Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase.
Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.
Brod
Fábio Cristiano Angonesi
FC
Depto de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil.
Pelisser
Márcia Regina
MR
Bertoldo
Jean Borges
JB
Vernal
Javier
J
Bloch
Carlos
C
Terenzi
Hernán
H
Arisi
Ana Carolina Maisonnave
AC
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase