Utilization of vegetable oil production waste for lipase production from Pseudomonas sp. HCU2-1 and enzyme characterization
Lipase-producing bacteria were successfully isolated and selected thorough oil contaminated wastewater and soil samples. Thirty-five bacterial strains were screened by activity on lipase test medium, and then further tested to determine their activity by using tributyrin agar. Consequently, a strain HCU2-1 was isolated as the best producer and finally identified as Pseudomonas sp. HCU2-1. Lipase production by Pseudomonas sp. HCU2-1 was optimized by varying environmental and nutritional conditions. The maximum lipase production was obtained using wastewater from palm oil refining process at concentration of 2% (v/v) of basal medium (70.29±0.09 U/mg), followed in order by wastewater from soybean oil refining process (62.59±0.38 U/mg) and crude acid oil from soybean oil refining process (62.48±0.43 U/mg), respectively. The highest lipase activity (77.14±0.33 U/mg) was obtained when peptone was used at concentration of 0.5% (v/v) of basal medium. The optimum temperature and pH for lipase production were 40°C and pH 7, respectively. Zymogram assay of crude enzyme on SDS-PAGE presented one band with lipase activity of molecular weight of 45 kDa. The optimum pH and temperature for activity were pH 11 and 60°C, respectively. The enzyme was stable in the pH range 9-11 for 60 min and at 40-60°C for 60 min. Higher activity was observed in the presence of surfactants, Ca2+, Mg2+, Mn2+ ions and strongly inhibited lipase activity by DTT and PMSF. Furthermore, this enzyme hydrolyzed synthetic triglycerides and ester of pNPP. The enzyme exhibited significant stability in the presence of commercial detergents and oxidizing agents. Hence, lipase from Pseudomonas sp. HCU2-1 could be considered to be suitable for a variety of industrial applications such as in detergent formulations.
2. Jaeger KE, Eggert T. Lipases for biotechnology. Curr Opin Biotech 2002;13:390-7.
3. Gao XG, Cao SG, Zhang KC. Production, properties and application to non-aqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain. Enzyme Microb Technol 2000;27:74-82.
4. Dalmou E, Montesinos JL, Lotti M, Casas C. Effect of different carbon sources on lipase production by Candida rugosa. Enzyme Microb Technol 2000;2:657-63.
5. Ferreira C, Maria A, Perolta RM. Production of lipase by soil fungi and partial characterization of lipase from a selected strains (Penicillium wortmanii). J Basic Microbiol 1999;39:11-5.
6. Ramani K, Kennedy LJ, Ramakrishnan M, Sekaran G. Purification, characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis. Process Biochem 2010;45:1683-91.
7. Jaina D, Mishraa S. Multifunctional solvent stable Bacillus lipase mediated biotransformations in the context of food and fuel. J Mol Catal B: Enzym 2015;117:21-30.
8. Palekar AA, Vasudevan PT, Yan S. Purification of lipase: a review. Biocatal Biotransform 2000;18:177-200.
9. Alonso FOM, Oliveira EBL, Dellamora-Ortiz GM, Pereira-Meirelles FV. Improvement of lipase production at different stirring speeds and oxygen levels. Braz J Chem Eng 2005;22:9-18.
10. Cheirsilp B, Louhasakul Y. Industrial waste as a promising renewable source for production of microbial lipid and direct transesterification of the lipid into biodiesel. Bioresour Technol 2013;142:329-37.
11. Prasad MP, Manjunath K. Comparative study on biodegradation of lipid-rich wastewater using lipase producing bacterial species. Indian J Biotechnol 2011;10:121-4.
12. D’Annibale A, Sermanni GG, Federici F, Petruccioli M. Olive-mill wastewaters: a promising substrate for microbial lipase production. Bioresour Technol 2006;97:1828-33.
13. Salihu A, Alam MZ, Karim MIA, Salleh HM. Optimization of lipase production by Candida cylindracea in palm oil mill effluent based medium using statistical experimental design. J Mol Catal B: Enzym 2011;69:66-73.
14. Scioli C, Vollaro L. The use of Yarrowia lipolytica to reduce pollution in olive mill wastewaters. Water Res 1997;31:2520-4.
15. Jaeger KE, Ransa S, Dijkstra BW, Colson C, van Heuvel M, Misset O. Bacterial lipases. FEMS Microbiol Rev 1994;15:29-63.
16. Rathi P, Saxena RK, Gupta R. A novel alkaline lipase from Burkholderia cepacia for detergent formulation. Process Biochem 2001;37:187-92.
17. Li XL, Zhang WH, Wang YD, Dai YJ, Zhang HT, Wang Y, et al. A high-detergent-performance, cold-adapted lipase from Pseudomonas stutzeri PS59 suitable for detergent formulation. J Mol Catal B: Enzym 2014;102:16-24.
18. Wang YX, Srivastava KC, Shen GJ, Wang HY. Thermostable alkaline lipase from a newly isolated thermophilic Bacillus, strain A30-1 (ATCC53841). J Ferment Bioeng 1995;79:433-8.
19. Silva WOB, Mitidieri S, Schrank A, Vainstein MH. Production and extraction of an extracellular lipase from the entomopathogenic fungus Metarhizium anisopliae. Process Biochem 2005;40:321-6.
20. Maia MMD, Heasley A, Camargo de Morais MM, Melo EHM, Morais Jr MA, Ledingham WM. Effect of culture conditions on lipase production by Fusarium solani in batch fermentation. Bioresour Technol 2001;76:23-7.
21. Bradford MM. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-52.
22. Laemmli UK. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nat 1970;227:680-5.
23. Lima VGM, Krieger N, Mitchell DA, Baratti JC, de Filippis I, Fontana JD. Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium. J Mol Catal B: Enzym 2004;31:53-64.
24. Brozzoli V, Crognate S, Sampedro I, Federici F, D’Annibale A, Petruccioli M. Assessment of olive-mill wastewater as a growth medium for lipase production by Candida cylindracea in bench-top reactor. Bioresour Technol 2009;100:3395-3402.
25. Haba E, Bresco O, Ferrer C, Marques A, Busquets M, Manresa A. Isolation of lipase-secreting bacteria by deploying used frying oil as selective substrate. Enzyme Microb Technol 2000;26:40-4.
26. Kanmani P, Kumaresan K, Aravind J. Utilization of coconut oil mill waste as a substrate for optimized lipase production, oil biodegradation and enzyme purification studies in Staphylococcus pasteuri. Electron J Biotech 2015;18:20-8.
27. Dharmsthiti S, Luchai S. Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027. FEMS Microb Lett 1999;179:241-6.
28. Supakdamrongkul P, Bhumiratana A, Wiwat C. Optimization of extracellular lipase production
fromthe biocontrol fungus Nomuraea rileyi. Biocontrol Sci Techn 2010;20:595-604.
29. Ramani K, Kennedy LJ, Ramakrishnan M, Sekaran G. Purification, characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis. Process Biochem 2010;45:1683-91.
30. Bose A, Keharia H. Production, characterization and applications of organic solvent tolerant lipase by Pseudomonas aeruginosa AAU2. Biocatal Agric Biotechnol 2013;2:255-66.
31. Patel V, Nambiar S, Madamwar D. An extracellular solvent stable alkaline lipase from Pseudomonas sp. DMVR46: partial purification, characterization and application in non-aqueous environment. Process Biochem
32. Hemachander C, Puvanakrishnan R. Lipase from Ralstonia pickettii as an additive in laundry detergent formulations. Process Biochem 2000;35(8):809-14.
33. Velu N, Divakar K, Nandhinidevi G, Gautam P. Lipase from Aeromonas caviae AU04: isolation, purification and protein aggregation. Biocatal Agric Biotechnol 2012;1:45-50.
34. Li M, Yang LR, Xu G, Wu JP. Screening, purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC
4254. Bioresour Technol 2013,148:114-20.
35. Ayaz B, Ugur A, Boran R. Purification and characterization of organic solvent-tolerant lipase from Streptomyces sp. OC119-7 for biodiesel production. Biocatal Agric Biotechnol 2015;4(1):103-8.
36. Sarkar P, Yamasaki S, Basak S, Bera A, Bag PK. Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere. Process Biochem 2012;47:858-66.
37. Van Oort MG, Deveer AMTJ, Dijkman R, Tjeenk ML, Verheij HM, De Haas GH, et al. Purification and substrate specificity of Staphylococcus hycius lipase. Biochemistry 1989;28:9278-85.
38. Dandavate V, Jinjala J, Keharia H, Madamwar D. Production, partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis. Bioresour Technol 2009;100:3374-81.
39. Liu R, Jiang X, Mou H, Guan H, Hwang HM, Li X. A novel low-temperature resistance alkaline lipase from a soda lake fungus strain Fusarium solani N4-2 for detergent formulation. Biochem Eng J 2009;46:265-70.
40. Jaeger KE, Dijkstra BW, Reetz MT. Bacterial biocatalyst: molecular biology, three dimensional structures and biotechnological applications of lipases. Annu Rev Microbiol 1999;53:315-51.
41. Saxena RK, Davidson WS, Sheoran A, Giri B. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem 2003;39:239-47.