Evolution of a Second Gene for β-Galactosidase in
            <i>Escherichia coli</i>

Campbell, John H.; Lengyel, Judith A.; Langridge, J.

doi:10.1073/pnas.70.6.1841

Cited by 103 publications

(56 citation statements)

References 18 publications

(3 reference statements)

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“…The E. coli Ebg protein shows characteristics different from those of the classic ␤-galactosidase, LacZ (2,11,12,13,27): (i) Ebg ␤-galactosidase has insufficient catalytic activity toward lactose compared to the LacZ enzyme. In addition, the K m value of Ebg toward the synthetic substrate ONPG is four times higher than that of LacZ.…”

Section: Discussionmentioning

confidence: 99%

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Distinct Roles of β-Galactosidase Paralogues of the Rumen Bacterium Mannheimia succiniciproducens

Lee

Kim

et al. 2012

J Bacteriol

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Mannheimia succiniciproducens, a rumen bacterium belonging to the family Pasteurellaceae, has two putative ␤-galactosidase genes, bgaA and bgaB, encoding polypeptides whose deduced amino acid sequences share 56% identity with each other and show approximately 30% identity to the Escherichia coli gene for LacZ. The M. succiniciproducens bgaA (MsbgaA) gene-deletion mutant was not able to grow on lactose as the sole carbon source, suggesting its essential role in lactose metabolism, whereas the MsbgaB gene-deletion mutant did not show any growth defect on a lactose medium. Furthermore, the expression of the MsbgaA gene was induced by the addition of lactose in the growth medium, whereas the MsbgaB gene was constitutively expressed independently of a carbon source. Biochemical characterization of the recombinant proteins revealed that MsBgaA is more efficient than MsBgaB in hydrolyzing o-nitrophenyl-␤-D-galactopyranoside and p-nitrophenyl-␤-D-galactopyranoside. MsBgaA was highly specific for the hydrolysis of lactose, with a catalytic efficiency of 46.9 s ؊1 mM ؊1 . However, MsBgaB was more efficient for the hydrolysis of lactulose than lactose, and the catalytic efficiency was 10.0 s ؊1 mM ؊1 . Taken together, our results suggest that the ␤-galactosidase paralogues of M. succiniciproducens BgaA and BgaB play a critical role in lactose metabolism and in an unknown but likely specific function for rumen bacteria, respectively. R uminant animals digest cellulosic biomass through their digestive tract and metabolize this material to produce milk, meat, wool, and hides. The rumen is the first of four stomachs in ruminant animals (5). Plant biomass is fermented by ruminal bacteria in the rumen under a relatively stable environment, characterized by a stable temperature of 39°C, a near neutral pH, and a constant oxidation-reduction potential (35, 37). More than 200 species of microorganisms in the rumen, through symbiosis or commensalism, convert plant polysaccharides to monosaccharides or disaccharides (5, 16). The resulting carbohydrates are further metabolized during fermentation by rumen bacteria into organic acids, including acetic, lactic, propionic, butyric, and succinic acids, and into methane and CO 2 (37). Among these organic acids, succinic acid is an attractive chemical building block for the manufacture of various substances, such as biodegradable polymers, surfactants, synthetic resins, and additives in food and pharmaceuticals (36).Several microorganisms, including Actinobacillus succinogenes, which enable the biotechnological production of succinic acid have been isolated from the rumen and in other habitats (8). Recently, Mannheimia succiniciproducens, which produces a large amount of succinic acid with a 67.5% yield from glucose under an anaerobic condition, was isolated from a bovine rumen (23). The Gram-negative rod, nonmotile, mesophilic, and capnophilic bacterium M. succiniciproducens belongs to the family Pasteurellaceae (23). M. succiniciproducens is unable to degrade the complex polysaccharides as...

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Section: Discussionmentioning

confidence: 99%

“…2 O, and 1 g Na 2 S · 9H 2 O. The cultures were grown in a sidearm flask containing 50 ml of MH3 medium at 39°C without shaking in a 10% CO 2 atmosphere.…”

mentioning

confidence: 99%

Distinct Roles of β-Galactosidase Paralogues of the Rumen Bacterium Mannheimia succiniciproducens

Lee

Kim

et al. 2012

J Bacteriol

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“…Perhaps the earliest example of in vivo evolution is that of the E. coli protein, EbgA, with little or no b-galactosidase activity to one with weak but detectable activity (Campbell et al, 1973). This concept of evolving proteins under controlled conditions in the laboratory (spearheaded by the Frances Arnold group at California Institute of Technology and by W.P.…”

Section: Molecular Evolution Strategiesmentioning

confidence: 99%

The Outlook for Protein Engineering in Crop Improvement

Rao

2008

Plant Physiology

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“…One early example is the evolution of the EbgA protein from Escherichia coli, an enzyme having almost no ␤-galactosidase activity. Through intensive selection of a LacZ Ϫ deletion strain of E. coli for growth on lactose as a sole carbon source, the wild-type EbgA was "evolved" as a ␤-galactosidase sufficient to replace the lacZ gene function (39). Perhaps surprisingly, the evolution of new functions of an enzyme can require few mutations, as was the case for the EbgA protein.…”

Section: Introductionmentioning

confidence: 99%

“…Systematic approaches to directed evolution of proteins have been documented since the 1970s (39,106,110). One early example is the evolution of the EbgA protein from Escherichia coli, an enzyme having almost no ␤-galactosidase activity.…”

Section: Introductionmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

171

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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Evolution of a Second Gene for β-Galactosidase in Escherichia coli

Cited by 103 publications

References 18 publications

Distinct Roles of β-Galactosidase Paralogues of the Rumen Bacterium Mannheimia succiniciproducens

Distinct Roles of β-Galactosidase Paralogues of the Rumen Bacterium Mannheimia succiniciproducens

The Outlook for Protein Engineering in Crop Improvement

Laboratory-Directed Protein Evolution

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