Characterization of novel metagenomic–derived lipase from Indian hot spring

  • Rajesh Kumar Sahoo
  • Aradhana Das
  • Kalpana Sahoo
  • Anshuman Sahu
  • Enketeswara SubudhiEmail author
Original Article


Extreme environments are the main source of industrially suitable biocatalysts. The non-cultivable approach of searching enzymes is known to provide ample scope to accomplish novelty for their industrial applications. Lip479 clone out of seven lipase-producing clones obtained from Taptapani hot spring was found to be optimally active at pH 8.0 and temperature 65 °C. The recombinant Lip479 was highly stable in organic solvents, methanol, DMF, DMSO, acetone, and dichloromethane. Lip479 lipase activity was enhanced in the presence of K+, Mn2+, Na+, Zn2+, and Ca2+ except for Fe3+. The ability of Lip479 lipase to act on long carbon chain of 4-nitrophenyl myristate suggests it might be a true lipase. Lip479 clone was found to have ORF of 1251 bp encoding 416 amino acid residues of 42.57 KDa size (theoretically calculated). The presence of conserved motif Ala-His-Ser-Gln-Gly and Zn2+-binding consensus sequence (GAAHAAKH) of the clone assigns the protein to lipase family 1.5. Phylogenetic lineage of the protein sequence of Lip479 was traced to family 1.5 as it was clubbed up with those of reported lipases of the same family. The above biochemical features indicated that Lip479 lipase can be a potential biocatalyst for its use in various industries.


Hot spring Metagenomic Thermostability Lipase 


Funding information

Present research work received the financial support from the DBT, New Delhi through project grants no: BT/PR7944/BCE/8/1036/2013).

Compliance with ethical standards

Conflict of interest

The authors declare that they do not have any conflict of interest.

Supplementary material

10123_2019_95_Fig4_ESM.png (1002 kb)
Fig. S1

Plate showing lipase positive clones. a LB agar containing olive oil and Rhodamine B, b LB agar containing tributyrin (PNG 1002 kb)

10123_2019_95_MOESM1_ESM.tif (3.7 mb)
High Resolution Image (TIF 3833 kb)
10123_2019_95_Fig5_ESM.png (1.7 mb)
Fig. S2

Lip479 clone (NCBI Accession No. KR232658) of 1251 bp length which codes 416 amino acid residues. Signal peptide was marked in red colour outline. Zinc-binding site and conserved motif sequence were marked in brown and blue colour outline respectively. The gene sequence has ATG as start codon (green outline) and TAA as stop codon (black outline). Small alphabets represent nucleotides and block alphabets represent the amino acids (PNG 1772 kb)

10123_2019_95_MOESM2_ESM.tif (7.3 mb)
High Resolution Image (TIF 7484 kb)
10123_2019_95_Fig6_ESM.png (132 kb)
Fig. S3

pH stability of Lip479. pH stability was investigated by incubating the crude enzyme at pH 7, 8, 9, 10, and 11 for a period ranging from 0 to 6 h (PNG 131 kb)

10123_2019_95_MOESM3_ESM.tif (499 kb)
High Resolution Image (TIF 498 kb)
10123_2019_95_Fig7_ESM.png (137 kb)
Fig. S4

Thermostability of Lip479. Thermostability was measured by incubating the crude enzyme at different temperatures 55, 65, 75, 85, and 95 °C for a period ranging from 0 to 6 h (PNG 137 kb)

10123_2019_95_MOESM4_ESM.tif (509 kb)
High Resolution Image (TIF 509 kb)


  1. 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 Biotechnol 160:2102–2113. CrossRefGoogle Scholar
  2. Aldercreutz P, Mattiasson B (1987) Aspects of biocatalyst stability in organic solvents. Biocatal Biotransform 1:99–108Google Scholar
  3. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343 Pt 1:177–183. CrossRefGoogle Scholar
  4. Bayer S, Kunert A, Ballschmiter M, Greiner-Stoeffele T (2010) Indication for a new lipolytic enzyme family: isolation and characterization of two esterases from a metagenomic library. J Mol Microbiol Biotechnol 18:181–187. CrossRefGoogle Scholar
  5. Biver S, Vandenbol M (2013) Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library. J Ind Microbiol Biotechnol 40:191–200. CrossRefGoogle Scholar
  6. Bose A, Keharia H (2013) Production, characterization and applications of organic solvent tolerant lipase by Pseudomonas aeruginosa AAU2. Biocatal Agric Biotechnol 2:255–266. CrossRefGoogle Scholar
  7. Castilho LR, Polato CMS, Baruque EA, Sant’Anna GL Jr, Freire DMG (2000) Economic analysis of lipase production by Penicillium restrictum in solidstate and submerged fermentations. Biochem Eng J 4(3):239–247CrossRefGoogle Scholar
  8. Castro GR, Knubovets T (2003) Homogeneous biocatalysis in organic solvents and water-organic mixtures. Crit Rev Biotechnol 23(3):195–231CrossRefGoogle Scholar
  9. Chakravorty D, Parameswaran S, Dubey VK, Patra S (2011) In silico characterization of thermostable lipases. Extremophiles 15:89–103. CrossRefGoogle Scholar
  10. Chen S, Qian L, Shi B (2007) Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71. Process Biochem 42:988–994. CrossRefGoogle Scholar
  11. Couto GH, Glogauer A, Faoro H, Chubatsu LS, Souza EM, Pedrosa FO (2010) Isolation of a novel lipase from a metagenomic library derived from mangrove sediment from the south Brazilian coast. Genet Mol Res 9:514–523. CrossRefGoogle Scholar
  12. Duan C-J, Feng J-X (2010) Mining metagenomes for novel enzymes. Biotechnol Lett 32:1765–1775CrossRefGoogle Scholar
  13. Fan X, Liu X, Wang K, Wang S, Huang R, Liu Y (2011) Highly soluble expression and molecular characterization of an organic solvent-stable and thermotolerant lipase originating from the metagenome. J Mol Catal B Enzym 72:319–325. CrossRefGoogle Scholar
  14. Ferrer M, Martínez-Abarca F, Golyshin PN (2005) Mining genomes and “metagenomes” for novel catalysts. Curr Opin Biotechnol 16:588–593. CrossRefGoogle Scholar
  15. Glogauer A, Martini VP, Faoro H, Couto GH, Müller-Santos M, Monteiro RA, Mitchell DA, de Souza EM, Pedrosa FO, Krieger N (2011) Identification and characterization of a new true lipase isolated through metagenomic approach. Microb Cell Factories 10:54. CrossRefGoogle Scholar
  16. Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification, and biotechnological properties. Appl Microbiol Biotechnol 64:763–781CrossRefGoogle Scholar
  17. Hårdeman F, Sjöling S (2007) Metagenomic approach for the isolation of a novel low-temperature active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol 59:524–534. CrossRefGoogle Scholar
  18. 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. CrossRefGoogle Scholar
  19. Jeon JH, Kim SJ, Lee HS, Cha SS, Lee JH, Yoon SH, Koo BS, Lee CM, Choi SH, Lee SH, Kang SG, Lee JH (2011) Novel metagenome-derived carboxylesterase that hydrolyzes β-lactam antibiotics. Appl Environ Microbiol 77:7830–7836CrossRefGoogle Scholar
  20. Ji Q, Xiao S, He B, Liu X (2010) Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production. J Mol Catal B Enzym 66:264–269. CrossRefGoogle Scholar
  21. Jinwal UK, Roy U, Chowdhury AR, Bhaduri AP, Roy PK (2003) Purification and characterization of an alkaline lipase from a newly isolated Pseudomonas mendocina PK-12CS and chemoselective hydrolysis of the fatty acid ester. Bioorg Med Chem 11:1041–1046. CrossRefGoogle Scholar
  22. Khan M, Jithesh K (2012) Expression and purification of organic solvent stable lipase from soil metagenomic library. World J Microbiol Biotechnol 28:2417–2424. CrossRefGoogle Scholar
  23. Khan M, Jithesh K, Mookambikay R (2013) Cloning and characterization of two functionally diverse lipases from soil metagenome. J Gen Appl Microbiol 31:21–31CrossRefGoogle Scholar
  24. Khmelnitsky YL, Levashov AV, Klyachko NL, Martinek K (1988) Engineering biocatalytic systems in organic media with low water content. Enzym Microb Technol 10(12):710–724CrossRefGoogle Scholar
  25. Korman TP, Sahachartsiri B, Charbonneau DM, Huang GL, Beauregard M, Bowie JU (2013) Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution. Biotechnol Biofuels 6:70. CrossRefGoogle Scholar
  26. Lee SW, Won K, Lim HK, Kim JC, Choi GJ, Cho KY (2004) Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl Microbiol Biotechnol 65:720–726. CrossRefGoogle Scholar
  27. Lee MH, Lee CH, Oh TK, Song JK, Yoon JH (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 Microbiol 72:7406–7409. CrossRefGoogle Scholar
  28. Lee MH, Hong KS, Malhotra S, Park JH, Hwang EC, Choi HK, Kim YS, Tao W, Lee SW (2010) A new esterase EstD2 isolated from plant rhizosphere soil metagenome. Appl Microbiol Biotechnol 88:1125–1134. CrossRefGoogle Scholar
  29. Liu K, Wang J, Bu D, Zhao S, McSweeney C, Yu P, Li D (2009) Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen. Biochem Biophys Res Commun 385:605–611. CrossRefGoogle Scholar
  30. López-López O, Knapik K, Cerdán M-E, González-Siso M-I (2015) Metagenomics of an alkaline hot spring in Galicia (Spain): microbial diversity analysis and screening for novel lipolytic enzymes. Front Microbiol 6:1291CrossRefGoogle Scholar
  31. Rahman RNZRA, Baharum SN, Basri M, Salleh AB (2005) High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5. Anal Biochem 341:267–274. CrossRefGoogle Scholar
  32. Sahoo RK (2016) Pure culture and metagenomic approaches to investigate Taptapani hot spring for lipase gene, Dissertation, Siksha O Anusnadhan University, IndiaGoogle Scholar
  33. Sahoo RK, Subudhi E, Kumar M (2014) Quantitative approach to track lipase producing Pseudomonas sp. S1 in nonsterilized solid state fermentation. Lett Appl Microbiol 58:610–616. CrossRefGoogle Scholar
  34. Salihu A, Alam MZ (2015) Solvent tolerant lipases: a review. Process Biochem 50:86–96. CrossRefGoogle Scholar
  35. Selvin J, Kennedy J, Lejon DPH, Kiran G, Dobson ADW (2012) Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans. Microb Cell Factories 11:72. CrossRefGoogle Scholar
  36. Sharma S, Kanwar SS (2014) Organic solvent tolerant lipases and applications. Sci World J 2014:1–15. Google Scholar
  37. Tirawongsaroj P, Sriprang R, Harnpicharnchai P, Thongaram T, Champreda V, Tanapongpipat S, Pootanakit K, Eurwilaichitr L (2008) Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library. J Biotechnol 133:42–49CrossRefGoogle Scholar
  38. Torres C, Otero C (1996) Influence of the organic solvents on the activity in water and the conformation of Candida rugosa lipase: description of a lipase activating pretreatment. Enzym Microb Technol 19(8):594–600CrossRefGoogle Scholar
  39. Yan W, Li F, Wang L, Zhu Y, Dong Z, Bai Z (2017) Discovery and characterizaton of a novel lipase with transesterification activity from hot spring metagenomic library. Biotechnol Rep 14:27–33CrossRefGoogle Scholar
  40. Zheng J, Liu C, Liu L, Jin Q (2013) Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach. Syst Appl Microbiol 36:197–204. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Centre For BiotechnologySiksha O Anusandhan Deemed to be UniversityBhubaneswarIndia

Personalised recommendations