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Received February 9, 2025
Revised April 14, 2025
Accepted April 24, 2025
Available online August 1, 2025
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Development of Cost-Effective Method for the Production and Purification of Microbial Lipase Enzyme Using Ion Exchange Chromatography
Industrial Biotechnology Laboratory, Department of Biotechnology, University of Malakand, Chakdara, Dir (Lower) 1Department of Economics, University of Malakand, Chakdara, Lower Dir
Korean Chemical Engineering Research, August 2025, 63(3), 105124
https://doi.org/10.9713/kcer.2025.63.3.105124
https://doi.org/10.9713/kcer.2025.63.3.105124
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Abstract
This study explores the economical production and purification of microbial lipase enzymes from Bacillus
subtilis, an enzyme with broad industrial applications in food, pharmaceuticals, and biofuels. Bacillus subtilis was cultured
in defined media and butter waste, supplemented with various salts for enhanced production. Among all the combinations
used for enzyme production, maximum lipase activity (18 U/ml) was achieved with butter waste supplemented with 1.0%
ammonium nitrate salt. Partial purification was conducted by anion exchange chromatography with 20 mM sodium
phosphate buffer (pH 8) as binding buffer. Elution profile showed the maximum enzyme recovery i.e., 4.22 U/ml and
5.22 U/ml in 50% and 30% of elution buffer, respectively. The collected fractions were desalted by commercial Sephadex G-
25 and were further analyzed for the presence of lipase enzyme and total protein content. The purified lipase demonstrated
significant activity when applied against various commercial oil samples compared to commercially available enzymes.
This work highlights a cost-effective approach for the production of microbial lipase enzyme with promising industrial
potential.
References
1. Gulhan, Y., Unzile, G. G., Elif, G. and Fatih, A., “Screening, Partial
Purification and Characterization of the Hyper-thermophilic
Lipase Produced by a New Isolate of Bacillus subtilis LP2,”
Biocatal. Biotransformation., 38(5), 367-375(2020).
2. Suci, M., Arbianti, R. and Hermansyah, H., “Lipase Production
from Bacillus Subtilis with Submerged Fermentation Using Waste
Cooking Oil,” IOP Conf. Ser.: Earth Environ. Sci., 105, 012126 (2018).
3. Li, P. L., Hudzaifa, M. K., Marimuthu, C., Subash, C. B. G.,
Thangavel, L. and Thean-Hock, T., “Lipase-secreting Bacillus
species in An Oil-contaminated Habitat: Promising Strains to Alleviate
Oil Pollution,” BioMed Res. Int., 2015, 1-9(2015).
4. Haniya, M., Naaz, A., S akhawat, A., Amir, S., Zahid, H . and
Syed, S. A., “Optimized Production of Lipase from Bacillus Subtilis
PCSIRNL-39,” Afr. J. Biotechnol., 16(19), 1106-1115(2017).
5. Rakesh, K., Arpit, S., Arun, K. and Deepak, S., “Lipase from Bacillus
Pumilus RK31: Production, Purification and Some Properties,”
World Appl. Sci. J., 16(7), 940-948(2012).
6. Femi-Ola, T. O., Odeyemi, A. T., Oliya, B. S. and Ojo, O. O.,
“Characterization of Lipase from Bacillus Subtilis Isolated from
Oil Contaminated Soil,” J. Appl. Environ. Microbiol., 6, 10-17(2018).
7. Sajid, A., Sumera, A. K., Muhammad, H. and In-Jung, L., “The
Recent Advances in the Utility of Microbial Lipases: A Review,”
Microorg., 11(2), 510(2023).
8. Myllena, R. S., Daniela, B. H. and Joelise, A. F. A., “Lipases:
Sources of Acquisition, Ways of Production, and Recent Applications,”
Catal. Res., 2(2), 1-43(2022).
9. Adegoke, I. A. and Ademola, O. O., “Production Strategies and
Biotechnological Relevance of Microbial Lipases: A Review,”
Braz. J. Microbiol., 52(3), 1257-1269(2021).
10. Tomasz, S., Justyna, C., Marcin, P. and Magdalena, F., “Various
Perspectives on Microbial Lipase Production Using Agri-food
Waste and Renewable Products,” Agriculture., 11(6), 540(2021).
11. Adyasa, B., Sudip, K. S., Geetanjali, R. and Sangeeta, R., “Purification
and Optimization of Extracellular Lipase from a Novel Strain
Kocuria Flava Y4,” Int. J. Anal. Chem., 2022(1), 6403090 (2022).
12. Naveen, P., Dhananjai, R., Shivam, Shraddha, S. and Umesh,
M., “Lipases: Sources, Production, Purification, and Applications,”
Recent Pat. Biotechnol., 13(1), 45-56(2019).
13. Justyna, P., Marek, T., Anna, M. S., Magdalena, K., Tadeusz, G.
and Jacek, N., “Green Chromatography,” J. Chromatogr. A., 1307,
1-20 (2013).
14. Szabolcs, F., Isabelle, K., Serge, R. and Davy, G., “Importance of
Instrumentation for Fast Liquid Chromatography in Pharmaceutical
Analysis,” J. Pharm. Biomed. Anal, 87, 105-119(2014).
15. Baraem, I. and Suzanne, N., “Basic Principles of Chromatography,”
Food Analysis, 27, 473-498(2010).
16. Szabolcs, F., Alain, B., Jean-Luc, V. and Davy, G., “Ion-exchange
Chromatography for the Characterization of Biopharmaceuticals,”
J. Pharm. Biomed. Anal., 113, 43-55(2015).
17. Ozlem, B. A., “Ion-exchange Chromatography and Its Applications,”
Column Chromatography, 10, 31-57(2013).
18. Philip, M. C., Keith, D. R. and Brendan, F. O., “Ion-exchange Chromatography:
Basic Principles and Application,” Protein Chromatography:
Methods and Protocols, 209-223(2017).
19. Michael, W., Peter, T. and Jurgin, P., “The Isoelectric Region of
Proteins: A Systematic Analysis,” PloS One, 5(5), 10546(2010).
20. Che, H. A. C. H., Raja, N. Z. R. A., Adam, L. T. C., Abu-Bakar, S.
and Mohd-Shukuri, M. A., “Enhancement of a Protocol Purifying
T1 Lipase Through Molecular Approach,” PeerJ., 6, e5833(2018).
21. Eldbjorg, B. V., Daina, R. and Marianne, T., “Biowaste Valorisation
in a Future Circular Bioeconomy,” Procedia Cirp., 69, 591-596(2018).
22. Sanjeev, K. A., Surendra, S., Vinay, K., Preeti, C., Raveendran,
S., Parameswaran, B., Zhengqiang, Z., Ashok, P. and Mukesh,
K. A., “Processing of Municipal Solid Waste Resources for a Circular
Economy in China: An Overview,” Fuel, 317, 123478 (2022).
23. Yuwen, Z., Vinay, K., Sharareh, H., Vigneswaran, V., Karthik, R.,
Pooja, S., Yen, W. T., Parameswaran, B., Raveendran, S., Surendra,
S., Deepanraj, B., Mofijur, M., Zhengqiang, Z., Mohammad, J.
T. and Mukesh, K. A., “Recovery of Value-added Products from
Biowaste: A Review,” Bioresour. Technol., 360, 127565(2022).
24. Temoor, A., Muhammad, S., Farrukh, A., Ijaz, R., Asad, A. S.,
Muhammad, N., Amir, H., Natasha, M., Irfan, M. and Sher, M.,
“Biodegradation of Plastics: Current Scenario and Future Prospects
for Environmental Safety,” Environ. Sci. Pollut. Res., 25,
7287-7298(2018).
25. Giovanna, T. B., Nicole, M., Francisco, L. C. A. and Marcus, B.
S. F., “Lipases: Market Study and Potential Applications of Immobilized
Derivatives,” Biofpr., 18(5), 1676-1689(2024).
26. Muthu, K. S., Pooja, C. A., Hrithik, B. and Soham, C., “Process
Optimization and Techno-economic Analysis for the Production of
Lipase from Bacillus sp,” J. Taibah Univ. Sci., 17(1), 2198925(2023).
27. Cesar, A. G., Juan, S. P. T. and Oveimar, B., “Microbial Lipases
and Their Potential in the Production of Pharmaceutical Building
Blocks,” Int. J. Mol. Sci., 23(17), 9933(2022).
28. David, G., “Lipases Industrial Applications: Focus on Food and
Agroindustries,” Ol., Corps Gras, Lipides., 24(4), D403(2017).
29. Richard, G. V. T. and Sheilah, S., “The Use of Tween 20 in a Sensitive
Turbidimetric Assay of Lipolytic Enzymes,” Can. J. Microbiol.,
35(4), 511-514(1989).
30. Mohd-Yusof, A. S., C-Nyonya, A. R., Abu-Bakar, S., Zin, W. Y.,
Kamaruzaman, A. and Mahiran, B., “A Plate Assay for Primary
Screening of Lipase Activity,” J. Microbiol. Methods, 9(1), 51-
56(1989).
31. Lucreti, R., Bruce, S. and Roshini, G., “Identification of Lipolytic
Enzymes Isolated from Bacteria Indigenous to Eucalyptus
Wood Species for Application in the Pulping Industry,” Biotechnol
Rep., 15, 114-124(2017).
32. Fariha, H., Aamer, A. S. and Abdul, H., “Influence of Culture
Conditions on Lipase Production by Bacillus sp. FH5,” Ann. Microbiol.,
56, 247-252(2006).
33. Rajendra, K. S., Anita, S., Bhoopander G. and Winlet, S. D.,
“Purification Strategies for Microbial Lipases,” J. Microbiol. Methods,
52(1), 1-18(2003).
34. Chris, J. F. and Phoebe, E. F., “Mechanism of Salt-mediated Inhibition
of Lipoprotein Lipase,” J. Lipid. Res., 17(3), 248-256(1976).
35. Fariha, H., Aamer, A. S. and Abdul, H., “Purification and Characterization
of a Mesophilic Lipase from Bacillus Subtilis FH5
Stable at High Temperature and pH,” Acta Biol. Hung., 58, 115-
132(2007).
36. Dag, R. G., Dominique, L. and Walther B. T., “Pancreatic Bile Salt
Dependent Lipase From Cod (Gadus morhua): Purification and
Properties,” BBA-Lipids and Lipid Metabolism, 1124(2), 123-134
(1992).
37. Esra, B. and Orkun, P., “A Preliminary Study on Purification
and Characterization of Lipase (s) Produced by Cryptococcus
Diffluens D44,” Int. J. Adv. Eng. Pure. Sci, 35(2), 203-212(2023).
38. Carolina, M. V-P., Paulo, R. R., Cintia, B. G. and Alessandra L.
D. O., “Determination of Free Fatty Acids in Crude Vegetable Oil
Samples Obtained by High-pressure Processes,” Food Chem.: X.,
12, 100166(2021).
39. You, L. L. and Badlishah, B., “Effects of Enzymatic Hydrolysis
on Crude Palm Olein by Lipase from Candida Rugosa,” J. Food
Lipids., 13(1), 73-87(2006).
Purification and Characterization of the Hyper-thermophilic
Lipase Produced by a New Isolate of Bacillus subtilis LP2,”
Biocatal. Biotransformation., 38(5), 367-375(2020).
2. Suci, M., Arbianti, R. and Hermansyah, H., “Lipase Production
from Bacillus Subtilis with Submerged Fermentation Using Waste
Cooking Oil,” IOP Conf. Ser.: Earth Environ. Sci., 105, 012126 (2018).
3. Li, P. L., Hudzaifa, M. K., Marimuthu, C., Subash, C. B. G.,
Thangavel, L. and Thean-Hock, T., “Lipase-secreting Bacillus
species in An Oil-contaminated Habitat: Promising Strains to Alleviate
Oil Pollution,” BioMed Res. Int., 2015, 1-9(2015).
4. Haniya, M., Naaz, A., S akhawat, A., Amir, S., Zahid, H . and
Syed, S. A., “Optimized Production of Lipase from Bacillus Subtilis
PCSIRNL-39,” Afr. J. Biotechnol., 16(19), 1106-1115(2017).
5. Rakesh, K., Arpit, S., Arun, K. and Deepak, S., “Lipase from Bacillus
Pumilus RK31: Production, Purification and Some Properties,”
World Appl. Sci. J., 16(7), 940-948(2012).
6. Femi-Ola, T. O., Odeyemi, A. T., Oliya, B. S. and Ojo, O. O.,
“Characterization of Lipase from Bacillus Subtilis Isolated from
Oil Contaminated Soil,” J. Appl. Environ. Microbiol., 6, 10-17(2018).
7. Sajid, A., Sumera, A. K., Muhammad, H. and In-Jung, L., “The
Recent Advances in the Utility of Microbial Lipases: A Review,”
Microorg., 11(2), 510(2023).
8. Myllena, R. S., Daniela, B. H. and Joelise, A. F. A., “Lipases:
Sources of Acquisition, Ways of Production, and Recent Applications,”
Catal. Res., 2(2), 1-43(2022).
9. Adegoke, I. A. and Ademola, O. O., “Production Strategies and
Biotechnological Relevance of Microbial Lipases: A Review,”
Braz. J. Microbiol., 52(3), 1257-1269(2021).
10. Tomasz, S., Justyna, C., Marcin, P. and Magdalena, F., “Various
Perspectives on Microbial Lipase Production Using Agri-food
Waste and Renewable Products,” Agriculture., 11(6), 540(2021).
11. Adyasa, B., Sudip, K. S., Geetanjali, R. and Sangeeta, R., “Purification
and Optimization of Extracellular Lipase from a Novel Strain
Kocuria Flava Y4,” Int. J. Anal. Chem., 2022(1), 6403090 (2022).
12. Naveen, P., Dhananjai, R., Shivam, Shraddha, S. and Umesh,
M., “Lipases: Sources, Production, Purification, and Applications,”
Recent Pat. Biotechnol., 13(1), 45-56(2019).
13. Justyna, P., Marek, T., Anna, M. S., Magdalena, K., Tadeusz, G.
and Jacek, N., “Green Chromatography,” J. Chromatogr. A., 1307,
1-20 (2013).
14. Szabolcs, F., Isabelle, K., Serge, R. and Davy, G., “Importance of
Instrumentation for Fast Liquid Chromatography in Pharmaceutical
Analysis,” J. Pharm. Biomed. Anal, 87, 105-119(2014).
15. Baraem, I. and Suzanne, N., “Basic Principles of Chromatography,”
Food Analysis, 27, 473-498(2010).
16. Szabolcs, F., Alain, B., Jean-Luc, V. and Davy, G., “Ion-exchange
Chromatography for the Characterization of Biopharmaceuticals,”
J. Pharm. Biomed. Anal., 113, 43-55(2015).
17. Ozlem, B. A., “Ion-exchange Chromatography and Its Applications,”
Column Chromatography, 10, 31-57(2013).
18. Philip, M. C., Keith, D. R. and Brendan, F. O., “Ion-exchange Chromatography:
Basic Principles and Application,” Protein Chromatography:
Methods and Protocols, 209-223(2017).
19. Michael, W., Peter, T. and Jurgin, P., “The Isoelectric Region of
Proteins: A Systematic Analysis,” PloS One, 5(5), 10546(2010).
20. Che, H. A. C. H., Raja, N. Z. R. A., Adam, L. T. C., Abu-Bakar, S.
and Mohd-Shukuri, M. A., “Enhancement of a Protocol Purifying
T1 Lipase Through Molecular Approach,” PeerJ., 6, e5833(2018).
21. Eldbjorg, B. V., Daina, R. and Marianne, T., “Biowaste Valorisation
in a Future Circular Bioeconomy,” Procedia Cirp., 69, 591-596(2018).
22. Sanjeev, K. A., Surendra, S., Vinay, K., Preeti, C., Raveendran,
S., Parameswaran, B., Zhengqiang, Z., Ashok, P. and Mukesh,
K. A., “Processing of Municipal Solid Waste Resources for a Circular
Economy in China: An Overview,” Fuel, 317, 123478 (2022).
23. Yuwen, Z., Vinay, K., Sharareh, H., Vigneswaran, V., Karthik, R.,
Pooja, S., Yen, W. T., Parameswaran, B., Raveendran, S., Surendra,
S., Deepanraj, B., Mofijur, M., Zhengqiang, Z., Mohammad, J.
T. and Mukesh, K. A., “Recovery of Value-added Products from
Biowaste: A Review,” Bioresour. Technol., 360, 127565(2022).
24. Temoor, A., Muhammad, S., Farrukh, A., Ijaz, R., Asad, A. S.,
Muhammad, N., Amir, H., Natasha, M., Irfan, M. and Sher, M.,
“Biodegradation of Plastics: Current Scenario and Future Prospects
for Environmental Safety,” Environ. Sci. Pollut. Res., 25,
7287-7298(2018).
25. Giovanna, T. B., Nicole, M., Francisco, L. C. A. and Marcus, B.
S. F., “Lipases: Market Study and Potential Applications of Immobilized
Derivatives,” Biofpr., 18(5), 1676-1689(2024).
26. Muthu, K. S., Pooja, C. A., Hrithik, B. and Soham, C., “Process
Optimization and Techno-economic Analysis for the Production of
Lipase from Bacillus sp,” J. Taibah Univ. Sci., 17(1), 2198925(2023).
27. Cesar, A. G., Juan, S. P. T. and Oveimar, B., “Microbial Lipases
and Their Potential in the Production of Pharmaceutical Building
Blocks,” Int. J. Mol. Sci., 23(17), 9933(2022).
28. David, G., “Lipases Industrial Applications: Focus on Food and
Agroindustries,” Ol., Corps Gras, Lipides., 24(4), D403(2017).
29. Richard, G. V. T. and Sheilah, S., “The Use of Tween 20 in a Sensitive
Turbidimetric Assay of Lipolytic Enzymes,” Can. J. Microbiol.,
35(4), 511-514(1989).
30. Mohd-Yusof, A. S., C-Nyonya, A. R., Abu-Bakar, S., Zin, W. Y.,
Kamaruzaman, A. and Mahiran, B., “A Plate Assay for Primary
Screening of Lipase Activity,” J. Microbiol. Methods, 9(1), 51-
56(1989).
31. Lucreti, R., Bruce, S. and Roshini, G., “Identification of Lipolytic
Enzymes Isolated from Bacteria Indigenous to Eucalyptus
Wood Species for Application in the Pulping Industry,” Biotechnol
Rep., 15, 114-124(2017).
32. Fariha, H., Aamer, A. S. and Abdul, H., “Influence of Culture
Conditions on Lipase Production by Bacillus sp. FH5,” Ann. Microbiol.,
56, 247-252(2006).
33. Rajendra, K. S., Anita, S., Bhoopander G. and Winlet, S. D.,
“Purification Strategies for Microbial Lipases,” J. Microbiol. Methods,
52(1), 1-18(2003).
34. Chris, J. F. and Phoebe, E. F., “Mechanism of Salt-mediated Inhibition
of Lipoprotein Lipase,” J. Lipid. Res., 17(3), 248-256(1976).
35. Fariha, H., Aamer, A. S. and Abdul, H., “Purification and Characterization
of a Mesophilic Lipase from Bacillus Subtilis FH5
Stable at High Temperature and pH,” Acta Biol. Hung., 58, 115-
132(2007).
36. Dag, R. G., Dominique, L. and Walther B. T., “Pancreatic Bile Salt
Dependent Lipase From Cod (Gadus morhua): Purification and
Properties,” BBA-Lipids and Lipid Metabolism, 1124(2), 123-134
(1992).
37. Esra, B. and Orkun, P., “A Preliminary Study on Purification
and Characterization of Lipase (s) Produced by Cryptococcus
Diffluens D44,” Int. J. Adv. Eng. Pure. Sci, 35(2), 203-212(2023).
38. Carolina, M. V-P., Paulo, R. R., Cintia, B. G. and Alessandra L.
D. O., “Determination of Free Fatty Acids in Crude Vegetable Oil
Samples Obtained by High-pressure Processes,” Food Chem.: X.,
12, 100166(2021).
39. You, L. L. and Badlishah, B., “Effects of Enzymatic Hydrolysis
on Crude Palm Olein by Lipase from Candida Rugosa,” J. Food
Lipids., 13(1), 73-87(2006).
