Random mutagenesis of a plasmid-borne glycosidase gene and phenotypic selection of mutants in Escherichia coli.
The bglA gene from Bacillus polymyxa encodes a beta-glucosidase able to hydrolyze p-nitrophenyl-beta-D-glucopyranoside (PNPG), and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), chromogenic substrates for beta-glucosidases and beta-galactosidases respectively. A plasmid carrying the blgA gene inserted in vector pUC18 was mutagenized in vitro with hydroxylamine, and subsequently used to transform E. coli selecting for the ampicillin resistance conferred by the cloning vector. The transformants were screened on petri dishes for mutations causing inability to hydrolyze either one of the two substrates, and for mutations increasing resistance of the enzyme to thermal inactivation. The isolation of several mutants with such characteristics suggests that the simple procedure used here can be applied to generate modifications of enzymatic properties that fit specific industrial requirements.
Lopez-Camacho
C
C
Instituto de Agroquímica y Tecnología de Alimentos, C.S.I.C., Valencia, Spain.
Polaina
J
J
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000633:toxicity; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D014170:Transformation, Genetic; D001617:beta-Glucosidase / Q000235:genetics*
D006898:Hydroxylamines; D019811:Hydroxylamine; D001617:beta-Glucosidase