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Purification and Properties of Extracellular Lipases with Transesterification Activity and 1,3-Regioselectivity from Rhizomucor miehei and Rhizopus oryzae

  • Tako, Miklos (Department of Microbiology, Faculty of Science and Informatics, University of Szeged) ;
  • Kotogan, Alexandra (Department of Microbiology, Faculty of Science and Informatics, University of Szeged) ;
  • Papp, Tamas (Department of Microbiology, Faculty of Science and Informatics, University of Szeged) ;
  • Kadaikunnan, Shine (Department of Botany and Microbiology, College of Science, King Saud University) ;
  • Alharbi, Naiyf S. (Department of Botany and Microbiology, College of Science, King Saud University) ;
  • Vagvolgyi, Csaba (Department of Microbiology, Faculty of Science and Informatics, University of Szeged)
  • Received : 2016.08.03
  • Accepted : 2016.10.11
  • Published : 2017.02.28

Abstract

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at $40^{\circ}C$ and pH 7.0 for the R. miehei lipase, and at $30^{\circ}C$ and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the $V_{max}$ of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM $Hg^{2+}$, $Zn^{2+}$, or $Mn^{2+}$, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.

Keywords

References

  1. Jaeger KE, Reetz MT. 1998. Microbial lipases form versatile tools for biotechnology. Trends Biotechnol. 16: 396-403. https://doi.org/10.1016/S0167-7799(98)01195-0
  2. Kazlauskas RJ, Bornscheuer UT. 1998. Biotransformations with lipases, pp. 37-191. In Rehm HJ, Reed G (eds.). Biotechnology: Biotransformations I, 2nd Ed., Vol. 8a. Wiley-VCH Verlag GmbH, Weinheim
  3. Verma ML, Azmi W, Kanwar SS. 2008. Microbial lipases: at the interface of aqueous and non-aqueous media. Acta Microbiol. Immunol. Hung. 55: 265-294. https://doi.org/10.1556/AMicr.55.2008.3.1
  4. Masse L, Kennedy KJ, Chou SP. 2001. The effect of an enzymatic pretreatment on the hydrolysis and size reduction of fat particles in slaughterhouse wastewater. J. Chem. Technol. Biotechnol. 76: 629-635. https://doi.org/10.1002/jctb.428
  5. Takamoto T, Shirasaka H, Uyama H, Kobayashi S. 2001. Lipase-catalyzed hydrolytic degradation of polyurethane in organic solvent. Chem. Lett. 30: 492-493. https://doi.org/10.1246/cl.2001.492
  6. Ferreira JA, Lennartsson PR, Edebo L, Taherzadeh MJ. 2013. Zygomycetes-based biorefinery: present status and future prospects. Bioresour. Technol. 135: 523-532. https://doi.org/10.1016/j.biortech.2012.09.064
  7. Papp T, Nyilasi I, Csernetics A, Nagy G, Tako M, Vagvolgyi C. 2016. Improvement of industrially relevant biological activities in Mucoromycotina fungi, pp. 97-118. In Schmoll M, Dattenbock C (eds.). Gene Expression Systems in Fungi: Advancements and Applications. Fungal Biology Series. Springer International Publishing, Switzerland.
  8. Hasan F, Shah AA, Hameed A. 2006. Industrial applications of microbial lipases. Enzyme Microb. Technol. 39: 235-251. https://doi.org/10.1016/j.enzmictec.2005.10.016
  9. Sharma D, Sharma B, Shukla AK. 2011. Biotechnological approach of microbial lipase: a review. Biotechnology 10: 23-40. https://doi.org/10.3923/biotech.2011.23.40
  10. Singh AK, Mukhopadhyay M. 2012. Overview of fungal lipase: a review. Appl. Biochem. Biotechnol. 166: 486-520. https://doi.org/10.1007/s12010-011-9444-3
  11. Rodrigues RC, Fernandez-Lafuente R. 2010. Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process. J. Mol. Catal. B Enzym. 64: 1-22. https://doi.org/10.1016/j.molcatb.2010.02.003
  12. Kotogan A, Nemeth B, Vagvolgyi C, Papp T, Tako M. 2014. Screening for extracellular lipase enzymes with transesterification capacity in Mucoromycotina strains. Food Technol. Biotechnol. 52: 73-82.
  13. Kotogan A, Kecskemeti A, Szekeres A, Papp T, Chandrasekaran M, Kadaikunnan S, et al. 2016. Characterization of transesterification reactions by Mucoromycotina lipases in non-aqueous media. J. Mol. Catal. B Enzym. 127: 47-55. https://doi.org/10.1016/j.molcatb.2016.02.008
  14. Mateos Diaz JC, Rodriguez JA, Roussos S, Cordova J, Abousalham A, Carriere F, Baratti J. 2006. Lipase from the thermotolerant fungus Rhizopus homothallicus is more thermostable when produced using solid state fermentation than liquid fermentation procedures. Enzyme Microb. Technol. 39: 1042-1050. https://doi.org/10.1016/j.enzmictec.2006.02.005
  15. Chattopadhyay M, Banik AK, Raychaudhuri S. 1999. Production and purification of lipase by a mutant strain of Rhizopus arrhizus. Folia Microbiol. 44: 37-40. https://doi.org/10.1007/BF02816218
  16. Yasuda M, Ogino H, Kiguchi T, Kotani T, Takakura S, Ishibashi T, et al. 1999. Purification and characterization of lipase from Rhizopus chinensis cells. J. Biosci. Bioeng. 88: 571-573. https://doi.org/10.1016/S1389-1723(00)87678-1
  17. Sun SY, Xu Y. 2008. Solid-state fermentation for 'whole-cell synthetic lipase' production from Rhizopus chinensis and identification of the functional enzyme. Process Biochem. 43: 219-224. https://doi.org/10.1016/j.procbio.2007.11.010
  18. Sun SY, Xu Y, Wang D. 2009. Novel minor lipase from Rhizopus chinensis during solid-state fermentation: biochemical characterization and its esterification potential for ester synthesis. Bioresour. Technol. 100: 2607-2612. https://doi.org/10.1016/j.biortech.2008.11.006
  19. Sun SY, Xu Y. 2009. Membrane-bound 'synthetic lipase' specifically cultured under solid-state fermentation and submerged fermentation by Rhizopus chinensis: a comparative investigation. Bioresour. Technol. 100: 1336-1342. https://doi.org/10.1016/j.biortech.2008.07.051
  20. Haas MJ, Cichowicz DJ, Bailey DG. 1992. Purification and characterization of an extracellular lipase from the fungus Rhizopus delemar. Lipids 27: 571-576. https://doi.org/10.1007/BF02536112
  21. Suzuki M, Yamamoto H, Mizugaki M. 1986. Purification and general properties of a metal-insensitive lipase from Rhizopus japonicus NR 400. J. Biochem. 100: 1207-1213. https://doi.org/10.1093/oxfordjournals.jbchem.a121825
  22. Kohno M, Kugimiya W, Hashimoto Y, Morita Y. 1994. Purification, characterization and crystallization of two types of lipases from Rhizpous niveus. Biosci. Biotechnol. Biochem. 58: 1007-1012. https://doi.org/10.1271/bbb.58.1007
  23. Hiol A, Jonzo MD, Rugani N, Druet D, Sarda L, Comeau LC. 2000. Purification and characterization of an extracellular lipase f rom a t hermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb. Technol. 26: 421-430. https://doi.org/10.1016/S0141-0229(99)00173-8
  24. Kantak JB, Prabhune AA. 2012. Characterization of smallest active monomeric lipase from novel Rhizopus strain: application in transesterification. Appl. Biochem. Biotechnol. 166: 1769-1780. https://doi.org/10.1007/s12010-012-9584-0
  25. Ben Salah R, Mosbah H, Fendri A, Gargouri A, Gargouri Y, Mejdoub H. 2006. Biochemical and molecular characterization of a lipase produced by Rhizopus oryzae. FEMS Microbiol. Lett. 260: 241-248. https://doi.org/10.1111/j.1574-6968.2006.00323.x
  26. Wu XY, Jaaskelainen S, Linko YY. 1996. Purification and partial characterization of Rhizomucor miehei lipase for ester synthesis. Appl. Biochem. Biotechnol. 59: 145-158. https://doi.org/10.1007/BF02787816
  27. Gulyamova KA, Davranov KD. 1995. Properties of two lipases from the fungus Mucor miehei. Chem. Nat. Compd. 31: 372-375.
  28. Noel M, Combes D. 2003. Effects of temperature and pressure on Rhizomucor miehei lipase stability. J. Biotechnol. 102: 23-32. https://doi.org/10.1016/S0168-1656(02)00359-0
  29. Reetz MT, Zonta A, Simpelkamp J. 1996. Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials. Biotechnol. Bioeng. 49: 527-534. https://doi.org/10.1002/(SICI)1097-0290(19960305)49:5<527::AID-BIT5>3.0.CO;2-L
  30. Monteiro JB, Nascimento MG, Ninow JL. 2003. Lipasecatalyzed synthesis of monoacylglycerol in a homogeneous system. Biotechnol. Lett. 25: 641-644. https://doi.org/10.1023/A:1023016215537
  31. Namboodiri VM, Chattopadhyaya R. 2000. Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger. Lipids 35: 495-502. https://doi.org/10.1007/s11745-000-549-3
  32. Ulker S, Karaoglu SA. 2012. Purification and characterization of an extracellular lipase from Mucor hiemalis f. corticola isolated from soil. J. Biosci. Bioeng. 114: 385-390. https://doi.org/10.1016/j.jbiosc.2012.04.023
  33. Yu XW, Wang LL, Xu Y. 2009. Rhizopus chinensis lipase: gene cloning, expression in Pichia pastoris and properties. J. Mol. Catal. B Enzym. 57: 304-311. https://doi.org/10.1016/j.molcatb.2008.10.002
  34. Saxena RK, Agarwal L, Meghwanshi GK. 2005. Diversity of fungal and yeast lipases: present and future scenario for the 21st century, pp. 791-814. In Satyanarayana T, Johri BN (eds.). Microbial Diversity: Current Perspectives and Potential Applications. I. K. International Publishing House Pvt Ltd, New Delhi.
  35. Hiol A, Jonzo MD, Druet D, Comeau L. 1999. Production, purification and characterization of an extracellular lipase from Mucor hiemalis f. hiemalis. Enzyme Microb. Technol. 25: 80-87. https://doi.org/10.1016/S0141-0229(99)00009-5
  36. Koblitz MGB, Pastore GM. 2006. Purification and biochemical characterization of an extracellular lipase produced by a new strain of Rhizopus sp. Cienc. Agrotec. 30: 494-502. https://doi.org/10.1590/S1413-70542006000300016
  37. Iftikhar T, Niaz M, Jabeen R, Haq IU. 2011. Purification and characterization of extracellular lipases. Pak. J. Bot. 43: 1541-1545.
  38. Huang Y, Locy R, Weete JD. 2004. Purification and characterization of an extracellular lipase from Geotrichum marinum. Lipids 39: 251-257. https://doi.org/10.1007/s11745-004-1227-1
  39. Noel M, Combes D. 2003. Rhizomucor miehei lipase: differential scanning calorimetry and pressure/temperature stability studies in presence of soluble additives. Enzyme Microb. Technol. 33: 299-308. https://doi.org/10.1016/S0141-0229(03)00123-6
  40. Salis A, Monduzzi M, Solinas V. 2007. Use of lipases for the production of biodiesel, pp. 317-339. In Polaina J, Maccabe AP (eds.). Industrial Enzymes. Structure, Function and Applications. Springer Netherlands, Dordrecht.
  41. Li Z, Li X, Wang Y, Wang Y, Wang F, Jiang J. 2011. Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production. Bioresour. Technol. 102: 9810-9813. https://doi.org/10.1016/j.biortech.2011.07.070
  42. Markweg-Hanke M, Lang S, Wagner F. 1995. Dodecanoic acid inhibition of a lipase from Acinetobacter sp. OPA 55. Enzyme Microb. Technol. 17: 512-516. https://doi.org/10.1016/0141-0229(94)00067-2
  43. Ruiz C, Falcocchio S, Xoxi E, Pastor FJ, Diaz P, Saso L. 2004. Activation and inhibition of Candida rugosa and Bacillusrelated lipases by saturated fatty acids, evaluated by a new colorimetric microassay. Biochim. Biophys. Acta 1672: 184-191. https://doi.org/10.1016/j.bbagen.2004.03.010
  44. Razak CN, Salleh AB, Musani R, Samad MY, Basri M. 1997. Some characteristics of lipases from thermophilic fungi isolated from palm oil mill effluent. J. Mol. Catal. B Enzym. 3: 153-159. https://doi.org/10.1016/S1381-1177(96)00035-5
  45. Toida J, Arikawa Y, Kondou K, Fukuzawa M, Sekiguchi J. 1998. Purification and characterization of triacylglycerol lipase from Aspergillus oryzae. Biosci. Biotechnol. Biochem. 62: 759-763. https://doi.org/10.1271/bbb.62.759
  46. Mhetras NC, Bastawde KB, Gokhale DV. 2009. Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207. Bioresour. Technol. 100: 1486-1490. https://doi.org/10.1016/j.biortech.2008.08.016
  47. Bakir ZB, Metin K. 2016. Purification and characterization of an alkali-thermostable lipase from thermophilic Anoxybacillus flavithermus HBB 134. J. Microbiol. Biotechnol. 26: 1087-1097. https://doi.org/10.4014/jmb.1512.12056
  48. Ebrahimpour A , Rahman RN, Basri M , Salleh AB. 2011. High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM. Bioresour. Technol. 102: 6972-6981. https://doi.org/10.1016/j.biortech.2011.03.083
  49. Lotrakul P, Dharmsthiti S. 1997. Purification and characterization of lipase from Aeromonas sobria LP004. J. Biotechnol. 54: 113-120. https://doi.org/10.1016/S0168-1656(97)01696-9
  50. Alnoch RC, Martini VP, Glogauer A, dos Santos Costa AC, Piovan L, Muller-Santos M, et al. 2015. Immobilization and characterization of a new regioselective and enantioselective lipase obtained from a metagenomic library. PLoS One 10: e0114945. https://doi.org/10.1371/journal.pone.0114945
  51. Yucel S, Terzioglu P, Ozcimen D. 2012. Lipase applications in biodiesel production, pp. 209-250. In Fang Z (ed.). Biodiesel - Feedstocks, Production and Applications. InTech, Croatia.

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