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Microbial Biotechnology

Microbiol. Biotechnol. Lett. 2020; 48(4): 491-505

https://doi.org/10.48022/mbl.2003.03010

Received: March 27, 2020; Accepted: September 7, 2020

Purification, Characterization and Immobilization of Lipase from Proteus vulgaris OR34 for Synthesis of Methyl Oleate

Asmae misbah1*, Saad Ibnsouda Koraichi1, 2 and Mohamed Ali Tahri Jouti1

1Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University, B.P 2202, Fez, Morocco 2Regional University Center of Interface, Sidi Mohamed Ben Abdellah University, B.P. 2626, 30000 Fez, Morocco

Correspondence to :
Asmae Misbah,     asmae.misbah@usmba.ac.ma

A newly isolated strain, Proteus vulgaris OR34, from olive mill waste was found to secrete an alkaline extracellular lipase at 11 U·ml-1 when cultivated on an optimized liquid medium. This lipase was purified 94.64- fold with a total yield of 9.11% and its maximal specific activity was shown to be 3232.58 and 1777.92 U·mg-1 when evaluated using the pH-stat technique at 55℃ and pH 9 and Tributyrin TC4 or olive oil as the substrate. The molecular mass of the pure OR34 lipase was estimated to be around 31 kDa, as revealed by SDSPAGE and its substrate specificity was investigated using a variety of triglycerides. This assay revealed that OR34 lipase preferred short and medium chain fatty acids. In addition, this lipase was stable in the presence of high concentrations of bile salt (NaDC) and calcium ions appear not to be necessary for its activity. This lipase was inhibited by THL (Orlistat) which confirmed its identity as a serine enzyme. In addition, the immobilization of OR34 lipase by adsorption onto calcium carbonate increased its stability at higher temperatures and within a larger pH range. The immobilized lipase exhibited a high tolerance to organic solvents and retained 60% of its activity after 10 months of storage at 4℃. Finally, the OR34 lipase was applied in biodiesel synthesis via oleic acid mediated esterification of methanol when using hexane as solvent. The best conversion yield (67%) was obtained at 12 h and 40℃ using the immobilized enzyme and this enzyme could be reused for six cycles with the same efficiency.

Keywords: Lipase, Proteus vulgaris, purification, immobilization, biodiesel

  1. Patel MT, Nagarajan R, Kilara A. 1996. Lipase-catalyzed biochemical reactions in novel media: A review. Chem. Eng. Commun. 152 : 365-404.
    CrossRef
  2. Reis P, Holmberg K, Watzke H, Leser ME, Miller R. 2009. Lipases at interfaces: a review. Adv. Colloid Interf. Sci. 147: 237-250.
    Pubmed CrossRef
  3. Pahoja VM, Sethar MA. 2002. A review of enzymatic properties of lipase in plants, animals and microorganisms. J. Appl. Sci. 2: 474-484.
    CrossRef
  4. Agobo KU, Arazu VA, Uzo K, Igwe CN. 2017. Microbial lipases: a prospect for biotechnological industrial catalysis for green products:a review. J. Ferment. Technol. 6: 1-12.
  5. Thakur S. 2012. Lipases, its sources, properties and applications: a review. Int. J. Sci. Eng. Res. 3: 1-29.
  6. Treichel H, de Oliveira D, Mazutti MA, Di Luccio M, Oliveira JV. 2010. A review on microbial lipases production. Food. Bioprocess. Tech. 3: 182-196.
    CrossRef
  7. Ji X, Chen G, Zhang Q, Lin L, Wei Y. 2015. Purification and characterization of an extracellular cold-adapted alkaline lipase produced by psychrotrophic bacterium Yersinia enterocolitica strain KM1. J. Basic. Microbial. 55: 718-728.
    Pubmed CrossRef
  8. Shao H, Xu L, Yan Y. 2014. Thermostable lipases from extremely radioresistant bacterium Deinococcus radiodurans: cloning, expression, and biochemical characterization J. Basic. Microbiol. 54: 984-995.
    Pubmed CrossRef
  9. Melani NB, Tambourgi EB, Silveira E. 2020. Lipases: From production to applications. Sep. Purif. Rev. 49: 143-158.
    CrossRef
  10. Robinson PK. 2015. Enzymes: principles and biotechnological applications. Ess. Biochem. 59: 1-41.
    Pubmed KoreaMed CrossRef
  11. Javed S, Azeem F, Hussain S, Rasul I, Siddique MH, Riaz M, et al. 2018. Bacterial lipases: A review on purification and characterization. Prog. Biophys. Mol. Bio. 132: 23-34.
    Pubmed CrossRef
  12. Nisha S, Karthick SA, Gobi N. 2012. A review on methods, application and properties of immobilized enzyme. Chem. Sci. Rev. Lett. 1: 148-155.
  13. Khan AK, Mubarak NM, Abdullah EC, Khalid M, Nizamuddin S, Baloch HA, et al. 2019. Immobilization of Lipase Enzyme Carbon Nanotubes via Adsorption. IOP Conf. Ser. Mater. Sci. Eng. 495: 012055.
    CrossRef
  14. Pereira DS, Fraga JL, Diniz MM, Fontes-Sant’Ana GC, Amaral PFF 2018. High catalytic activity of lipase from Yarrowia lipolytica immobilized by microencapsulation. Int. J. Mol. Sci. 19: 3393.
    Pubmed KoreaMed CrossRef
  15. Bhushan I, Parshad R, Qazi GN, Gupta VK. 2008. Immobilization of lipase by entrapment in Ca-alginate beads. J. Bioact. Compat. Pol. 23: 552-562.
    CrossRef
  16. Carvalho NB, Vidal BT, Barbosa AS, Pereira MM, Mattedi S, Freitas LDS, et al. 2018. Lipase immobilization on silica xerogel treated with protic ionic liquid and its application in biodiesel production from different oils. Int. J. Mol. Sci. 19: 1829.
    Pubmed KoreaMed CrossRef
  17. Adlercreutz P. 2013. Immobilization and application of lipases in organic media. Chem. Soc. Rev. 42: 6406-6436.
    Pubmed CrossRef
  18. Kim HK, Lee JK, Kim H, Oh TK. 1996. Characterization of an alkaline lipase from Proteus vulgaris K80 and the DNA sequence of the encoding gene. FEMS Microbiol. Lett. 135: 117-121.
    Pubmed CrossRef
  19. Natalia A, Kristiani L, Kim HK. 2014. Characterization of Proteus vulgaris k80 lipase immobilized on amine-terminated magnetic microparticles. J. Microbiol. Biotechnol. 24: 1382-1388.
    Pubmed CrossRef
  20. Whangsuk W, Sungkeeree P, Thiengmag S, Kerdwong J, Sallabhan R, Mongkolsuk S, 2013. Gene cloning and characterization of a novel highly organic solvent tolerant lipase from Proteus sp. SW1 and its application for biodiesel production. Mol. Biotechnol. 53: 55-62.
    Pubmed CrossRef
  21. 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.
    Pubmed KoreaMed CrossRef
  22. Misbah A, Aouine M, Er raouan S, Lekbach Y, Ettadili H, Ibnsouda Koraichi S, et al. 2019. Microorganisms isolated from Moroccan olive-mill wastes: Screening of their enzymatic activities for biotechnological use. Eur. Sci. J. 15: 464-494.
    CrossRef
  23. Rathelot J, Julien R, Canioni P, Coeroli C, Sarda L. 1976. Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase. Biochimie 57: 1117-1122.
    CrossRef
  24. Gargouri Y, Pieroni G, Lowe PA, Sarda L, Verger R. 1986. Human gastric lipase. The effect of amphiphiles. Eur. J. Biochem. 156 : 305-310.
    Pubmed CrossRef
  25. 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: 248-254.
    CrossRef
  26. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
    Pubmed CrossRef
  27. Ornstein L. 1964. Disc electrophoresis. I. Background and theory. Ann. NY Acad. Sci. 121: 321.
    Pubmed CrossRef
  28. Davis BJ. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. NY Acad. Sci. 121: 404-427.
    Pubmed CrossRef
  29. Singh R, Gupta N, Goswami VK, Gupta R. 2006. A simple activity staining protocol for lipases and esterases. Appl. Microbiol. Biotechnol. 70: 679-682.
    Pubmed CrossRef
  30. Gargouri Y, Chahinian H, Moreau H, Ransac S, Verger R. 1991. Inactivation of pancreatic and gastric lipases by THL and C12: 0TNB: a kinetic study with emulsified tributyrin. Biochim. Biophys. Acta (BBA) - Lipids and Lipid Metabolism 1085: 322-328.
    CrossRef
  31. Ghamgui H, Karra chaabouni M, Gargouri Y. 2004. 1-Butyl oleate synthesis by immobilized lipase from Rhizopus oryzae: a comparative study between n-hexane and solvent-free system. Enzyme. Microb. Technol. 35: 355-363.
    CrossRef
  32. Kaur M, Mehta A, Gupta R. 2019. Synthesis of methyl butyrate catalyzed by lipase from Aspergillus fumigatus. J. Oleo Sci. 68: 989-993.
    Pubmed CrossRef
  33. Ghamgui H, Miled N, Karra-chaâbouni M, Gargouri Y. 2007. Immobilization studies and biochemical properties of free and immobilized Rhizopus oryzae lipase onto CaCO3: A comparative study. Biochem. Eng. J. 37: 34-41.
    CrossRef
  34. Deng L, Tan T, Wang F, Xu X. 2003. Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp. 99-125. Eur. J. Lipid Sci. Technol. 105: 727-734.
    CrossRef
  35. Priyanka P, Kinsella G, Henehan GT, Ryan BJ. 2019. Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei. Protein Express. Purif. 153: 121-130.
    Pubmed CrossRef
  36. Maraite A, Hoyos P, Carballeira JD, Cabrera ÁC, AnsorgeSchumacher MB, Alcántara AR. 2013. Lipase from Pseudomonas stutzeri: purification, homology modelling and rational explanation of the substrate binding mode. J. Mol. Catal B- Enzym. 87: 88-98.
    CrossRef
  37. Mohammadi M, Sepehrizadeh Z, Ebrahim-Habibi A, Shahverdi AR, Faramarzi MA, Setayesh N. 2015. Bacterial expression and characterization of an active recombinant lipase A from Serratia marcescens with truncated C-terminal region. J. Mol. Catal BEnzym. 120: 84-92.
    CrossRef
  38. Abdou AM. 2003. Purification and partial characterization of psychrotrophic Serratia marcescens lipase. J. Dairy. Sci. 86: 127-132.
    CrossRef
  39. Bouaziz A, Horchani H, Salem NB, Gargouri Y, Sayari A. 2011. Expression, purification of a novel alkaline Staphylococcus xylosus lipase acting at high temperature. Biochem. Eng. J. 54: 93-102.
    CrossRef
  40. Bacha AB, Al-Assaf A, Moubayed NM, Abid I. 2018. Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations. Saudi. J. Biol. Sci. 25: 409-417.
    Pubmed KoreaMed CrossRef
  41. Oliveira AF, Bastos RG, Lucimara G. 2019. Bacillus subtilis immobilization in alginate microfluidic-based microparticles aiming to improve lipase productivity. Biochem. Eng. J. 143: 110-120.
    CrossRef
  42. Musa H, Kasim FH, Gunny AAN, Gopinath SC, Ahmad MA. 2018. Biosecretion of higher halophilic lipase by a novel Bacillus amyloliquefaciens AIKK2 using agro-waste as supporting substrate. Process. Biochem. 72: 55-62.
    CrossRef
  43. Jadhav VV, Pote SS, Yadav A, Shouche YS, Bhadekar RK. 2013. Extracellular cold active lipase from the psychrotrophic Halomonas sp. BRI 8 isolated from the Antarctic sea water. Songklanakarin J. Sci. Technol. 35: 623-630.
  44. Gutiérrez-Arnillas E, Arellano M, Deive FJ, Rodríguez A, Sanromán MÁ. 2017. Unravelling the suitability of biological induction for halophilic lipase production by Halomonas sp. LM1C cultures. Bioresour. Technol. 239: 368-377.
    Pubmed CrossRef
  45. Gao B, Su E, Lin J, Jiang Z, Ma Y, Wei D. 2009. Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production. J. Biotechnol. 139: 169-175.
    Pubmed CrossRef
  46. Korman TP, Bowie JU. 2012. Crystal Structure of Proteus mirabilis Lipase, a novel lipase from the proteus/psychrophilic subfamily of lipase family I.1. PLoS One 7: e52890.
    Pubmed KoreaMed CrossRef
  47. Kovacic F, Babic N, Krauss U, Jaeger K. 2019. Classification of lipolytic enzymes from bacteria. pp. 1-35. Aerobic utilization of hydrocarbons, oils and lipids, Springer.
    CrossRef
  48. Gao B, Xu T, Lin J, Zhang L, Su E, Jiang Z, et al. 2011. Improving the catalytic activity of lipase LipK107 from Proteus sp. by sitedirected mutagenesis in the lid domain based on computer simulation. J. Mol. Cataly. B- Enzym. 68: 286-291.
    CrossRef
  49. Kim HK, Park YS, Kim H, Oh TK. 1996. Partial interfacial activation of Proteus vulgaris lipase overexpressed in Escherichia coli. Biosci. Biotechnol. Biochem. 60: 1365-1367.
    Pubmed CrossRef
  50. Liu W, Li M, Yan Y. 2017. Heterologous expression and characterization of a new lipase from Pseudomonas fluorescens Pf0-1 and used for biodiesel production. Sci. Rep. 7: 1-11.
    Pubmed KoreaMed CrossRef
  51. Van Oort MG, Deveer AMTJ, Dijkman R, Tjeenk ML, Verheij HM, De Haas GH, et al. 1989. Purification and substrate specificity of Staphylococcus hyicus lipase. Biochem. 28: 9278-9285.
    Pubmed CrossRef
  52. Ayala-Bribiesca E, Turgeon SL, Britten M. 2017. Effect of calcium on fatty acid bioaccessibility during in vitro digestion of Cheddartype cheeses prepared with different milk fat fractions. J. Dairy. Sci. 100: 2454-2470.
    Pubmed CrossRef
  53. Torcello-Gómez A, Boudard C, Mackie AR. 2018. Calcium alters the interfacial organization of hydrolyzed lipids during intestinal digestion. Langmuir 34: 7536-7544.
    Pubmed CrossRef
  54. Alvarez FJ, Stella VJ. 1989. The role of calcium ions and bile salts on the pancreatic lipase-catalyzed hydrolysis of triglyceride emulsions stabilized with lecithin. Pharm. Res. 6: 449-457.
  55. El Khattabi M, Van Gelder P, Bitter W, Tommassen J. 2003. Role of the calcium ion and the disulfide bond in the Burkholderia glumae lipase. J. Mol. Catal. B: Enzym. 22: 329-338.
    CrossRef
  56. Hertadi R, Widhyastuti H. 2015. Effect of Ca2+ Ion to the activity and stability of lipase isolated from Chromohalobacter japonicus BK-AB18. Procedia. Chem. 16: 306-313.
    CrossRef
  57. Invernizzi G, Papaleo E, Grandori R, De Gioia L, Lotti M. 2009. Relevance of metal ions for lipase stability: Structural rearrangements induced in the Burkholderia glumae lipase by calcium depletion. J. Struct. Biol. 168: 562-570.
    Pubmed CrossRef
  58. Martigne M, Julien R, Sarda L. 1987. Studies on the effect of bile and lipolysis products on pancreatic lipase and colipase activity in vitro. Reprod. Nutr. Dev. 27: 1005-1012.
    Pubmed CrossRef
  59. Borgström B. 1977. The action of bile salts and other detergents on pancreatic lipase and the interaction with colipase. Biochim. Biophys. Acta (BBA) - Lipids Lipid Metabolism 488: 381-391.
    CrossRef
  60. Borkar PS, Bodade RG, Rao SR, Khobragade CN. 2009. Purification and characterization of extracellular lipase from a new strain:Pseudomonas aeruginosa SRT 9. Braz. J. Microbiol. 40: 358-366.
    Pubmed KoreaMed CrossRef
  61. Ye P, Xu YJ, Han ZP, Hu PC, Zhao ZL, Lu XL, et al. 2013. Probing effects of bile salt on lipase adsorption at air/solution interface by sum frequency generation vibrational spectroscopy. Biochem. Eng. J. 80: 61-67.
    CrossRef
  62. Hadvary P, Sidler W, Meister W, Vetter W, Wolfer H. 1991. The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase. J. Biol. Chem. 266: 2021-2027.
  63. Lüthi-Peng Q, Märki HP, Hadváry P. 1992. Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin. FEBS Lett. 299: 111-115.
    CrossRef
  64. Qi X. 2018. Review of the clinical effect of orlistat. IOP Conf. Ser. Mater. Sci. Eng. 301: 012063.
    CrossRef
  65. Sternby B, Hartmann D, Borgstroöm B, Nilsson Å. 2002. Degree of in vivo inhibition of human gastric and pancreatic lipases by Orlistat (Tetrahydrolipstatin, THL) in the stomach and small intestine. Clin. Nutr. 21: 395-402.
    Pubmed CrossRef
  66. Kelley DE, Bray GA, Pi-Sunyer FX, Klein S, Hill J, Miles J, et al. 2002. Clinical efficacy of orlistat therapy in overweight and obese patients with insulin-treated type 2 diabetes: a 1-year randomized controlled trial. Diabetes Care 25: 1033-1041.
    Pubmed CrossRef
  67. Ben Ayed S, Ali MB, Bali A, Gargouri Y, Laouini D, Ben Ali Y. 2018. Secretory lipase from the human pathogen Leishmania major:Heterologous expression in the yeast Pichia pastoris and biochemical characterization. Biochimie 146: 119-126.
    Pubmed CrossRef
  68. Salah RB, 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.
    Pubmed CrossRef
  69. Zouari N, Miled N, Cherif S, Mejdoub H, Gargouri Y. 2005. Purification and characterization of a novel lipase from the digestive glands of a primitive animal: the scorpion. Biochim. Biophys. Acta (BBA) - General Subjects 1726: 67-74.
    Pubmed CrossRef
  70. Luthi-peng Q, Winkler FK. 1992. Large spectral changes accompany the conformational transition of human pancreatic lipase induced by acylation with the inhibitor tetrahydrolipstatin. Eur. J. Biochem. 205: 383-390.
    Pubmed CrossRef
  71. Minovska V, Winkelhausen E, Kuzmanova S. 2005. Lipase immobilized by different techniques on various support materials applied in oil hydrolysis. J. Serbian Chem. Soc. 70: 609-624.
    CrossRef
  72. Rosu R, Uozaki Y, Iwasaki Y, Yamane T. 1997. Repeated use of immobilized lipase for monoacylglycerol production by solidphase glycerolysis of olive oil. J. Am. Oil. Chem. Soc. 74: 445-450.
    CrossRef
  73. Kharrat N, Ali YB, Marzouk S, Gargouri YT, Karra-Châabouni M. 2011. Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: Comparison with the free enzyme. Process. Biochem. 46: 1083-1089.
    CrossRef
  74. Egwim EC, Adesina AA, Oyewole OA, Okoliegbe IN. 2012. Optimization of lipase immobilized on chitosan beads for biodiesel production. Global. Res. J. Microbiol. 2: 103-112.
  75. Dong H, Li J, Li Y, Hu L, Luo D. 2012. Improvement of catalytic activity and stability of lipase by immobilization on organobentonite. Chem. Eng. J. 181: 590-596.
    CrossRef
  76. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, FernandezLafuente R. 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym. Microb. Technol. 40: 1451-1463.
    CrossRef
  77. Sankaran R, Show PL, Chang JS. 2016. Biodiesel production using immobilized lipase: feasibility and challenges. Biofuel. Bioprod. Bior. 10: 896-916.
    CrossRef
  78. Narwal SK, Gupta R. 2012. Biodiesel production by transesterification using immobilized lipase. Biotechnol. Lett. 35: 479-490.
    Pubmed CrossRef
  79. Taher H, Al-Zuhair S. 2016. The use of alternative solvents in enzymatic biodiesel production: a review. Biofuel. Bioprod. Bior. 11: 168-194.
    CrossRef
  80. Laane C, Boeren S, Vos K, Veeger C. 1987. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30: 81-87.
    Pubmed CrossRef
  81. Gorman LAS, Dordick JS. 1992. Organic solvents strip water off enzymes. Biotechnol. Bioeng. 39: 392-397.
    Pubmed CrossRef
  82. Religia P, Wijanarko A. 2015. Utilization of n-hexane as co-solvent to increase biodiesel yield on direct transesterification reaction from marine microalgae. Procedia Environ. Sci. 23: 412-420.
    CrossRef
  83. Yusuf M, Athar M. 2015. Biodiesel Production Using Hexane as Co-Solvent. J. Biofuels 6: 88-91.
    CrossRef

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