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Isolation of a thermostable enzyme variant by cloning and selection in a thermophile.

Abstract：

:We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47 degrees C but not at 55 degrees C and above. Variants that were kanamycin resistant at 63 degrees C were obtained by selection of spontaneous mutants, by passage of a shuttle plasmid through the Escherichia coli mutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the first variant by selecting for kanamycin resistance at 70 degrees C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Liao H,McKenzie T,Hageman R

doi

10.1073/pnas.83.3.576

subject

Has Abstract

pub_date

1986-02-01 00:00:00

pages

576-80

issue

3

eissn

0027-8424

issn

1091-6490

journal_volume

83

pub_type

杂志文章

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