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A new alkaline lipase obtained from the metagenome of marine sponge Ircinia sp.

  • Jing Su
  • Fengli Zhang
  • Wei Sun
  • Valliappan Karuppiah
  • Guangya Zhang
  • Zhiyong LiEmail author
  • Qun JiangEmail author
Original Paper

Abstract

Microorganisms associated with marine sponges are potential resources for marine enzymes. In this study, culture-independent metagenomic approach was used to isolate lipases from the complex microbiome of the sponge Ircinia sp. obtained from the South China Sea. A metagenomic library was constructed, containing 6568 clones, and functional screening on 1 % tributyrin agar resulted in the identification of a positive lipase clone (35F4). Following sequence analysis 35F4 clone was found to contain a putative lipase gene lipA. Sequence analysis of the predicted amino acid sequence of LipA revealed that it is a member of subfamily I.1 of lipases, with 63 % amino acid similarity to the lactonizing lipase from Aeromonas veronii (WP_021231793). Based on the predicted secondary structure, LipA was predicted to be an alkaline enzyme by sequence/structure analysis. Heterologous expression of lipA in E. coli BL21 (DE3) was performed and the characterization of the recombinant enzyme LipA showed that it is an alkaline enzyme with high tolerance to organic solvents. The isolated lipase LipA was active in the broad alkaline range, with the highest activity at pH 9.0, and had a high level of stability over a pH range of 7.0–12.0. The activity of LipA was increased in the presence of 5 mM Ca2+ and some organic solvents, e.g. methanol, acetone and isopropanol. The optimum temperature for the activity of LipA is 40 °C and the molecular weight of LipA was determined to be ~30 kDa by SDS-PAGE. LipA is an alkaline lipase and shows good tolerance to some organic solvents, which make it of potential utility in the detergent industry and enzyme mediated organic synthesis. The result of this study has broadened the diversity of known lipolytic genes and demonstrated that marine sponges are an important source for new enzymes.

Keywords

Lipase Metagenomic library Marine sponge Alkaline lipase Secondary structure amino acid composition 

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC) (31000062).

References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  2. Andree H, Mueller WR, Schmid RD (1980) Lipases as detergent components. J Appl Biochem 2:218–229Google Scholar
  3. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183CrossRefGoogle Scholar
  4. Bajaj A, Lohan P, Jha PN, Mehrotra R (2010) Biodiesel production through lipase catalyzed transesterification: an overview. J Mol Catal B Enzym 62:9–14CrossRefGoogle Scholar
  5. Beisson F, Tiss A, Rivière C, Verger R (2000) Methods for lipase detection and assay: a critical review. Eur J Lipid Sci Technol 102:133–153CrossRefGoogle Scholar
  6. 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 72:248–254CrossRefGoogle Scholar
  7. Cherif S, Mnif S, Hadrich F, Abdelkafi S, Sayadi S (2011) A newly high alkaline lipase: an ideal choice for application in detergent formulations. Lipids Health Dis 10:221CrossRefGoogle Scholar
  8. Colton I, Ahmed S, Kazlauskas R (1995) A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic-acids. J Org Chem 60:212–217CrossRefGoogle Scholar
  9. Dubnovitsky AP, Kapetaniou EG, Papageorgiou AC (2005) Enzyme adaptation to alkaline pH: atomic resolution (1.08 Å) structure of phosphoserine amino-transferase from Bacillus alcalophilus. Protein Sci 14:97–110CrossRefGoogle Scholar
  10. Feby A, Nair S (2010) Sponge-associated bacteria of Lakshadweep coral reefs, India: resource for extracellular hydrolytic enzymes. Adv Bios Biotechnol 1:330–337CrossRefGoogle Scholar
  11. Frishman D, Argos P (1997) Seventy-five percent accuracy in protein secondary structure prediction. Proteins 27:329–335CrossRefGoogle Scholar
  12. Fullbrook PD (1996) Kinetics. In: Godfrey T, Reichelt J (eds) Industrial enzymology: the application of enzymes in industry, 2nd edn. Nature, New YorkGoogle Scholar
  13. Geierstanger B, Jamin M, Volkman BF, Baldwin RL (1998) Protonation behavior of histidine 24 and histidine 119 in forming the pH 4 folding intermediate of apomyoglobin. Biochemistry 37:4254–4265CrossRefGoogle Scholar
  14. Glogauer A, Martini V, Faoro H, Couto G, Muller-Santos M, Monteiro R, Mitchell D, Souza E, Pedrosa F, Kireger N (2011) Identification and characterization of a new true lipase isolated through metagenomic approach. Microb Cell Fact 10:54CrossRefGoogle Scholar
  15. Gombert AK, Pinto AL, Castilho LR, Freirc DMG (1999) Lipase by Penicillium restrictum in solid state fermentation using Babassu oil cake as substrate. Process Biochem 35:85–90CrossRefGoogle Scholar
  16. Grochulski P, Li Y, Schrag J, Bouthillier F, Smith P (1993) Insights into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem 268:12843Google Scholar
  17. Gupta M, Batra R, Tyagi R, Sharma A (1997) Polarity index: the guiding solvent parameter for enzyme stability in aqueous-organic cosolvent mixtures. Biotechnol Prog 13:284–288CrossRefGoogle Scholar
  18. Gupta R, Rathi P, Gupta N, Bradoo S (2003) Lipase assays for conventional and molecular screening: an overview. Biotechnol Appl Biochem 37:63–71CrossRefGoogle Scholar
  19. Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Mocrobiol Biotechnol 64:763–781CrossRefGoogle Scholar
  20. Han Y, Yang B, Zhang F, Miao X, Li Z (2009) Characterization of antifungal chitinase from marine Streptomyces sp. DA11 associated with South China Sea sponge Craniella australiensis. Mar Biotechnol 11:132–140CrossRefGoogle Scholar
  21. Hu Y, Fu C, Huang Y, Yin Y, Cheng G, Lei F, Lu N, Li J, Ashforth E, Zhang L, Zhu B (2010) Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment. FEMS Microbiol Ecol 72:228–237CrossRefGoogle Scholar
  22. Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16:396–403CrossRefGoogle Scholar
  23. Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351CrossRefGoogle Scholar
  24. Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ, Lee JH (2009) Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol 81:865–874CrossRefGoogle Scholar
  25. Kelch BA, Eagen KP, Erciyas FP, Humphris EL, Thomason AR, Mitsuiki S (2007) Structural and mechanistic exploration of acid resistance: kinetic stability facilitates evolution of extremophilic behavior. J Mol Biol 368:870–883CrossRefGoogle Scholar
  26. Kennedy J, Marchesi JR, Dobson ADW (2008) Marine Metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine ecosystems. Microb Cell Fact 7:27CrossRefGoogle Scholar
  27. Kim E, Oh K, Lee M, Kang C, Oh T, Yoon J (2009) Novel cold-adapted alkaline lipase from an intertidal flat metagenome and proposal for a new family of bacterial lipases. Appl Environ Microbiol 75(1):257–260CrossRefGoogle Scholar
  28. Kiran GS, Shanmughapriya S, Jayalakshmi J, Selvin J, Gandhimathi R, Sivaramakrishnan S, Arunkumar M, Thangavelu T, Natarajaseenivasan K (2008) Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI057). Bioprocess Biosyst Eng 31:483–492CrossRefGoogle Scholar
  29. Kiran GS, Lipton AN, Kennedy J, Dobson ADW, Selvin J (2014) A halotolerant thermostable lipase from the marine bacterium Oceanobacillus sp. PUMB02 with an ability to disrupt bacterial biofilms. Bioengineered 5(5):305–318CrossRefGoogle Scholar
  30. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  31. Lailaja VP, Chandrasekaran M (2013) Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11. World J Microbiol Biotechnol 29:1349–1360CrossRefGoogle Scholar
  32. Lee M-H, Lee CH, Oh T-K, Song JK, Yoon J-H (2006) Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microb 72:7406–7409CrossRefGoogle Scholar
  33. Muralidhar RV, Marchant R, Nigam P (2001) Lipases in racemic resolutions. J Chem Technol Biotechnol 76:3–8CrossRefGoogle Scholar
  34. Nardini M, Lang DA, Liebeton K, Jaeger KE, Dijkstra BW (2000) Crystal structure of Pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases. J Biol Chem 275:31219–31225CrossRefGoogle Scholar
  35. Okamura Y, Kimura T, Yokouchi H, Meneses-Osori M, Katoh M, Matsunga T, Takeyama H (2010) Isolation and characterization of a GDSL esterase from the metagenome of a marine sponge-associated bacteria. Mar Biotechnol 12:395–402CrossRefGoogle Scholar
  36. Ranjan R, Grover A, Kapardar RK, Sharma R (2005) Isolation of novel lipolytic genes from uncultured bacteria of pond water. Biochem Bioph Res Co 335:57–65CrossRefGoogle Scholar
  37. Salameh MA, Wiegel J (2007) Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl Environ Microb 73:7725–7731CrossRefGoogle Scholar
  38. Saxena RK, Sheoran A, Giri B, Davidson S (2003) Purification strategies for microbial lipases. J Microbiol Meth 52:1–18CrossRefGoogle Scholar
  39. Selvin J, Kennedy J, Lejon D, Kiran G, Dobson A (2012) Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans. Microb Cell Fact 11:72CrossRefGoogle Scholar
  40. Settembre EC, Chittuluru JR, Mill CP, Kappock TJ, Ealick SE (2004) Acidophilic adaptations in the structure of Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase (PurE). Acta Crystallogr D Biol Crystallogr 60:1753–1760CrossRefGoogle Scholar
  41. Shanmughapriya S, Kiran GS, Selvin J, Thomas TA, Rani C (2010) Optimization, purification, and characterization of extracellular mesophilic alkaline cellulase from sponge-associated Marinobacter sp. MSI032. Appl Biochem Biotechnol 162:625–640CrossRefGoogle Scholar
  42. Shirai T, Suzuki A, Yamane T, Ashida T, Kobayashi T, Hitomi J (1997) High resolution crystal structure of M-protease: phylogeny aided analysis of the high-alkaline adaptation mechamism. Protein Eng 10:627–634CrossRefGoogle Scholar
  43. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  44. Torres S, Castro G (2004) Non-aqueous biocatalysis in homogenous solvent systems. Food Technol Biotechnol 42(4):271–277Google Scholar
  45. 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(10):6235–6242CrossRefGoogle Scholar
  46. Wang Y, Srivastava KC, Shen G-J, Wang HY (1995) Thermostable alkaline lipase from a newly isolated thermophilic Bacillus, strain A30-1 (ATCC 53841). J Ferment Bioeng 79:433–438CrossRefGoogle Scholar
  47. Watanabe Y, Shimada Y, Sugihara A, Tominaga Y (2002) Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. J Mol Catal B Enzym 17:151–155CrossRefGoogle Scholar
  48. Wei P, Bai L, Song W, Hao G (2009) Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate. Arch Microbiol 191:233–240CrossRefGoogle Scholar
  49. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, Dicuccio M, Edgar R, Federhen S, Feolo LYG, Helberg W, Kapustin Y, Khovayko O, Landsman D, Lipman DJ, Madden TL, Maglott DR, Miller V, Ostell J, Pruitt KD, Schuler GD, Shumway M, Sequeira E, Sherry ST, Sirotkin K, Souvorov A, Starchenko R, Tatusov L, Tatusova TA (2008) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 36:D13–D21CrossRefGoogle Scholar
  50. Zhang GY, Li HC, Fang BS (2009a) Discriminating acidic and alkaline enzymes using a random forest model with secondary structure amino acid composition. Process Biochem 44(6):654–660CrossRefGoogle Scholar
  51. Zhang H, Zhang F, Li Z (2009b) Gene analysis, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa. World J Microb Biotechol 25:1267–1274CrossRefGoogle Scholar
  52. Zheng X, Chu X, Zhang W, Wu N, Fan Y (2011) A novel cold-adapted lipase from Acinetobacter sp.XMZ-26: gene cloning and characterization. Appl Microbiol Biotechnol 90:971–978CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism and School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic China
  2. 2.Department of Biotechnology and BioengineeringHuaqiao UniversityXiamenPeople’s Republic China

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