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Microbiology and Biotechnology Letters

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Bioactive Compounds / Food Microbiology

Microbiol. Biotechnol. Lett. 2016; 44(2): 130-139

https://doi.org/10.4014/mbl.1604.04001

Received: April 1, 2016; Accepted: April 27, 2016

Aeromonas hydrophila PL43이 생산하는 지질분해 효소의 정제 및 특성

Purification and Characterization of a Lipolytic Enzyme Produced by Aeromonas hydrophila PL43

Yong-Woo Kim 1, Sung Wook Hong 2 and Kun Sub Chung 1*

1Division of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea, 2World Institute of Kimchi, Gwangju 61755, Republic of Korea

A bacterial strain, producing an excellent lipolytic enzyme, was isolated from the intestinal tracts of an earthworm (Eisenia fetida). The strain was identified as Aeromonas hydrophila by phenotypic, chemotaxonomic characteristics and 16S ribosomal DNA analysis, and was designated as Aeromona hydrophila PL43. The lipolytic enzyme from A. hydrophila PL43 was purified via 35−45% ammonium sulfate precipitation, DEAE-sepharose fast flow ion-exchange, and sephacryl S-300HR gel filtration chromatography. The yield of the purified enzyme was 3.7% and 2.5% of the total activity of crude extracts with p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as substrates, respectively. The molecular weight of the purified enzyme was approximately 74 kDa using gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and zymography. The optimal activity of purified enzyme was observed at 50°C and pH 8.0 using pNPB, and 60°C and pH 8.0 using pNPP. The purified enzyme was stable in the ranges 20− 60°C and pH 7.0−10.0. The activity of purified enzyme was inhibited by PMSF, pepstatin A, Co2+, Cu2+, and Fe2+, but was recovered by metal chelating of EDTA. The Km and Vmax values of the purified enzyme were 1.07 mM and 7.27 mM/min using pNPB and 1.43 mM and 2.72 mM/min using pNPP, respectively.

Keywords: Purification, lipolytic enzyme, earthworm, Aeromonas hydrophila

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