Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.


Abstract

We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme. Study holds ProTherm entries: 9413, 9414, 9415, 9416 Extra Details:

Submission Details

ID: TUxh2rRT

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Akanuma S;Yamagishi A;Tanaka N;Oshima T,Protein Sci. (1998) Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution. PMID:9541402
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 3-isopropylmalate dehydrogenase P05645 LEU3_BACSU