Molecular Identification of Lipase LipA from Pseudomonas protegens Pf-5 and Characterization of Two Whole-Cell Biocatalysts Pf-5 and Top10lipA
2
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
To identify lipase LipA (PFL_0617) from Pseudomonas protegens Pf-5, a lipA deletion mutant (Pf0617) and a complementary strain (Pf0617lipA) were constructed, and their effects on the lipase production were examined. Pf0617 remarkably decreased its whole-cell lipase activity, whereas Pf0617lipA made its whole-cell lipase activity not only restore to wild-type level but also get a further increment. However, the deletion and overexpression of lipA did not affect the extracellular lipase activity. In addition, the unbroken whole cells of these strains were able to catalyze the hydrolysis of membrane-permeable p-nitrophenyl esters, but could not hydrolyze the membrane-impermeable olive oil. These results confirmed that LipA was an intracellular lipase and Pf-5 could also be used as a natural whole-cell biocatalyst. To evaluate the potential of Pf-5 as a whole-cell biocatalyst and separately characterize the whole-cell LipA, the properties of the whole-cell lipases from Pf-5 and Top10lipA were characterized. The results demonstrated that both Pf-5 and Top10lipA exhibited high tolerance to alkaline condition, high temperature, heavy metal ions, surfactants, and organic solvents. Taken together, lipA can realize functional expression in E. coli Top10, and Pf-5 and Top10lipA as whole-cell biocatalysts may have enormous potential in applications.
Abstract
To identify lipase LipA (PFL_0617) from Pseudomonas protegens Pf-5, a lipA deletion mutant (Pf0617) and a complementary strain (Pf0617lipA) were constructed, and their effects on the lipase production were examined. Pf0617 remarkably decreased its whole-cell lipase activity, whereas Pf0617lipA made its whole-cell lipase activity not only restore to wild-type level but also get a further increment. However, the deletion and overexpression of lipA did not affect the extracellular lipase activity. In addition, the unbroken whole cells of these strains were able to catalyze the hydrolysis of membrane-permeable p-nitrophenyl esters, but could not hydrolyze the membrane-impermeable olive oil. These results confirmed that LipA was an intracellular lipase and Pf-5 could also be used as a natural whole-cell biocatalyst. To evaluate the potential of Pf-5 as a whole-cell biocatalyst and separately characterize the whole-cell LipA, the properties of the whole-cell lipases from Pf-5 and Top10lipA were characterized. The results demonstrated that both Pf-5 and Top10lipA exhibited high tolerance to alkaline condition, high temperature, heavy metal ions, surfactants, and organic solvents. Taken together, lipA can realize functional expression in E. coli Top10, and Pf-5 and Top10lipA as whole-cell biocatalysts may have enormous potential in applications.
참고문헌 (34)
- Angkawidjaja C, "Family I.3 lipase: bacterial lipases secreted by the type I secretion system", Cell Mol. Life Sci, 63, pp.2804~2817 (2006)
- Arpigny JL, "Bacterial lipolytic enzymes: classification and properties", Biochem. J, 343, pp.177~183 (1999)
- Busto E, "Kinetic resolution of 4-chloro-2-(1-hydroxyalkyl)pyridines using Pseudomonas cepacia lipase", Nat. Protoc, 1, pp.2061~2067 (2006)
- Chakraborty K, "Purification and biochemical characterization of an extracellular lipase from Pseudomonas fluorescens MTCC 2421", J. Agric. Food Chem, 57, pp.3859~3866 (2009)
- Grbavcic S, "Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives:compatibility study with detergent ingredients and washing performance", Bioresour. Technol, 102, pp.11226~11233 (2011)
- Gupta R, "Bacterial lipases: an overview of production, purification and biochemical properties", Appl. Microbiol. Biotechnol, 64, pp.763~781 (2004)
- Howell CR, "Control of Rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium", Phytopathology, 69, pp.480~482 (1979)
- Jeganathan J, "Pre-treatment of high oil and grease pet food industrial wastewaters using immobilized lipase hydrolyzation", J. Hazard. Mater, 137, pp.121~128 (2006)
- Jin Z, "Combined utilization of lipase-displaying Pichia pastoris whole-cell biocatalysts to improve biodiesel production in co-solvent media", Bioresour. Technol, 130, pp.102~109 (2013)
- Karadzic I, "Purification and characterization of an alkaline lipase from Pseudomonas aeruginosa isolated from putrid mineral cutting oil as component of metalworking fluid", J. Biosci. Bioeng, 102, pp.82~89 (2006)
- Khan M, "Expression and purification of organic solvent stable lipase from soil metagenomic library", World J. Microbiol. Biotechnol, 28, pp.2417~2424 (2012)
- Kim J, "An organic solvent-tolerant alkaline lipase from cold-adapted Pseudomonas mandelii:cloning, expression, and characterization", Biosci. Biotechnol. Biochem, 77, pp.320~323 (2013)
- Kim KR, "Purification, refolding, and characterization of recombinant Pseudomonas fluorescens lipase", Protein Expr. Purif, 39, pp.124~129 (2005)
- Kim YO, "Gene cloning and catalytic characterization of cold-adapted lipase of Photobacterium sp. MA1-3 isolated from blood clam", J. Biosci. Bioeng, 114, pp.589~595 (2012)
- Kobayashi DY, "A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3", Appl. Environ. Microbiol, 71, pp.261~269 (2005)
- Krzeslak J, "Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase sigma54 and response regulator LipR", FEMS Microbiol. Lett, 329, pp.146~153 (2012)
- Leong SA, "Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti", J. Biol. Chem, 257, pp.8724~8730 (1982)
- Liu W, "Preparation of a whole-cell biocatalyst of Aspergillus niger lipase and its practical properties", J. Agric. Food Chem, 58, pp.10426~10430 (2010)
- Liu Y, "Enhancing effect of Tween-80 on lipase performance in enantioselective hydrolysis of ketoprofen ester", J. Mol. Catal. B Enzym, 10, pp.523~529 (2000)
- McCarthy CN, "Regulation of the aprX-lipA operon of Pseudomonas fluorescens B 52:differential regulation of the proximal and distal genes, encoding protease and lipase, by ompR-envZ", FEMS Microbiol. Lett, 241, pp.243~248 (2004)
- Omar IC, "Characteristics of cell-bound lipase production by a newly isolated strain of Aspergillus flavus", Pertanika J. Sci. Technol, 4, pp.1~9 (1996)
- Pan XX, "Efficient display of active Geotrichum sp. lipase on Pichia pastoris cell wall and its application as a whole-cell biocatalyst to enrich EPA and DHA in fish oil", J. Agric. Food Chem, 60, pp.9673~9679 (2012)
- Panizza P, "Acidic lipase Lip I.3 from a Pseudomonas fluorescens-like strain displays unusual properties and shows activity on secondary alcohols", J. Appl. Microbiol, 114, pp.722~732 (2013)
- Peng R, "Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa CS-2", Appl. Biochem. Biotechnol, 162, pp.733~743 (2010)
- Quandt J, "Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria", Gene, 127, pp.15~21 (1993)
- Ramette A, "Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin", Syst. Appl. Microbiol, 34, pp.180~188 (2011)
- Rosenau F, "Bacterial lipases from Pseudomonas:regulation of gene expression and mechanisms of secretion", Biochimie, 82, pp.1023~1032 (2000)
- Shu ZY, "Production and characteristics of the whole-cell lipase from organic solvent tolerant Burkholderia sp. ZYB002", Appl. Biochem. Biotechnol, 166, pp.536~548 (2012)
- Talukder MMR, "Potential use of whole cell lipase from a newly isolated Aspergillus nomius for methanolysis of palm oil to biodiesel", J. Mol. Catal. B Enzym, 89, pp.108~113 (2013)
- Tanaka D, "Characterization of a new cold-adapted lipase from Pseudomonas sp. TK-3", Appl. Biochem. Biotechnol, 168, pp.327~338 (2012)
- Wu SS, "Markerless deletions of pil genes in Myxococcus xanthus generated by counterselection with the Bacillus subtilis sacB gene", J. Bacteriol, 178, pp.5817~5821 (1996)
- Yang J, "Cloning and expression of Pseudomonas fluorescens 26-2 lipase gene in Pichia pastoris and characterizing for transesterification", Appl. Biochem. Biotechnol, 159, pp.355~365 (2009)
- Zhang A, "Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963", J. Mol. Catal. B Enzym, 56, pp.78~84 (2009)
- Zheng X, "A novel cold-adapted lipase from Acinetobacter sp. XMZ-26: gene cloning and characterisation", Appl. Microbiol. Biotechnol, 90, pp.971~980 (2011)