Browse > Article
http://dx.doi.org/10.48022/mbl.2003.03010

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

Misbah, Asmae (Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University)
Koraichi, Saad Ibnsouda (Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University)
Jouti, Mohamed Ali Tahri (Microbial Biotechnology Laboratory, Faculty of Sciences and Technologies, Sidi Mohamed Ben Abdellah University)
Publication Information
Microbiology and Biotechnology Letters / v.48, no.4, 2020 , pp. 491-505 More about this Journal
Abstract
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 SDS-PAGE 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;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 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.   DOI
2 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.   DOI
3 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.   DOI
4 Maraite A, Hoyos P, Carballeira JD, Cabrera AC, Ansorge-Schumacher MB, Alcantara 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.   DOI
5 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 B-Enzym. 120: 84-92.   DOI
6 Abdou AM. 2003. Purification and partial characterization of psychrotrophic Serratia marcescens lipase. J. Dairy. Sci. 86: 127-132.   DOI
7 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.   DOI
8 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.   DOI
9 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.   DOI
10 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.   DOI
11 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.
12 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.
13 Gutierrez-Arnillas E, Arellano M, Deive FJ, Rodriguez A, Sanroman MA. 2017. Unravelling the suitability of biological induction for halophilic lipase production by Halomonas sp. LM1C cultures. Bioresour. Technol. 239: 368-377.   DOI
14 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.   DOI
15 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.   DOI
16 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 site-directed mutagenesis in the lid domain based on computer simulation. J. Mol. Cataly. B- Enzym. 68: 286-291.   DOI
17 Ayala-Bribiesca E, Turgeon SL, Britten M. 2017. Effect of calcium on fatty acid bioaccessibility during in vitro digestion of Cheddar-type cheeses prepared with different milk fat fractions. J. Dairy. Sci. 100: 2454-2470.   DOI
18 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.   DOI
19 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.   DOI
20 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.   DOI
21 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.
22 Patel MT, Nagarajan R, Kilara A. 1996. Lipase-catalyzed biochemical reactions in novel media: A review. Chem. Eng. Commun. 152 : 365-404.   DOI
23 Reis P, Holmberg K, Watzke H, Leser ME, Miller R. 2009. Lipases at interfaces: a review. Adv. Colloid Interf. Sci. 147: 237-250.   DOI
24 Pahoja VM, Sethar MA. 2002. A review of enzymatic properties of lipase in plants, animals and microorganisms. J. Appl. Sci. 2: 474-484.   DOI
25 Thakur S. 2012. Lipases, its sources, properties and applications: a review. Int. J. Sci. Eng. Res. 3: 1-29.
26 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.   DOI
27 Torcello-Gomez A, Boudard C, Mackie AR. 2018. Calcium alters the interfacial organization of hydrolyzed lipids during intestinal digestion. Langmuir 34: 7536-7544.   DOI
28 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.   DOI
29 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.   DOI
30 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.   DOI
31 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.   DOI
32 Borgstrom 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.   DOI
33 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.   DOI
34 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.   DOI
35 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.   DOI
36 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.   DOI
37 Luthi-Peng Q, Marki HP, Hadvary P. 1992. Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin. FEBS Lett. 299: 111-115.   DOI
38 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.   DOI
39 Qi X. 2018. Review of the clinical effect of orlistat. IOP Conf. Ser. Mater. Sci. Eng. 301: 012063.
40 Sternby B, Hartmann D, Borgstroöm B, Nilsson A. 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.   DOI
41 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.   DOI
42 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.   DOI
43 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.   DOI
44 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.   DOI
45 Robinson PK. 2015. Enzymes: principles and biotechnological applications. Ess. Biochem. 59: 1-41.   DOI
46 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.   DOI
47 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.   DOI
48 Melani NB, Tambourgi EB, Silveira E. 2020. Lipases: From production to applications. Sep. Purif. Rev. 49: 143-158.   DOI
49 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.   DOI
50 Nisha S, Karthick SA, Gobi N. 2012. A review on methods, application and properties of immobilized enzyme. Chem. Sci. Rev. Lett. 1: 148-155.
51 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.
52 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.   DOI
53 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.   DOI
54 Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R. 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym. Microb. Technol. 40: 1451-1463.   DOI
55 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.   DOI
56 Rosu R, Uozaki Y, Iwasaki Y, Yamane T. 1997. Repeated use of immobilized lipase for monoacylglycerol production by solid-phase glycerolysis of olive oil. J. Am. Oil. Chem. Soc. 74: 445-450.   DOI
57 Kharrat N, Ali YB, Marzouk S, Gargouri YT, Karra-Chaabouni M. 2011. Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: Comparison with the free enzyme. Process. Biochem. 46: 1083-1089.   DOI
58 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.   DOI
59 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.   DOI
60 Sankaran R, Show PL, Chang JS. 2016. Biodiesel production using immobilized lipase: feasibility and challenges. Biofuel. Bioprod. Bior. 10: 896-916.   DOI
61 Narwal SK, Gupta R. 2012. Biodiesel production by transesterification using immobilized lipase. Biotechnol. Lett. 35: 479-490.   DOI
62 Taher H, Al-Zuhair S. 2016. The use of alternative solvents in enzymatic biodiesel production: a review. Biofuel. Bioprod. Bior. 11: 168-194.   DOI
63 Laane C, Boeren S, Vos K, Veeger C. 1987. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30: 81-87.   DOI
64 Gorman LAS, Dordick JS. 1992. Organic solvents strip water off enzymes. Biotechnol. Bioeng. 39: 392-397.   DOI
65 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.   DOI
66 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.   DOI
67 Adlercreutz P. 2013. Immobilization and application of lipases in organic media. Chem. Soc. Rev. 42: 6406-6436.   DOI
68 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.   DOI
69 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.   DOI
70 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.   DOI
71 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.
72 Singh R, Gupta N, Goswami VK, Gupta R. 2006. A simple activity staining protocol for lipases and esterases. Appl. Microbiol. Biotechnol. 70: 679-682.   DOI
73 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.   DOI
74 Gargouri Y, Pieroni G, Lowe PA, Sarda L, Verger R. 1986. Human gastric lipase. The effect of amphiphiles. Eur. J. Biochem. 156 : 305-310.   DOI
75 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.   DOI
76 Yusuf M, Athar M. 2015. Biodiesel Production Using Hexane as Co-Solvent. J. Biofuels 6: 88-91.   DOI
77 Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.   DOI
78 Ornstein L. 1964. Disc electrophoresis. I. Background and theory. Ann. NY Acad. Sci. 121: 321.   DOI
79 Davis BJ. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. NY Acad. Sci. 121: 404-427.   DOI
80 Gargouri Y, Chahinian H, Moreau H, Ransac S, Verger R. 1991. Inactivation of pancreatic and gastric lipases by THL and C12: 0-TNB: a kinetic study with emulsified tributyrin. Biochim. Biophys. Acta (BBA) - Lipids and Lipid Metabolism 1085: 322-328.   DOI
81 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.   DOI
82 Kaur M, Mehta A, Gupta R. 2019. Synthesis of methyl butyrate catalyzed by lipase from Aspergillus fumigatus. J. Oleo Sci. 68: 989-993.   DOI
83 Ghamgui H, Miled N, Karra-chaabouni 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.   DOI