Development of Biocatalysts Using Organized Nano-reactors

• Project/Area Number: 15550093
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Synthetic chemistry
• Research Institution: OKAYAMA UNIVERSITY
• Principal Investigator: EMA Tadashi OKAYAMA UNIVERSITY, Faculty of Engineering, Associate Professor, 工学部, 助教授 (20263626)
• Co-Investigator(Kenkyū-buntansha): SAKAI Takashi OKAYAMA UNIVERSITY, Faculty of Engineering, Professor, 工学部, 教授 (00170556) ; KORENAGA Toshinobu OKAYAMA UNIVERSITY, Faculty of Engineering, Assistant Professor, 工学部, 助手 (70335579)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,800,000 (Direct Cost: ¥3,800,000); Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000); Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
• Keywords: Biocatalyst / Enzyme / Lipase / Immobilization / High temperature / Enantioselectivity / Kinetic resolution / Asymmetric reduction / 超臨界二酸化炭素

Research Abstract

Kinetic resolution of 1,1-diphenyl-2-propanol was performed at high temperatures using a lipase immobilized on porous ceramics, Toyonite 200M. Reactions proceeded even at 120℃, giving enantiomerically pure ester product. In contrast, the reaction at 100℃ using the same lipase immobilized on Celite resulted in no reaction. The high-temperature biocatalysis in supercritical CO_2 medium at 80℃ gave 42% conversion with an E value of 211. The lipase-catalyzed acylation of a secondary alcohol having tetraphenylporphyrin as a substituent proceeded at 60℃ most efficiently, giving enantiomerically pure ester and alcohol.
Lipase mutant was immobilized on the porous ceramics to study how far the enantioselectivity can be controlled on the basis of a reaction mechanism. Substitution of some amino acid residue in the proximity of the active site with another one resulted in a change in enantioselectivity, as expected. Thermodynamic parameters suggested that the change in enantioselectivity was driven by the change in activation enthalpy.
A new method for the prediction of absolute configurations of secondary alcohols using immobilized lipase was invented. Using the E values for two kinds of 1-substituted ethanols having different substituents, the E value for another secondary alcohol having the two substituents on both sides was predicted and determined. The experimental results for 19 out of 20 examples were consistent with the prediction.
A carbonyl reductase obtained with recombinant E.coli was immobilized on the porous ceramics, which unfortunately showed no remarkable effect. Rather, whole-cell reductions using E.coli coexpressing two genes encoding the carbonyl reductase and a glucose dehydrogenase gave optically active alcohols much more easily and efficiently. Highly enantioselective asymmetric reductions were achieved, and 8 out of 16 ketones were reduced to optically active alcohols with >98% ee.

Research Products

• [Journal Article] Lipase-Catalyzed Resolution of (2R^*,3S^*)- and (2R^*,3R^*)-3-Methyl-3-phenyl-2-aziridinemethanol at Low Temperatures and Determination of the Absolute Configurations of the Four Stere oisomers. (2005)
  • Author(s): Sakai, T., Liu, Y., Ohta, H., Korenaga, T., Ema, T.
  • Journal Title: The Journal of Organic Chemistry 70 Pages: 1369-1375
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Asymmetric Reduction of a Variety of Ketones with a Recombinant Carbonyl Reductase : Identification of the Gene Encoding a Versatile Biocatalyst. (2005)
  • Author(s): Ema, T., Yagasaki, H., Okita, N., Nishikawa, K., Korenaga, T., Sakai, T.
  • Journal Title: Tetrahedron : Asymmetry 16 Pages: 1075-1078
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Lipase-Catalyzed Resolution of (2R^*,3S^*)-and (2R^*,3R^*)-3-Methyl-3-phenyl-2-aziridinemethanol at Low Temperatures and Determination of the Absolute Configurations of the Four Stereoisomers. (2005)
  • Author(s): Sakai, T., Liu, Y., Ohta, H., Korenaga, T., Ema, T.
  • Journal Title: The Journal of Organic Chemistry 70 Pages: 1369-1375
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Asymmetric Reduction of a Variety of Ketones with a Recombinant Carbonyl Reductase : Identification of the Gene Encoding a Versatile Biocatalyst. (2005)
  • Journal Title: Tetrahedron : Asymmetry 16 Pages: 1075-1078
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Lipase-Catalyzed Resolution of (2R^*,3S^*)- and (2R^*,3R^*)-3-Methyl-3-phenyl-2-aziridinemethanol at Low Temperatures and Determination of the Absolute Configurations of the Four Stereoisomers. (2005)
  • Author(s): Sakai, T., Lin, Y., Ohta, H., Korenaga, T., Ema, T.
  • Journal Title: The Journal of Organic Chemistry 70・4 Pages: 1369-1375
• [Journal Article] The Effect of Temperature on the Lipase-Catalyzed Asymmetric Protonation of 1-Acetoxy-2-methylcyclohexene Giving (R)-2-Methylcyclohexanone. (2004)
  • Author(s): Sakai, T., Matsuda, A., Tanaka, Y., Korenaga, T., Ema, T.
  • Journal Title: Tetrahedron : Asymmetry 15 Pages: 1929-1932
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The Effect of Temperature on the Lipase-Catalyzed Asymmetric Protonation of 1-Acetoxy-2-methylcyclohexene Giving (R)-2-Methylcyclohexanone. (2004)
  • Author(s): Sakai, T., Matsuda, A., Tanaka, Y., Korenaga, T., Ema, T.
  • Journal Title: Tetrahedron : Asymmetry 15・12 Pages: 1929-1932
• [Journal Article] Highly Enantioselective Lipase-Catalyzed Reactions at High Temperatures up to 120℃ in Organic Solvent. (2003)
  • Author(s): Ema, T., Kageyama, M., Korenaga, T., Sakai, T.
  • Journal Title: Tetrahedron : Asymmetry 14 Pages: 3943-3947
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Asymmetric Reduction of a Variety of Ketones with a Recombinant Carbonyl Reductase : Identification of the Gene Encoding a Versatile Biocatalyst.
  • Author(s): Ema, T., Yagasaki, H., Okita, N., Nishikawa, K., Korenaga, T., Sakai, T.
  • Journal Title: Tetrahedron : Asymmetry (印刷中)
• [Publications] Ema, T., Kageyama, M., Korenaga, T., Sakai, T.: "Highly Enantioselective Lipase-Catalyzed Reactions at High Temperatures up to 120℃ in Organic Solvent"Tetrahedron : Asymmetry. 14・24. 3943-3947 (2003)