The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase.
The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.
Simons
J W
JW
Department of Enzymology and Protein Engineering, CBLE, Utrecht University, The Netherlands.
Adams
H
H
Cox
R C
RC
Dekker
N
N
Götz
F
F
Slotboom
A J
AJ
Verheij
H M
HM
D004952:Esters / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals; D010743:Phospholipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013210:Staphylococcus / Q000201:enzymology*; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism
D004952:Esters; D008670:Metals; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D008049:Lipase