Influence of cultivation conditions on the production of a thermostable extracellular lipase from Amycolatopsis mediterranei DSM 43304

  • Dharmendra S. Dheeman
  • Jesus M. Frias
  • Gary T. M. Henehan
Original Article


Among several lipase-producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture conditions and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28°C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. Under optimal culture conditions, the lipase activity was enhanced 12-fold with a twofold increase in lipase specific activity. The lipase showed maximum activity at 60°C and pH 8.0. The enzyme was stable between pH 5.0 and 9.0 and temperatures up to 60°C. Lipase activity was significantly enhanced by Fe3+ and strongly inhibited by Hg2+. Li+, Mg2+ and PMSF significantly reduced lipase activity, whereas other metal ions and effectors had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24 h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions.


Amycolatopsis mediterranei Screening Organic solvent-tolerant Thermostable Lipase 



This work was supported by ABBEST Research Scholarship from Dublin Institute of Technology (PB 03557/2007). The authors want to express their thanks to Prof. Michael Goodfellow and Dr. Amanda L. Jones, School of Biology, Newcastle University, UK for providing the strains for this study.


  1. 1.
    Abramic M, Lescic I, Korica T, Vitale L, Saenger W, Pigac J (1999) Purification and properties of extracellular lipase from Streptomyces rimosus. Enzym Microb Technol 25:522–529CrossRefGoogle Scholar
  2. 2.
    Aryee ANA, Simpson BK, Villalonga R (2007) Lipase fraction from the viscera of grey mullet (Mugil cephalus) isolation, partial purification and some biochemical characteristics. Enzym Microb Technol 40:394–402CrossRefGoogle Scholar
  3. 3.
    Bapiraju KVVSN, Sujatha P, Ellaiah P, Ramana T (2005) Sequential parametric optimisation of lipase production by a mutant strain Rhizopus sp, BTNT-2. J Basic Microbiol 45:257–273CrossRefPubMedGoogle Scholar
  4. 4.
    Benjamin S, Pandey A (1996) Optimization of liquid media for lipase production by Candida rugosa. Bioresour Technol 55:167–170CrossRefGoogle Scholar
  5. 5.
    Bielen A, Cetkovic H, Long PF, Schwab H, Abramic M, Vujaklija D (2009) The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity. Biochimie 91:390–400CrossRefPubMedGoogle Scholar
  6. 6.
    Bormann C, Nikoleit K, Potgeter M, Tesch C, Sommer P, Goetz F (1993) Investigation of lipolytic enzymes from Streptomyces. In: DECHEMA-monographs. VCH, Weinheim, Germany, pp 237–247Google Scholar
  7. 7.
    Cardenas F, Alvarez E, De Castro-Alvarez MS, Sanchez-Montero JM, Elson S, Sinistera JV (2001) Three new lipases from actinomycetes and their use in organic reactions. Biocatal Biotransfor 19:315–329CrossRefGoogle Scholar
  8. 8.
    Cardenas F, Castro MS, Sanchez-Montero JM, Sinisterra JV, Valmaseda M, Elson SW, Alvarez E (2001) Novel microbial lipases: catalytic activity in reactions in organic media. Enzym Microb Technol 28:145–154CrossRefGoogle Scholar
  9. 9.
    Clausen IG (1997) Aspects in lipase screening. J Mol Catal B: Enzymatic 3:139–146CrossRefGoogle Scholar
  10. 10.
    Costas M, Deive FJ, Longo MA (2004) Lipolytic activity in submerged cultures of Issatchenkia orientalis. Process Biochem 39:2109–2114CrossRefGoogle Scholar
  11. 11.
    Cote A, Shareck F (2008) Cloning, purification and characterization of two lipases from Streptomyces coelicolor A3(2). Enzym Microb Technol 42:381–388CrossRefGoogle Scholar
  12. 12.
    Cruz H, Perez C, Wellington E, Castro C, Servin-Gonzalez L (1994) Sequence of the Streptomyces albus G lipase-encoding gene reveals the presence of a prokaryotic lipase family. Gene 144:141–142CrossRefPubMedGoogle Scholar
  13. 13.
    Dominguez A, Deive FJ, Sanroman MA, Longo MA (2003) Effect of lipids and surfactants on extracellular lipase production by Yarrowia lipolytica. J Chem Technol Biotechnol 78:1166–1170CrossRefGoogle Scholar
  14. 14.
    Enea O, Jolicoeur C (1982) Heat capacities and volumes of several oligopeptides in urea-water mixtures at 25°C. Some implications for protein unfolding. J Phys Chem 86:3870–3881CrossRefGoogle Scholar
  15. 15.
    Ertugrul S, Donmez G, Takac SS (2007) Isolation of lipase producing Bacillus sp. from olive mill wastewater and improving its enzyme activity. J Hazard Mater 149:720–724CrossRefPubMedGoogle Scholar
  16. 16.
    Faber K, Franssen MCR (1993) Prospects for the increased application of biocatalysts in organic transformations. Trends Biotechnol 11:461–470CrossRefPubMedGoogle Scholar
  17. 17.
    Gaur R, Gupta A, Khare SK (2008) Purification and characterization of lipase from solvent tolerant Pseudomonas aeruginosa PseA. Process Biochem 43:1040–1046CrossRefGoogle Scholar
  18. 18.
    Ghanem EH, Al-Sayeed HA, Saleh KM (2000) An alkalophilic thermostable lipase produced by a new isolate of Bacillus alcalophilus. World J Microb Biot 16:459–464CrossRefGoogle Scholar
  19. 19.
    Gulati R, Saxena RK, Gupta R, Yadav RP, Davidson WS (1999) Parametric optimisation of Aspergillus terreus lipase production and its potential in ester synthesis. Process Biochem 35:459–464CrossRefGoogle Scholar
  20. 20.
    Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial α-amylases: a biotechnological perspective. Process Biochem 38:1599–1616CrossRefGoogle Scholar
  21. 21.
    Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64:763–781CrossRefPubMedGoogle Scholar
  22. 22.
    Hazarika S, Goswami P, Dutta NN, Hazarika AK (2002) Ethyl oleate synthesis by porcine pancreatic lipase in organic solvents. Chem Eng J 85:61–68CrossRefGoogle Scholar
  23. 23.
    Hun CJ, Rahman RNZA, Salleh AB, Basri M (2003) A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase. Biochem Eng J 15:147–151CrossRefGoogle Scholar
  24. 24.
    Izumi T, Nakamura K, Fukase T (1990) Purification and characterization of a thermostable lipase from newly isolated Pseudomonas sp. KWI-56. Agri Biol Chem 54:1253–1258Google Scholar
  25. 25.
    Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16:396–403CrossRefPubMedGoogle Scholar
  26. 26.
    Jaeger KE, Ransac S, Dijkstra BW, Colson C, Van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15:29–63CrossRefPubMedGoogle Scholar
  27. 27.
    Janssen PH, Monk CR, Morgan HW (1994) A thermophilic, lipolytic Bacillus sp., and continuous assay of its p-nitrophenyl-palmitate esterase activity. FEMS Microbiol Lett 120:195–200CrossRefGoogle Scholar
  28. 28.
    Kanwar L, Gogoi BK, Goswami P (2002) Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulphate precipitation technique. Bioresour Technol 84:207–211CrossRefPubMedGoogle Scholar
  29. 29.
    Kouker G, Jaeger KE (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53:211–213PubMedGoogle Scholar
  30. 30.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  31. 31.
    Large KP, Mirjalili N, Osborne M, Peacock LM, Zormpaidis V, Walsh M, Cavanagh ME, Leadlay PF, Ison AP (1999) Lipase activity in Streptomycetes. Enzym Microb Technol 25:569–575CrossRefGoogle Scholar
  32. 32.
    Lescic I, Vukelic B, Majeric-Elenkov M, Saenger W, Abramic M (2001) Substrate specificity and effects of water-miscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus. Enzym Microb Technol 29:548–553CrossRefGoogle Scholar
  33. 33.
    Lima VMG, Krieger N, Mitchell DA, Baratti JC, Filippis I, Fontana JD (2004) Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium. J Mol Catal B Enzym 31:53–61CrossRefGoogle Scholar
  34. 34.
    Lima VMG, Krieger N, Mitchell DA, Fontana JD (2004) Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochem Eng J 18:65–71CrossRefGoogle Scholar
  35. 35.
    Lin ES, Ko HC (2005) Glucose stimulates production of the alkaline-thermostable lipase of the edible basidiomycete Antrodia cinnamomea. Enzyme Microb Technol 37:261–265CrossRefGoogle Scholar
  36. 36.
    Lin ES, Wang CC, Sung SC (2006) Cultivating conditions influence lipase production by the edible Basidiomycete Antrodia cinnamomea in submerged culture. Enzyme Microb Technol 39:98–102CrossRefGoogle Scholar
  37. 37.
    Lin SF, Chiou CM, Tsai YC (1995) Effect of Triton X-100 on alkaline lipase production by Pseudomonas pseudoalkaligenes F-111. Biotechnol Lett 17:959–962CrossRefGoogle Scholar
  38. 38.
    Macris JB, Kourentzi E, Hatzinikiolaou DG (1996) Studies on localization and regulation of lipase production by Aspergillus niger. Process Biochem 31:807–812CrossRefGoogle Scholar
  39. 39.
    Mahadik ND, Bastawde KB, Puntambekar US, Khire JM, Gokhale DV (2004) Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation. Process Biochem 39:2031–2034CrossRefGoogle Scholar
  40. 40.
    Maia MMD, Heasley A, Camargo de Morais MM, Melo EHM, Morais MA Jr, Ledingham WM, Filho JLL (2001) Effect of culture conditions on lipase production by Fusarium solani in batch fermentations. Bioresour Technol 76:23–27CrossRefPubMedGoogle Scholar
  41. 41.
    Makhzoum A, Knapp JS, Owusu RK (1995) Factor affecting growth and lipase production by Pseudomonas fluorescens 2D. Food Microbiol 12:277–290CrossRefGoogle Scholar
  42. 42.
    Miranda OA, Salgueiro AA, Pimentel MCB, Filho JLL, Melo EHM, Duran N (1999) Lipase production by a Brazilian strain of Penicillium citrinum using an industrial residue. Bioresour Technol 69:145–147CrossRefGoogle Scholar
  43. 43.
    Moon SH, Parulekar SJ (1991) A parametric study of protease production on batch and fed-batch cultures of Bacillus firmus. Biotechnol Bioeng 37:467–483CrossRefPubMedGoogle Scholar
  44. 44.
    Mudgetti RE (1986) Solid-state fermentations. In: Demain AL, Solmen NA (eds) Manual of industrial biotechnology. American Society for Microbiology. Washington, DC, pp 66–83Google Scholar
  45. 45.
    Muralidhar RV, Chirumamila RR, Marchant R, Nigam P (2001) A response surface approach for the comparison of lipase production from Candida cylindracea using two different carbon sources. Biochem Eng J 9:17–23CrossRefGoogle Scholar
  46. 46.
    Nawani N, Dosanjh NS, Kaur J (1998) A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies. Biotechnol Lett 20:997–1000CrossRefGoogle Scholar
  47. 47.
    O’Fagain C (2003) Enzyme stabilization-recent experimental progress. Enzym Microb Technol 33:137–149CrossRefGoogle Scholar
  48. 48.
    Obradors N, Montesinos JL, Valero F, Lafuente FJ, Sola C (1993) Effect of different fatty acids in lipase production by Candida rugosa. Biotechnol Lett 15:357–360CrossRefGoogle Scholar
  49. 49.
    Ohnishi K, Yoshida Y, Sekiguchi J (1994) Lipase production of Aspergillus oryzae. J Ferment Bioeng 77:490–495CrossRefGoogle Scholar
  50. 50.
    Paetzel M, Karla A, Strynadka NCJ, Dalbey RE (2002) Signal peptidases. Chem Rev 102:4549–4579CrossRefPubMedGoogle Scholar
  51. 51.
    Prim N, Sanchez M, Ruiz C, Pastor FI, Diaz P (2003) Use of methylumbeliferyl-derivative substrates for lipase activity characterization. J Mol Catal B Enzymatic 22:339–346CrossRefGoogle Scholar
  52. 52.
    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 341:267–274CrossRefPubMedGoogle Scholar
  53. 53.
    Rathi P, Sapna B, Sexena R, Gupta R (2000) A hyperthermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation. Biotechnol Lett 22:495–498CrossRefGoogle Scholar
  54. 54.
    Rathi P, Saxena RK, Gupta R (2001) A novel alkaline lipase from Burkholderia cepacia for detergent formulation. Process Biochem 37:187–192CrossRefGoogle Scholar
  55. 55.
    Rua ML, Diaz-Maurino T, Fernandez VM, Otero C, Ballesteros A (1993) Purification and characterization of two distinct lipases from Candida cylindracea. Biochim Biophys Acta 1156:181–189PubMedGoogle Scholar
  56. 56.
    Sanchez A, Ferrer P, Serrano A, Valero F, Sola C, Pemas M, Rua ML, Femandez-Lafuente R, Guisan JM, de la Casa RM, Sinisterra JV, Sinchez-Montero JM (1999) A controlled fed-batch cultivation for the production of new crude lipases from Candida rugosa with improved properties in fine chemistry. J Biotechnol 69:169–182CrossRefGoogle Scholar
  57. 57.
    Schmid RD, Verger R (1998) Lipases: interfacial enzymes with attractive applications. Angew Chem Int Ed Engl 37:1608–1633CrossRefGoogle Scholar
  58. 58.
    Servin-Gonzalez L, Castro C, Perez C, Rubio M, Valdez F (1997) bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator. J Bacteriol 179:7816–7826PubMedGoogle Scholar
  59. 59.
    Shirazi SH, Rahman SR, Rahman MM (1998) Production of extracellular lipases by Saccharomyces cerevisiae. World J Microbiol Biotechnol 14:595–597CrossRefGoogle Scholar
  60. 60.
    Simon LM, Laszlo K, Vertesi A, Bagi K, Szajani B (1998) Stability of hydrolytic enzymes in water-organic solvent systems. J Mol Catal B Enzym 4:41–45CrossRefGoogle Scholar
  61. 61.
    Sugihara A, Ueshima M, Shimada Y, Tsunasawa S, Tominaga Y (1992) Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia. J Biochem 112:598–603PubMedGoogle Scholar
  62. 62.
    Swift S, Throup JP, Williams P, Salmond GP, Stewart GS (1996) Quorum sensing: a population-density component in the determination of bacterial phenotype. Trend Biochem Sci 21:214–219PubMedGoogle Scholar
  63. 63.
    Sztajer H, Maliszewska I (1988) The effect of culture conditions on lipolytic productivity of microorganisms. Biotechnol Lett 10:199–204CrossRefGoogle Scholar
  64. 64.
    Sztajer H, Maliszewska I (1989) The effect of culture conditions on lipolytic productivity of Penicillium citrinum. Biotechnol Lett 11:895–898CrossRefGoogle Scholar
  65. 65.
    Sztajer H, Maliszewska I, Wierczorek J (1988) Production of exogenous lipases by bacteria, fungi, and actinomycetes. Enzyme Microbiol Technol 10:492–497CrossRefGoogle Scholar
  66. 66.
    Tan T, Zhang M, Xu J, Zhang J (2004) Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3. Process Biochem 39:1495–1502CrossRefGoogle Scholar
  67. 67.
    Winkler UK, Stuckman M (1979) Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–679PubMedGoogle Scholar
  68. 68.
    Yang FC, Huang HC, Yang MJ (2003) The influence of environmental conditions on the mycelial growth of Antrodia cinnamomea in submerged cultures. Enzyme Microb Technol 33:395–402CrossRefGoogle Scholar
  69. 69.
    Yu L, Xu Y, Yu X (2009) Purification and properties of a highly enantioselective L-menthyl acetate hydrolase from Burkholderia cepacia. J Mol Catal B Enzym 57:27–33CrossRefGoogle Scholar
  70. 70.
    Zhang Y, Meg K, Wang Y, Luo H, Yang P, Shi P, Wu N, Fan Y, Li J, Yao B (2008) A novel proteolysis-resistant lipase from keratinolytic Streptomyces fradiae var. k11. Enzym Microb Technol 42:346–352CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2009

Authors and Affiliations

  • Dharmendra S. Dheeman
    • 1
  • Jesus M. Frias
    • 1
  • Gary T. M. Henehan
    • 1
  1. 1.School of Food Science and Environmental HealthDublin Institute of TechnologyDublin 1Ireland

Personalised recommendations