Construction of a chimeric aminopeptidase by a combination of gene shuffling and mutagenesis.
Three chimeric genes were constructed by gene shuffling of aminopeptidases from Aeromonas caviae and Vibrio proteolyticus. Although expressed chimeric enzymes formed inclusion bodies in Escherichia coli, the introduction of two amino acid mutations into the chimeric genes by site-saturated mutagenesis and a random mutation on error-prone PCR resulted in solubilization of the chimeric enzyme. In addition, active chimeric enzyme showed a different thermostability and thermoactivity to parental enzymes.
Nirasawa
Satoru
S
Enzyme Laboratory, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8642, Japan. stnirasa@affrc.go.jp
Hayashi
Kiyoshi
K
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics*; D032621:DNA Shuffling / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D012995:Solubility
D011994:Recombinant Proteins; D000626:Aminopeptidases