Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method

Kotsuka, Takashi; Akanuma, Satoshi; Tomuro, Masaaki; Yamagishi, Akihiko; Oshima, Tairo

doi:10.1128/jb.178.3.723-727.1996

Cited by 60 publications

(29 citation statements)

References 37 publications

(38 reference statements)

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“…All the enzymes used in this study were purified to homogeneity as judged by SDS-gel electrophoresis. The remaining activities of the purified enzymes after heat treatment over temperature range 40-63 "C were measured at 40 "C as described previously (Kotsuka et al, 1996).…”

Section: Pcr and Sequencingmentioning

confidence: 99%

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

et al. 1998

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We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Themus themophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a ZeuB-deficient strain of Z themophilus. The resulting transformant showed a leucine-autotrophy at 56 "C but not at 61 "C and above. Phenotypically thermostabilized strains that can grow at 61 "C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 "C and mutants that showed a leucine-autotrophic growth at 70 "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.

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Section: Pcr and Sequencingmentioning

confidence: 99%

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

et al. 1998

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“…We started with proteins which form 4-helix bundle structure [4] by two proteins, each having two helices. In our previous studies [5][6] of molecular dynamics simulation of all-atom model, we have found that IPMDH (3-isopropylmalate dehydrogenase) [7][8][9] is a good candidate for a building unit of constructing nano-fibers because of the high stability of the mutated IPMDH. In this study, we have performed molecular dynamics simulation of coarse-grained model of IPMDH proteins.…”

Section: Introductionmentioning

confidence: 99%

Coarse-Grained Molecular Dynamics Simulation of IPMDH Proteins

Hirata¹,

Komatsu²,

Fukuda³

et al. 2014

Proceedings of the 12th Asia Pacific Physics Conference (APPC12)

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We have performed molecular dynamics simulation of coarse-grained model of IPMDH protein to study the effect of mutation to the binding sites of protein. We replace original amino acids on the surface by negatively or positively charged amino acids. We also study the effect of solvent to the binding of proteins. Our computational study may be useful for future experimental study of protein bindings.

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“…We chose Thermus thermophilus as a host due to its high transformation ability (17) and ability to use melibiose (␣-galactoside) as a sole carbohydrate source (10). Thermus species have been used for expression of heterologous genes and selection of thermostable enzyme variants (16,19,34,36). They possess a natural transformation system (17) and are competent regardless of their growth phase (12).…”

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Production of Recombinant α-Galactosidases in Thermus thermophilus

Friðjónsson¹,

Mattes

2001

Appl Environ Microbiol

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A Thermus thermophilus selector strain for production of thermostable and thermoactive ␣-galactosidase was constructed. For this purpose, the native ␣-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (␣-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal ؉ phenotype was assumed to be essential for growth on melibiose. In a Gal ؊ background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant ␣-galactosidase. Moreover, the AgaT ؊ strain had to be Km s for establishment of the plasmids containing ␣-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT ؊ Gal ؉ Km s ) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous ␣-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.␣-Galactosidases catalyze the hydrolysis of ␣-1,6-linked ␣-galactose residues from oligosaccharides and polymeric galactomannans (9,25,26,42). They have considerable potential in various industrial applications, e.g., in the sugar industry for the elimination of D-raffinose from sugar beet syrup. Due to the elevated temperatures used during the sugar manufacturing process, as well as in other industrial applications, stability and activity at high temperatures are important properties of ␣-galactosidases.We have been studying ␣-galactosidases from various bacteria with regard to their application as oligosaccharidehydrolyzing enzymes (9,11,14). Our intention was to subject ␣-galactosidase to thermoadaptation by introducing genes encoding enzymes inactive at high temperatures into a thermophilic bacterium for subsequent selection of enzyme variants active at high temperatures. We chose Thermus thermophilus as a host due to its high transformation ability (17) and ability to use melibiose (␣-galactoside) as a sole carbohydrate source (10). Thermus species have been used for expression of heterologous genes and selection of thermostable enzyme variants (16,19,34,36). They possess a natural transformation system (17) and are competent regardless of their growth phase (12). Genetic systems based on the applic...

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Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method

Cited by 60 publications

References 37 publications

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

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

Coarse-Grained Molecular Dynamics Simulation of IPMDH Proteins

Production of Recombinant α-Galactosidases in Thermus thermophilus

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