Purification and Characterization of tert-Butyl Ester-hydrolyzing Lipase from Burkholderia sp. YY62

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概要

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• An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) was purified to homogeneity from crude cell-free extracts of Burkholderia (formerly Pseudomonas) sp. YY62 with 9% overall yield. Seventy-four-fold purification was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filtration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G2000SW_<XL> gel-filtration and phenyl 5PW hydrophobic interaction columns). Enzymes hydrolyzing p-nitrophenyl acetate were separated into two peaks (peak I and II) on the hydrophobic HPLC, and only peak II was found to have TBO-hydrolyzing activity. The peak preparation showed a single band of 40 kDa on SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denatured conditions, indicating the monomeric nature of the TBO-hydrolyzing lipase. The lipase showed maximum activity at pH 7.0 and 28℃. The N-terminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-Asn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known proteins, suggesting that the purified enzyme may belong to a novel class of hydrolase.

• 社団法人日本農芸化学会の論文
• 1998-12-23

著者

• Nihira Takuya

  Department Of Biotechnology Graduate School Of Engineering Osaka University

• Nihira Takuya

  Department Of Biotechnology Faculty Of Engineering Osaka University

• Nakamura Takefumi

  Department Of Applied Biological Chemistry Graduate School Of Agricultural And Life Sciences The Uni

• Yamada Yasuhiro

  Department Of Biotechnology Graduate School Of Engineering Osaka University

• Yamada Yasuhiro

  Department Of Agricultural Chemistry Faculty Of Agriculture University Of Tokyo

• YEO SooHwan

  Department of Biotechnology, Graduate School of Engineering, Osaka University

• Yeo Soohwan

  Department Of Biotechnology Graduate School Of Engineering Osaka University

• Nihira T

  Department Of Applied Biological Chemistry Graduate School Of Agricultural And Life Sciences The Uni

• Yeo Soo-Hwan

  Department of Biotechnology, Graduate School of Engineering, Osaka University

• Nihira Takuay

  Department of Biotechnology, Graduate School of Engineering, Osaka University

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