KEGG(Kyoto Encyclopedia of Genes and Genomes)〔和文〕 (特集 ゲノム医学の現在と未来--基礎と臨床) -- (データベース)

基礎講座 電気泳動(Electrophoresis)

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Biological control of soilborne pathogens by introduced microorganisms has been studied for over 65 years (9, 49), but during most of that time it has not been considered commercially feasible. Since about 1 965, however, interest and research in this area have increased steadily (9), as reflected by the number of books (10, 47,49, 152) and reviews about it (11,26,30, 106, 143, 153, 173, 174, 183) that have appeared . Concurrently, there has been a shift to the opinion that biological control can have an important role in agriculture in the future, and it is encouraging that several companies now have programs to develop biocontrol agents as commercial products. This renewed interest in biocontrol is in part a response to public concern about hazards associated with chemical pesticides. Microorganisms that can grow in the rhizosphere are ideal for use as biocontrol agents, since the rhizosphere provides the front-line defense for roots against attack by pathogens. Pathogens encounter antagonism from rhizosphere microorganisms before and during primary infection and also during secondary spread on the root. In some soils described as microbiologi­ cally suppressive to pathogens (172), microbial antagonism of the pathogen is especially great, leading to substantial disease control. Although pathogen­ suppressive soils are rare, those identified are excellent examples of the full potential of biological control of soilborne pathogens.

Biological control of soilborne plant pathogens in the rhizosphere with bacteria

Each recent report of liquid water existing elsewhere in the Solar System has reverberated through the international press and excited the imagination of humankind. Why? Because in the past few decades we have come to realize that where there is liquid water on Earth, virtually no matter what the physical conditions, there is life. What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring 'extremophiles'. This realization, coupled with new data on the survival of microbes in the space environment and modelling of the potential for transfer of life between celestial bodies, suggests that life could be more common than previously thought. Here we examine critically what it means to be an extremophile, and the implications of this for evolution, biotechnology and especially the search for life in the Universe.

/pdf/life-in-extreme-environments-qdlikt0yc0.pdf

Life in Extreme Environments

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.

Prospects for a bio-based succinate industry

Thermozymes: Identifying molecular determinants of protein structural and functional stability

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).

https://aem.asm.org/content/aem/63/9/3569.full.pdf

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.

Claire Vieille

Papers

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Prospects for a bio-based succinate industry

Thermozymes: Identifying molecular determinants of protein structural and functional stability

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli