High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis.
Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70 degrees C) at acidic pH (pH 3.5-6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g(-1) wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg(-1) protein.
Durban
Markus A
MA
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Friedrich-Ludwig-Jahn-Str. 18c, 17487, Greifswald, Germany.
Silbersack
Jörg
J
Schweder
Thomas
T
Schauer
Frieder
F
Bornscheuer
Uwe T
UT
D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial; D010738:Type C Phospholipases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
C033523:4-nitrophenylphosphorylcholine; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010767:Phosphorylcholine; D010738:Type C Phospholipases