Isolation and Characterization of a Novel Triolein Selective Lipase from Soil Environmental Genes
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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.
Abstract
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.
참고문헌 (37)
- Gupta R, "Bacterial lipases : an overview of production, purification and biochemical properties", Appl. Mocrobiol. Biotechnol, 64, pp.763~781 (2004)
- Kumar A, "Lipase catalysis in organic solvents : advantages and applications", Biol. Proced. Online, 18, pp.2~ (2016)
- Bajaj A, "Biodiesel production through lipase catalyzed transesterification", J. Mol. Catal. B : Enzym, 62, pp.9~14 (2010)
- Torsvik V, "High diversity in DNA of soil bacteria", Appl. Environ. Microbiol, 56, pp.782~787 (1990)
- Steele HL, "Advances in recovery of novel biocatalysts from metagenomes", J. Mol. Microbiol. Biotechnol, 16, pp.25~37 (2009)
- Riesenfeld CS, "Metagenomics : genomic analysis of microbial communities", Annu. Rev. Genet, 38, pp.525~552 (2004)
- Ranjan R, "Isolation of novel lipolytic genes from uncultured bacteria of pond water", Biochem. Bioph. Res. Commun, 335, pp.57~65 (2005)
- Lee MH, "A new esterase EstD2 isolated from plant rhizosphere soil metagenome", Appl. Microbiol. Biotechnol, 88, pp.1125~1134 (2010)
- Glogauer A, "Identification and characterization of a new true lipase isolated through metagenomic approach", Microb. Cell Fact, 10, pp.54~69 (2011)
- Su J, "A new alkaline lipase obtained from the metagenome of marine sponge Ircinia sp", World J. Microbiol. Biotechnol, 31, pp.1093~1102 (2015)
- Selvin J, "Isolation identification and biochemical characterization of a novel halotolerant lipase from the metagenome of the marine sponge Haliclona simulans", Microb. Cell Fact, 11, pp.72~86 (2012)
- Wang Y, "Thermostable alkaline lipase from a newly isolated thermophilic Bacillus, strain A30-1(ATCC 53841)", J. Ferment. Bioeng, 79, pp.433~438 (1995)
- Hoshino T, "Purification and some characteristics of extracellular lipase from Fusarium oxysporum f. sp. lini", Biosci. Biotechnol. Biochem, 56, pp.660~664 (1992)
- Hasan F, "Industrial applications of microbial lipases", Enzyme Microb. Technol, 39, pp.235~251 (2005)
- Ochoa LC, "Screening, purification and characterization of the thermoalkalophilic lipase produced by bacillus thermoleovorans CCR11", Enzyme Microb. Technol, 37, pp.648~654 (2005)
- Ericks SA, "Transesterification activity of a novel lipase from acinetobacter venetianus RAG-1", Antonie Van Leeuwenhoek, 94, pp.621~625 (2008)
- Rondon MR, "Cloning the soil metagenome : a strategy for accessing the genetic and functional diversity of uncultured microorganisms", Appl. Environ. Microbiol, 66, pp.2541~2547 (2000)
- Lee SW, "Screening for novel lipolytic enzymes from uncultured soil microorganisms", Appl. Microbiol. Biotechnol, 65, pp.720~726 (2004)
- Vieira J, "Production of single-stranded plasmid DNA", Methods Enzymol, 153, pp.3~11 (1987)
- Sambrook J, "Molecular cloning. A laboratory manual", pp.1549~ (1989), Cold Spring Harbor Laboratory Press
- Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 227, pp.680~685 (1970)
- Wu S, "LOMETS : a local meta-threading-server for protein structure prediction", Nucleic Acids Res, 35, pp.3375~3382 (2007)
- Roy A, "I-TASSER : a unified platform for automated protein structure and function prediction", Nat. Protoc, 5, pp.725~738 (2010)
- Ngo TD, "Structural and functional analyses of a bacterial homologue of hormonesensitive lipase from a metagenomics library", Acta Crystallogr. D Biol. Crystallogr, 69, pp.1726~1737 (2013)
- Zhang Y, "SPICKER : a clustering approach to identify near-native protein folds", J. Comput. Chem, 25, pp.865~871 (2004)
- Handelsman J, "Metagenomics : application of genomics to uncultured microorganisms", Microbiol. Mol. Biol. Rev, 68, pp.669~685 (2004)
- Simon C, "Achievements and new knowledge unraveled by metagenomic approaches", Appl. Microbiol. Biotechnol, 85, pp.265~276 (2009)
- Sandkvist M, "Biology of type II secretion", Mol. Microbiol, 40, pp.271~283 (2001)
- Wang X, "Lipid signaling", Curr. Opin. Plant Biol, 7, pp.329~336 (2004)
- Ramnath L, "Classification of lipolytic enzymes and their biotechnological applications in the pulping industry", Can. J. Microbiol, 63, pp.179~192 (2017)
- Mohamed YM, "Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea brine pool", Sci. Rep, 3, pp.3358~3366 (2013)
- Haki GD, "Developments in industrially important thermostable enzymes : a review", Bioresour. Technol, 89, pp.17~34 (2003)
- Joseph B, "Standard review cold-active microbial lipases : a versatile toosul for industrial applications", Biotechnol. Mol. Biol. Rev, 2, pp.39~48 (2007)
- Cherif S, "A newly high alkaline lipase : an ideal choice for application in detergent formulations", Lipids Health Dis, 10, pp.221~229 (2011)
- Lailaja VP, "Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11", World J. Microbiol. Biotechnol, 29, pp.1349~1360 (2013)
- López-López O, "New extremophilic lipases and esterases from metagenomics", Curr. Protein Pept. Sci, 15, pp.445~455 (2014)
- Jeon JY, "Feasibility of unsaturated fatty acid feedstocks as green alternatives in bio-oil refinery", Biofuels Bioprod. Biorefin, 13, pp.690~722 (2019)