Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144.
The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.
Feller
G
G
University of Liege, Institute of Chemistry, Belgium.
Thiry
M
M
Arpigny
J L
JL
Gerday
C
C
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009016:Moraxella / Q000201:enzymology / Q000235:genetics*; D009596:Nitrophenols / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D009596:Nitrophenols; D011994:Recombinant Proteins; D008049:Lipase; C024836:4-nitrophenol