Purification and Characterization of the Lipase from Serratia marcescens Sr41 8000 Responsible for Asymmetric Hydrolysis of 3-Phenylglycidic Acid Esters

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概要

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• A new lipase which enantioselectively hydrolyzes (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride, was purified from the culture supernatant of Serratia marcescens Sr41 8000. The apparent kinetic constants (K_m, V_<max>) for hydrolysis of (2S, 3R)-MPGM [(+)-MPGM] were 350 mM and 1.7×10^<-3> mol/min/mg protein in a toluene-water (1 : 1) emulsion system. The lipase did not attack (2R, 3S)-MPGM [(-)-MPGM], and (-)-MPGM acted as a competitive inhibitor. The molecular mass was estimated to be 62,000±2,000 from SDS-PAGE. The lipase preferentially hydrolyzed (2S, 3R)-3-phenylglycidic acid esters, but did not hydrolyze cinnamic acid esters. The lipase released glycerol and fatty acid from olive oil, and the optimum pH and temperature for hydrolysis of olive oil were pH8 and 45℃, respectively. The lipase was inhibited by Co^<2+>, Ni^<2+>, Fe^<2+>, Fe^<3+> and EDTA, and activated by Ca^<2+>, Li^+ and SDS. It was presumed that the lipase was a metalloenzyme containing approximately one gram atom of calcium per molecular mass of 62,000. The lipase selectively hydrolyzed the 1,3 ester of triglycerides. Sequencing of the N-terminal amino acids revealed that this lipase was distinct from other known lipases.

• 公益社団法人日本生物工学会の論文
• 1994-02-25

著者

• Shibatani Takeji

  Research Laboratory Of Applied Biochemistry Tanabe Seiyaku Co. Ltd.

• Matsumae Hiroaki

  Research Laboratory Of Applied Biochemistry Tanabe Seiyaku Co. Ltd.

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• Microbial Asymmetric Reduction of (RS)-2-(4-Methoxyphenyl)-1,5-benzothiazepin-3,4(2H, 5H)-dione, the Key Intermediate in the Synthesis of Diltiazem Hydrochloride
• Production of Optically Active 3-Phenylglycidic Acid Ester by the Lipase from Serratia marcescens on a Hollow-Fiber Membrane Reactor
• Lipase-Catalyzed Asymmetric Hydrolysis of 3-Phenylglycidic Acid Ester, the Key Intermediate in the Synthesis of Diltiazem Hydrochloride
• Purification and Characterization of the Lipase from Serratia marcescens Sr41 8000 Responsible for Asymmetric Hydrolysis of 3-Phenylglycidic Acid Esters

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