Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.
Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.
Lopez-Camacho
C
C
Instituto de Agroquímica y Tecnología de Alimentos, Cosejo Superior de Investigaciones Científicas, Paterna (Valencia), Spain.
Salgado
J
J
Lequerica
J L
JL
Madarro
A
A
Ballestar
E
E
Franco
L
L
Polaina
J
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D019811:Hydroxylamine; D006898:Hydroxylamines; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism*
D006898:Hydroxylamines; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019811:Hydroxylamine; D001617:beta-Glucosidase