Purification and biochemical characterization of an extracellular lipase from psychrotolerant Pseudomonas fluorescens KE38

An extracellular lipase producing bacterium was isolated from a soil sample, and identified as a strain of Pseudomonas fluorescens by 16S rRNA gene sequencing. It was named Pseudomonas fluorescens KE38. KE38 showed psychrotolerant properties with an optimum growth temperature of 25 °C. The lipase enzyme secreted by KE38 was purified 41.13-fold with an overall yield of 54.99%, and a specific activity of 337.3 U/mg. The molecular mass of purified lipase was estimated to be approximately 43 kDa by SDS-PAGE. Although the lipase was active at a temperature range of 1565 °C, it exhibited maximum activity at 45 °C, at pH 8.0. The enzyme exhibited high stability retaining 100% and 70% of its activity after an incubation period of 45 and 100 min at 45 °C and pH 8.0 respectively. It also showed a broad substrate specificity acting on p-nitrophenyl esters with C8-C18 acyl groups as substrates and was activated by Ca2+ and Ni2+ at 1 mM. While the enzyme retained its activity levels in the presence of a variety of organic solvents, DMSO and dimethylformamide enhanced this. High stability, broad substrate specificity and activity at cold temperatures in the presence of organic solvents, and metal ions make the extracellular lipase of KE38 a candidate for industrial applications.

Anahtar Kelimeler:

Key words: Pseudomonas fluorescens, extracellular lipase, enzyme purification

Purification and biochemical characterization of an extracellular lipase from psychrotolerant Pseudomonas fluorescens KE38

Keywords:

Key words: Pseudomonas fluorescens, extracellular lipase, enzyme purification,

PDF

___

Ahmed EH, Raghavendra T, Madamwar D (2010). A thermostable alkaline lipase from a local isolate Bacillus subtilis EH 37: characterization, partial purification, and application in organic synthesis. Appl Biochem Biotech 160: 2102–2113.
Alquati C, De Gioia L, Santarossa G, Alberghina L, Fantucci P, Lotti M (2002). The cold-active lipase of Pseudomonas fragi - Heterologous expression, biochemical characterization and molecular modeling. Eur J Biochem 269: 3321–3328.
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.
Arpigny JL, Jaeger KE (1999). Bacterial lipolytic enzymes: classification and properties. Biochem J 343: 177–183.
Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–54.
Cai YJ, Wang L, Liao XR, Ding YR, Sun J (2009). Purification and partial characterization of two new cold-adapted lipases from mesophilic Geotrichum sp SYBC WU-3. Process Biochem 44: 786–790.
Choo DW, Kurihara T, Suzuki T, Soda K, Esaki N (1998). A coldadapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: Gene cloning and enzyme purification and characterization. Appl Environ Microb 64: 486–491.
Chung GH, Lee YP, Jeohn GH, Yoo OJ, Rhee JS (1991). Cloning and nucleotide-sequence of thermostable lipase gene from Pseudomonas fluorescens Sik-W1. Agr Biol Chem Tokyo 55: 2359–2365.
Gupta N, Rathi P, Gupta R (2002). Simplified para-nitrophenyl palmitate assay for lipases and esterases. Anal Biochem 311: 98–9.
Gupta R, Gupta N, Rathi P (2004). Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biot 64: 763–781.
Hasan F, Shah AA, Hameed A (2006). Industrial applications of microbial lipases. Enzyme Microb Tech 39: 235–251.
Jaeger KE, Ransac S, Dijkstra BW, Colson C, Vanheuvel M, Misset O (1994). Bacterial Lipases. Fems Microbiol Rev 15: 29–63.
Jaeger KE, Reetz MT (1998). Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16: 396–403.
Jaeger KE, Dijkstra BW, Reetz MT (1999). Bacterial biocatalysts: Molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53: 315–351.
Joseph B, Ramteke PW, Kumar PA (2006). Studies on the enhanced production of extracellular lipase by Staphylococcus epidermidis. J Gen Appl Microbiol 52: 315–320.
Joseph B, Ramteke PW, Thomas G (2008). Cold active microbial lipases: some hot issues and recent developments. Biotechnol Adv 26: 457–470.
Karadzic I, Masui A, Zivkovic LI, Fujiwara N (2006). 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: 82–89.
Konarzycka-Bessler M, Jaeger KE (2006). Select the best: novel biocatalysts for industrial applications. Trends Biotechnol 24: 248–250.
Kouker G, Jaeger KE (1987). Specific and sensitive plate assay for bacterial lipases. Appl Environ Microb 53: 211–213.
Laemmli UK (1970). Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227: 680–685.
Lee HK, Ahn MJ, Kwak SH, Song WH, Jeong BC (2003). Purification and characterization of cold active lipase from psychrotrophic Aeromonas sp LPB 4. J Microbiol 41: 22–27.
Lin SF, Chiou CM, Yeh CM, Tsai YC (1996). Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111. Appl Environ Microb 62: 1093–1095.
Mayordomo I, Randez-Gil F, Prieto JA (2000). Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans. J Agr Food Chem 48: 105–109.
Peng R, Lin JP, Wei DZ (2010). Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa CS-2. Appl Biochem Biotech 162: 733–743.
Rajan A, Nair AJ (2011). A comparative study on alkaline lipase production by a newly isolated Aspergillus fumigatus MTCC 9657 in submerged and solid-state fermentation using economically and industrially feasible substrate. Turk J Biol 35: 569–574.
Rashid N, Shimada Y, Ezaki S, Atomi H, Imanaka T (2001). Lowtemperature lipase from psychrotrophic Pseudomonas sp strain KB700A. Appl Environ Microb 67: 4064–4069.
Sakiyama T, Yoshimi T, Miyake A, Umeoka M, Tanaka A, Ozaki S, Nakanishi K (2001). Purification and characterization of a monoacylglycerol lipase from Pseudomonas sp LP7315. J Biosci Bioeng 91: 27–32.
Schmidt-Dannert C (1999). Recombinant microbial lipases for biotechnological applications. Bioorgan Med Chem 7: 2123– 2130.
Shimizu S, Kojima Y, Kobayashi M (2003). A novel lipase from Pseudomonas fluorescens HU380: Gene cloning, overproduction, renaturation-activation, two-step purification, and characterization. J Biosci Bioeng 96: 242–249.
Ülker S, Özel A, Çolak A, Karaoğlu ŞA (2011). Isolation, production, and characterization of an extracellular lipase from Trichoderma harzianum isolated from soil. Turk J Biol 35: 543–550.
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.). Agr Sci China 7: 1216–1221.
Wang TJ, Chen TL (1998). Lipase production by Acinetobacter radioresistens in a batch fill-and-draw culture. Appl Biochem Biotech 73: 185–194.
Wang SL, Lin YT, Liang TW, Chio SH, Ming LJ, Wu PC (2009). Purification and characterization of extracellular lipases from Pseudomonas monteilii TKU009 by the use of soybeans as the substrate. J Ind Microbiol Biot 36: 65–73.
Yadav KNS, Adsul MG, Bastawde KB, Jadhav DD, Thulasiram HV, Gokhale DV (2011). Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639. Bioresource Technol 102: 10663–10670.
Yuan BH, Cai YJ, Liao XR, Yun LH, Zhang F, Zhang DB (2010). Isolation and identification of a cold-adapted lipase producing strain from decayed seeds of Ginkgo biloba L. and characterization of the lipase. Afr J Biotechnol 9: 2661–2667.
Zhang JW, Zeng RY (2008). Molecular cloning and expression of a cold-adapted lipase gene from an Antarctic deep sea psychrotrophic bacterium Pseudomonas sp 7323. Mar Biotechnol 10: 612–621.