014170:Transformation, Genetic / Q000235:genetics; D010738:Type C Phospholipases / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D010738:Type C Phospholipases