High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis

• Published in: Applied microbiology and biotechnology Vol. 74; no. 3; pp. 634 - 639
• Main Authors: Durban, Markus A, Silbersack, Jörg, Schweder, Thomas, Schauer, Frieder, Bornscheuer, Uwe T
• Format: Journal Article
• Language: English
• Published: Berlin Berlin/Heidelberg : Springer-Verlag 01.03.2007; Springer; Springer Nature B.V
• Subjects: Amino acids; Bacillus cereus; Bacillus cereus - enzymology; Bacillus cereus - genetics; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacteria; Biological and medical sciences; Biotechnology; Cloning; Cloning, Molecular; DNA, Bacterial - genetics; Enzyme Stability; Expression; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; High temperature; Hot Temperature; Hydrogen-Ion Concentration; Microbiology; phospholipase C; Phosphorylcholine - analogs & derivatives; Phosphorylcholine - metabolism; Polymerase Chain Reaction; Recombinant Proteins - biosynthesis; Sequence Homology, Amino Acid; Transformation, Bacterial; Type C Phospholipases - biosynthesis; Type C Phospholipases - genetics; Type C Phospholipases - isolation & purification; Bacillus cereus; Bacillales; Enzyme; Gene overexpression; Bacteria; Hydrolases; Esterases; Phosphoric diester hydrolases; Bacillaceae; Phospholipase C
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-006-0712-z

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°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.