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Microbiology and Biotechnology Letters

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Microbial Biotechnology

Microbiol. Biotechnol. Lett. 2020; 48(4): 480-490

https://doi.org/10.48022/mbl.2007.07005

Received: July 16, 2020; Accepted: September 15, 2020

Isolation and Characterization of a Novel Triolein Selective Lipase from Soil Environmental Genes

Hee Kyung Lim1, Ye-Jin Han1, Moon-Sun Hahm2, Soo Youl Park1 and In Taek Hwang1*

1Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea 2Bioprogen, VentureTown Jangyoungsil, Daejeon 34324, Republic of Korea

Correspondence to :
In Taek Hwang,    ithwang@krict.re.kr

A novel lipase gene, Lip-1420, was isolated from a metagenomic library constructed from reed marsh from Mt. Jumbong in Korea, comprising 112,500 members of recombinant plasmids. The DNA sequence of Lip- 1420-subclone (5,513 bp) was found to contain at least 11 ORFs according to the GenBank database. The ORF-3 gene was inserted into the pET21a plasmid containing the C-terminal 6-His tag and transformed into E. coli BL21(DE3) to express the recombinant lipase protein. Lip-1420 was purified using a fast protein liquid chromatography system. The gene was registered in GenBank (MH628529). The values of Km and Vmax were determined as 0.268 mM and 1.821 units, respectively, at 40℃ and pH 8.0, using p-nitrophenyl palmitate as the substrate. This lipase belongs to family IV taxonomically because it has conserved HGGG and GDSAG motifs in the constitutive amino acid sequence. According to the predicted structural model, the binding sites are represented by residues H78, G81, D150, S151, A152, V181, and D236. Finally, Lip-1420 showed triolein selectivity for methanolysis between triolein (18:1) and tristearin (18:0) substrates. Further study of the selective mechanism and structure-function relationship of this new lipase could be useful for more practical applications.

Keywords: Lipase, rhizosphere, metagenomics, 3D-structure, triolein selectivity

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