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Enzymes from extremophiles -Protein science and protein engineering-

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Enzymes from extremophiles -Protein science and protein engineering-

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    1. Enzymes from extremophiles -Protein science and protein engineering- Extremophiles Thermophile enzymes Protein science Protein engineering Psychrophile enzymes

    2. 1 Extremophiles Organisms inhabiting extreme environments are called ‘extremophiles.’ Extreme environments include high temperature, low temperature, low pH (acid), high pH (alkaline), high salinity, high pressure , etc.

    3. Current record holders for extremophiles

    4. History of research on thermophiles in brief 1960s Dr. T. Brock isolated bacteria that can grow ?80?. 1970s Biochemical researches on heat stable biomolecules from thermophiles were activated. 1974 The term “extremophiles” dates from 1974 (MacElroy). 1975 International symposium on thermophiles was held. 1982 The first thermophile that can grow >100? was reported. 1988 ‘PCR’ method with thermophile enzyme was developed. 1995 Proteomics projects on two thermophiles started. 1996 First genome project on a thermophile was finished. 1997 The scientific journal ‘Extremophiles’ was started. 1999 Japanese Society for Extremophiles was founded. 2005 Genome projects on 21 thermophile spp. were finished.

    5. Classification of thermophiles on the basis of temperature for growth Highest temp. Typical species, for growth typical inhabitats Mesophiles <55? E. coli Moderate ?55? Bacillus stearothermophilus thermophiles Compost Extreme ?75? Thermus thermophilus thermophiles Hot spa Hyper- ?90? Pyrococcus furiosus thermophiles Hydrothermal vent

    6. Drawing hot water (Isolation of thermohpiles)

    7. 2 Thermophile enzymes Unusual high stability Capable of reaction at high temperature Highly preservative Tendency to easily form better crystals.

    8. 2 Thermophile enzymes Unusual high stability Capable of reaction at high temperature

    9. PCR Taq Tth Tfl : origins = extreme thermophiles Pfu Pwo Pyrobest Tli : hyperthermophiles Mixture of these enzymes

    10. Thermophiles as important sources of enzymes The extreme thermophile Thermus thermophilus Max temp for growth: 83? Genome:1.6Mb (GC-rich) A sole thermophile authorized as the host in gene engineering DNA polymerases and restriction endonucleases are on sale. The genome project has been finished in 1998, the proteomics project is in progress. The hyperthermophile Pyrococcus furiosus Max temp for growth: 103? Genome: 2.1Mb (AT-rich) An archaeon, material useful for evolution and bioscience researchs DNA polymerases are on sale. The genome project has been finished in 2000, the proteomics project is in progress.

    11. 3 Protein sciences

    12. Flow of protein science researches

    13. Flow of protein science researches

    14. Difficulty of every stage in protein science research E. coli genes E. coli proteins Crystals 3D structures

    15. Why is the expression level of the thermophile genes in recombinant E. coli poor?

    16. Stage 1 Overexpression of genes

    17. Stage 1 Overexpression of genes

    18. Flow of protein science researches

    21. Frow of protein science researchs

    24. Mechanisms for thermostability in thermophile proteins 1 A diverse range of structural changes may occur. Enhancement of interactions in hydrophobic core in the interior of proteins Enhancement of interactions, such as increase in ion-pair network, in the domain interface and the subunit interface Stabilization (Gly?Ala) in the secondary structures Increase in entropic stabilization, such as shortening the peptide chain, decrease in Gly, and increase in Pro Elimination of unstable amino acids, such as Cys 2 A significant change in stability may arise in response to small changes in energy.

    25. Flow of protein science researches

    26. The merits and demerits of thermophile enzymes

    27. 4 Protein engineering Definition: To synthesize a newly-designed protein by making the most of recombinant DNA technology and chemical synthesis of nucleic acids. In 1982, M. Smith altered a natural gene sequence by the site-directed mutagenesis method. In 1983, K. Ulmer advocated the new concept “Protein engineering.” Up to now, many improved methods other than site-directed mutagenesis are proposed. i.e. Evolutionally molecular engineering, phage display method, de novo design, and so on.

    28. Site-directed mutagenesis

    29. Evolutionally molecular engineering 10 PRODUCE A MUTANT SPECTRUM OF SELF-REPRODUCING TEMPRATES 20 SEPARATE AND CLONE INDIVIDUAL MUTANTS 30 AMPLIFY CLONES 40 EXPRESS CLONES 50 TEST FOR OPTIMAL PHENOTYPES 60 IDENTIFY OPTIMAL GENOTYPES 70 RETERN TO 10 WITH A SAMPLE OF OPTIMAL GENOTYPES

    30. Thermostabilization of enzyme by E.M.E.

    31. Thermostabilization of mesophile enzyme by evolutionally molecular engineering

    32. Adaptation of a thermophile enzyme to low temperature

    33. 5 Psychrophile enzymes 1)Protein science To elucidate the molecular mechanism of cold-adaptation. Why are the psychrophile enzymes highly active under low temperature? To improve the physicochemical properties of enzyme. If we stabilize a psychrophile enzyme by protein engineering, the super enzyme having both high cold-activity and high stability will be created. 2)Biotechnology To hunt up enzymes that are useful for industrial applications, such as research reagents, efficient fermentation in low temperature, etc.

    34. 1991 Shewanella frigidimarina was isolated from the body surface of a marine fish that spoiled quickly during chilled preservation, in collaboration with Drs. S. Kimura & T. Fujii. 1998 Psychrophilic Vibrio and Photobacterium spp. were isolated from surface water of the Sea of Okhotsk, Hokkaido prefecture. 2004 Some psychrophiles were isolated from deep seawater in Sagami Bay in collabo- ration with Drs. N. Urano & H. Kabasawa. 2005 Some psychrophiles were isolated from surface water of the Antarctic Sea in collabo- ration with Drs. N. Urano & T. Hayashi, and our vessel, the Umitakamaru.

    36. Nucleases from Vibrio logei and S. frigidimarina

    37. Cold-active enzymes from psychrophiles from deep seawater and the Antarctic seawater Promissing strains of enzyme-producing psychrophiles Protease-producing psychrophiles Five strains, such as Pr-76 Nuclease-producing psychrophiles Seven strains,such as Dn-1 Lipase-producing psychrophiles Five strains, such as 2A-25 Amylase-producing psychrophiles Strains Am-1 and Am-2

    38. Summary Overview of extremophiles, especially thermophiles The general characteristics of thermophile enzymes and their application to PCR The overall picture of protein science research that uses thermophile proteins as research materials The advances in proteomics projects and the elucidation of the thermostability in thermophile enzymes Protein engineering research for overcoming the disadvantages of the thermophilic enzymes Psychrophile enzymes as valuable resources for enzyme modification

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