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http://dx.doi.org/10.4014/jmb.1506.06011

Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil  

Yang, Wenjuan (Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology)
Xu, Li (Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology)
Zhang, Houjin (Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology)
Yan, Yunjun (Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.11, 2015 , pp. 1880-1893 More about this Journal
Abstract
In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/β hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60℃ with a Km of 0.37 mM and a kcat of 6.42 s-1. It retained over 90% of its original activity after incubation at 50℃ for 12 h. In addition, LipR was activated by Ca2+, Mg2+, Ba2+, and Sr2+, while strongly inhibited by Cu2+, Zn2+, Mn2+, and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.
Keywords
Genome walking; subfamily I.3 lipase; thermostability; hydrolysis; long-chain polyunsaturated fatty acids; diacylglycerols;
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1 Arpigny JL, Jaeger KE. 1999. Bacterial lipolytic enzymes: classification and properties. Biochem. J. 343: 177-183.   DOI
2 Amada K, Haruki M, Imanaka T, Morikawa M, Kanaya S. 2000. Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp. MIS38. Biochim. Biophys. Acta 1478: 201-210.   DOI
3 Angkawidjaja C, Kanaya S. 2006. Family I.3 lipase: bacterial lipases secreted by the type I secretion system. Cell. Mol. Life Sci. 63: 2804-2817.   DOI
4 Arifin AR, Kim SJ, Yim JH, Suwanto A, Kim HK. 2013. Isolation and biochemical characterization of Bacillus pumilus lipases from the Antarctic. J. Microbiol. Biotechnol. 23: 661-667.   DOI
5 Faoro H, Glogauer A, Couto GH, de Souza EM, Rigo LU, Cruz LM, et al. 2012. Characterization of a new Acidobacteria-derived moderately thermostable lipase from a Brazilian Atlantic Forest soil metagenome. FEMS Microbiol. Ecol. 81: 386-394.   DOI
6 Bradford MM. 1976. Arapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.   DOI
7 Christensen MS, Høy CE, Becker CC, Redgrave TG. 1995. Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids dependence on intramolecular triacyiglycerol structure. Am. J. Clin. Nutr. 61: 56-61.   DOI
8 Dyall SC. 2015. Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA. Front. Aging Neurosci. 7: 52.   DOI
9 Gil A. 2002. Polyunsaturated fatty acids and inflammatory diseases. Biomed. Pharmacother. 56: 388-396.   DOI
10 Glogauer A, Martini VP, Faoro H, Couto GH, Muller-Santos M, Monteiro RA, et al. 2011. Identification and characterization of a new true lipase isolated through metagenomic approach. Microb. Cell Fact. 10: 54.   DOI
11 Gökbulut AA, Arslanolu A. 2013. Purification and biochemical characterization of an extracellular lipase from psychrotolerant Pseudomonas fluorescens KE38. Turk. J. Biol. 37: 538-546.   DOI
12 Hills G. 2003. Industrial use of lipases to produce fatty acid esters. Eur. J. Lipid Sci. Technol. 105: 601-607.   DOI
13 Jaouani A, Neifar M, Hamza A, Chaabouni S, Martinez MJ, Gtari M. 2012. Purification and characterization of a highly thermostable esterase from the actinobacterium Geodermatophilus obscurus strain G20. J. Basic Microbiol. 52: 653-660.   DOI
14 Kim J, Lee C. 2012. Cloning, expression, and purification of a lipase from psychrotrophic Pseudomonas mandelii. J. Life Sci. 22: 306-311.   DOI
15 Kojima Y, Shimizu S. 2003. Purification and characterization of the lipase from Pseudomonas fluorescens HU380. J. Biosci. Bioeng. 96: 219-226.   DOI
16 Korbie DJ, Mattick JS. 2008. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat. Protoc. 3: 1452-1456.   DOI
17 Lo SK, Tan CP, Long K, Yusoff M, Lai OM. 2008. Diacylglycerol oil-properties, processes and products: a review. Food Bioprocess Technol. 1: 223-233.   DOI
18 Kuwahara K, Angkawidjaja C, Koga Y, Takano K, Kanaya S. 2011. Importance of an extreme C-terminal motif of a family I.3 lipase for stability. Protein Eng. Des. Sel. 24: 411-418.   DOI
19 Lee YP, Chung GH, Rhee JS. 1993. Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli. Biochim. Biophys. Acta 1169: 156-164.   DOI
20 Liu T , Zhao YD, Wang XF, Li X, Yan YJ. 2013. A novel oriented immobilized lipase on magnetic nanoparticles in reverse micelles system and its application in the enrichment of polyunsaturated fatty acids. Bioresour. Technol. 132: 99-102.   DOI
21 Meier R, Drepper T, Svensson V, Jaeger KE, Baumann U. 2007. A calcium-gated lid and a large beta-roll sandwich are revealed by the crystal structure of extracellular lipase from Serratia marcescens. J. Biol. Chem. 282: 31477-31483.   DOI
22 Okada T, Morrissey MT. 2007. Production of n-3 polyunsaturated fatty acid concentrate from sardine oil by lipase-catalyzed hydrolysis. Food Chem. 103: 1411-1419.   DOI
23 Prazeres JN, Cruz JAB, Pastore GM. 2006. Characterization of alkaline lipase from Fusarium oxysporum and the effect of different surfactants and detergents on the enzyme activity. Braz. J. Microbiol. 37: 505-509.   DOI
24 Panizza P, Syfantou N, Pastor FI, Rodriguez S, Diaz P. 2013. Acidic lipase Lip I.3 from a Pseudomonas fluorescens-like strain displays unusual properties and shows activity on secondary alcohols. J. Appl. Microbiol. 114: 722-732.   DOI
25 Rose TM, Henikoff JG, Henikoff S. 2003. CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 31: 3763-3766.   DOI
26 Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GS, Mavrodi DV, et al. 2005. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23: 873-878.   DOI
27 Potterton E, Briggs P, Turkenburg M, Dodson E. 2003. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59: 1131-1137.   DOI
28 Rahman RN, Kamarudin NH, Yunus J, Salleh AB, Basri M. 2010. Expression of an organic solvent stable lipase from Staphylococcus epidermidis AT2. Int. J. Mol. Sci. 11: 3195-3208.   DOI
29 Rost B. 2002. Enzyme function less conserved than anticipated. J. Mol. Biol. 318: 595-608.   DOI
30 Shao H, Xu L, Yan Y. 2013. Isolation and characterization of a thermostable esterase from a metagenomic library. J. Ind. Microbiol. Biotechnol. 40: 1211-1222.   DOI
31 Shao H, Xu L, Yan Y. 2013. Thermostable lipases from extremely radioresistant bacterium Deinococcus radiodurans: cloning, expression, and biochemical characterization. J. Basic Microbiol. 53: 1-12.   DOI
32 Weickert MJ, Doherty DH, Best EA, Olins PO. 1996. Optimization of heterologous protein production in Escherichia coli. Curr. Opin. Biotechnol. 7: 494-499.   DOI
33 Tirawongsaroj P, Sriprang R, Harnpicharnchai P, Thongaram T, Champreda V, Tanapongpipat S, et al. 2008. Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library. J. Biotechnol. 133: 42-49.   DOI
34 Yan Y, Zhang X, Chen D. 2013. Enhanced catalysis of Yarrowia lipolytica lipase LIP2 immobilized on macroporous resin and its application in enrichment of polyunsaturated fatty acids. Bioresour. Technol. 131: 179-187.   DOI
35 Voget S, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit WR. 2003. Prospecting for novel biocatalysts in a soil metagenome. Appl. Environ. Microbiol. 69: 6235-6242.   DOI
36 Wang CH, Guo RF, Yu HW, Jia YM. 2008. Cloning and sequence analysis of a novel cold-adapted lipase gene from strain lip35 (Pseudomonas sp.). Agric. Sci. China 7: 1216-1221.   DOI
37 Yan J, Liu S, Hu J, Gui X, Wang G, Yan Y. 2011. Enzymatic enrichment of polyunsaturated fatty acids using novel lipase preparations modified by combination of immobilization and fish oil treatment. Bioresour. Technol. 102: 7154-7158.   DOI
38 Zha D, Xu L, Zhang H, Yan Y. 2014. Molecular identification of lipase LipA from Pseudomonas protegens Pf-5 and characterization of two whole-cell biocatalysts Pf-5 and Top10lipA. J. Microbiol. Biotechnol. 24: 619-628.   DOI
39 Zheng X, Chu X, Zhang W, Wu N, Fan Y. 2011. A novel cold-adapted lipase from Acinetobacter sp. XMZ-26: gene cloning and characterisation. Appl. Microbiol. Biotechnol. 90: 971-980.   DOI