Enhancement of the stability and activity of aspartase by random and site-directed mutagenesis.
Enzymatic generation of mutant libraries for random mutagenesis of aspartase gene from E. coli J2 was made. A mutant enzyme with 4-fold increase in aspartase activity was found. It is stable at pH7.5-9.0 (wild-type: pH7.0-8.0); heat stability and alpha-helicity are higher than those of the wild-type. By using site directed mutagenesis, the aspartase was activated by replacement of Lys-126 with an arginine residue. The mutation produced functional alterations without appreciable structure changes. The optimum pH for the mutant enzyme is 8.5. The stable pH range is 7.0-9.0. Heat stability is higher than that of the wild-type one. Activity of the mutant enzyme is about 5-fold as much as that of wild-type one.
Zhang
H Y
HY
National Laboratory of Enzyme Engineering, Jilin University, Changchun, P.R. China.
Zhang
J
J
Lin
L
L
Du
W Y
WY
Lu
J
J
D000595:Amino Acid Sequence; D001220:Aspartate Ammonia-Lyase / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed
D001220:Aspartate Ammonia-Lyase