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Site‐directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis
Author(s) -
Chang ReyChang,
Chou ShuJen,
Shaw JeiFu
Publication year - 2000
Publication title -
journal of the american oil chemists' society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.512
H-Index - 117
eISSN - 1558-9331
pISSN - 0003-021X
DOI - 10.1007/s11746-000-0162-x
Subject(s) - site directed mutagenesis , lipase , mutagenesis , enzyme , biochemistry , active site , escherichia coli , chemistry , mutant , staphylococcus epidermidis , stereochemistry , biology , bacteria , gene , staphylococcus aureus , genetics
A fragment of Staphylococcus epidermidis lipase gene (Lys‐303 to Lys‐688) was inserted into plasmid pET‐20b(+). The resulting C‐terminal His‐tagged recombinant protein (43 kDa) was overexpressed in Escherichia coli BL21(DE3) as a highly active lipase and was purified with nickel‐coupled resin. Putative catalytic sites were determined by site‐directed mutagenesis. Mutant enzymes (S418C and H648K) lost enzyme activities, which strongly suggests that the proposed residues of Ser‐418 and His‐648 are involved in catalysis. Site‐directed mutagenesis showed that in comparison with wild‐type enzyme, the M419A and V649l enzymes showed a 2.0‐ and 4.0‐fold increase in the k cat / K m′ respectively, but the M419l, M419Q, V649A, and V649L variants lost enzyme activities. The wild‐type enzyme and the V649l mutant favored the hydrolysis of p ‐nitrophenyl esters of butyrate, but the M419A favored decanoate. The results suggested that the amino acid residues (Met‐419 and Val‐649), following the catalytic triad, could affect the substrate specificity and/or catalytic efficiency.