Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum
Background and Objectives: Due to the widespread use of lipase enzymes in various industries, finding native lipase producing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed.
Materials and Methods: Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in different media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20-90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads.
Results: Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respectively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%.
Conclusion: The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.
2. Javed S, Azeem F, Hussain S, Rasul I, Siddique MH, Riaz M, et al. Bacterial lipases: a review on purification and characterization. Prog Biophys Mol Biol 2018; 132: 23-34.
3. Chandra P, Enespa, Singh R, Arora PK. Microbial lipases and their industrial applications: a comprehensive review. Microb Cell Fact 2020; 19: 169.
4. Guan C, Tao Z, Wang L, Zhao R, Chen X, Huang X, et al. Isolation of novel Lactobacillus with lipolytic activity from the vinasse and their preliminary potential using as probiotics. AMB Express 2020; 10: 91.
5. Uppada S R, Akula M, Bhattacharya A, Dutta JR. (2017). Immobilized lipase from Lactobacillus plantarum in meat degradation and synthesis of flavor esters. J Genet Eng Biotechnol 2017; 15: 331-334.
6. Esteban-Torres M, Mancheno JM, de las Rivas B, Munoz R. Characterization of a halotolerant lipase from the lactic acid bacteria Lactobacillus plantarum useful in food fermentations. LWT - Food Sci Technol 2015; 60: 246-252.
7. Chiou SH, Hung TC, Giridhar R, Wu WT. Immobilization of lipase to chitosan beads using a natural cross-linker. Prep Biochem Biotechnol 2007; 37: 265-275.
8. Hanefeld U, Cao L, Magner E. Enzyme immobilisation: fundamentals and application. Chem Soc Rev 2013; 42: 6211-6212.
9. Homaei AA, Sariri R, Vianello F, Stevanato R. Enzyme immobilization: an update. J Chem Biol 2013; 6: 185-205.
10. Silva JA, Macedo GP, Rodrigues DS, Giordano RLC, Gonçalves LRB. Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies. Biochem Eng J 2019; 60: 16-24.
11. Yagar H, Balkan U. Entrapment of laurel lipase in chitosan hydrogel beads. Artif Cells Nanomed Biotechnol 2017; 45: 864-870.
12. Mobarak-Qamsari E, Kasra-Kermanshahi R, Erfan M, Ghadam P. Screening of potentially probiotic Lactobacillus possessing surface layer protein from Iranian traditional dairy products. J Sci Islam Repub Iran 2013; 27: 305-312.
13. Lanka S, Latha JNL. A short review on various screening methods to isolate potential lipase producers: lipases-the present and future enzymes of biotech industry. Int J Biol Chem 2015; 9: 207-219.
14. Marroki A, Zúñiga M, Kihal M, Pérez-Martínez G. Characterization of Lactobacillus from algerian goat’s milk based on phenotypic, 16S rDNA sequencing and their technological properties. Braz J Microbiol 2011;42:158-171.
15. Mobarak-Qamsari E, Kasra-Kermanshahi R, Moosavi-Nejad Z. Isolation and identification of a novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110. Iran J Microbiol 2011; 3: 92-98.
16. Rashmi BS, Gayathri D. Partial purification, characterization of Lactobacillus sp. G5 lipase and their probiotic potential. Int Food Res J 2014; 21: 1737-1743.
17. Goldring JP. Spectrophotometric methods to determine protein concentration. Methods Mol Biol 2015; 1312: 41-47.
18. Brunelle JL, Green R. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).
Methods Enzymol 2014; 541: 151-159.
19. Beisson F, Tiss A, Rivière C, Verger R. Methods for lipase detection and assay: a critical review. Eur J Lipid Sci Technol 2000; 102: 133-153.
20. Fojan P, Jonson PH. Petersen MT, Petersen SB. What distinguishes an esterase from a lipase: a novel structural approach. Biochimie 2000; 82: 1033-1041.
21. Stoytcheva M, Montero G, Zlatev R, Leon JA, Gochev V. Analytical methods for lipases activity determination: a review. Curr Anal Chem 2012; 8: 400-407.
22. Sooch BS, Kauldhar BS. Influence of multiple bioprocess parameters on production of lipase from Pseudomonas sp. BWS-5. Braz Arch Biol Technol 2013; 56: 711-721.
23. Iqbal AS, Rehman A. Characterization of lipase from Bacillus subtilis I-4 and its potential use in oil contaminated wastewater. Braz Arch Biol Technol 2015; 58: 789-797.
24. Lopes Mde F, Leitão AL, Regalla M, Marques JJ, Carrondo MJ, Crespo MT. Characterization of a highly thermostable extracellular lipase from Lactobacillus plantarum. Int J Food Microbiol 2002; 76: 107-115.
25. Fatima S, Faryad A, Ataa A, Joyia FA, Parvaiz A. Microbial lipase production: a deep insight into the recent advances of lipase production and purification techniques. Biotechnol Appl Biochem 2021; 68: 445-458.
26. Yagmurov ER, Kozlov GV, Pushkarev MA. Lipase purification: the review of conventional and novel methods. J Hyg Eng Des 2017; 20: 60-69.
27. Wingfield P. Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci 2001; Appendix 3: Appendix 3F.
28. Saxena RK, Sheoran A, Giri B, Davidson WS. Purification strategies for microbial lipases. J Microbiol Methods 2003; 52: 1-18.
29. Qu B, Luo Y. Chitosan-based hydrogel beads: preparations, modifications and applications in food and agriculture sectors–a review. Int J Biol Macromol 2020; 152: 437-448.
30. Rafiee F, Rezaee M. Different strategies for the lipase immobilization on the chitosan based supports and their applications. Int J Biol Macromol 2021; 179: 170-195.
|Issue||Vol 13 No 6 (2021)|
|Lipase; Dairy products; Olive oil; Lactobacillus fermentum; Adsoption; Chitosan|
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|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.|