Global Advanced Research Journal of Microbiology (GARJM) ISSN: 2315-5116
February 2020 Vol. 9(2): pp. 013-029
Copyright © 2020 Global Advanced Research Journals


Full Length Research Paper


Purification and Characterization of Bacterial Thermoalkalistable Lipase for Application in Bio-detergent Industry

Al-Dhumri, S.A.1 and Bayoumi, R.A.2

 1Biology Dept., Khormah University College, Taif University, KSA.

2Botany & Microbiology Dept., Faculty of Science (Boys)، Al-Azhar University, Cairo, Egypt, P.N. 11884. Biology Dept., Khormah University College, Taif University, KSA.


Accepted 13 February, 2020



Lipases with unique substrate specificity are highly desired in biotechnological applications. Lipases from microbial sources have received heightened attention for an array of industrial applications, and these enzymes have been well exploited in the environmental sector as well. Thermostable lipases occupy a prominent position in aqueous and non-aqueous biocatalysis. The primary goals of this research work are to isolate and identify a lipase producing thermoalkaliphilic Bacillus stearothermophilus- KKSA12 species from oil polluted soil samples collected from Khormah Governorate, KSA. The other purposes of this study are production, partial purification, characterization of lipase activity. The optimum conditions for Bacillus stearothermophilus- KKSA12 hyperthermoalkalistable lipase with slaughter house wastes and tap water, pH value: 11.5; incubation period (hours): 24; incubation temperature (⁰C): 70 ⁰C; substrate concentration (%) (slaughter house wastes):1% ; inoculum size(ml):2ml; incubation conditions: Shaking; best carbon source: Sucrose; Without investigated nitrogen source (Control);Yeast extract concentration(%):1(%); Surfactant: Tween 20; water content : 1 gm slaughter house wastes and 20 ml medium); 100 ml Erlenmeyer flask capacity and 1.25 gm sloid wastes and ZnSO4 (100ml) The batch produced by New Brunswick USA Bioreactor 3000 by submerged fermentation method. The partially purified up to 35.66 % saturation using ammonium sulphate precipitation. One active peak for lipase obtained between 5 to 10 fractions and the fraction number 8 reached the highest specific activity up to 249.28 (U/mg. Protein/ml). Fifteen amino acids were detected. The purified lipase showed the maximum activity at 80⁰C, pH 9.5; at 1.5 % substrate concentration after 60 hours. Chlorine concentration resulted in decreasing the enzyme activity by different ratios depending on the time of enzyme exposure to chlorine. The purified lipase was stable towards strong anionic surfactants. Finally, Bacillus stearothermophilus- KKSA12hyperthermoalkalistable lipase candidate for widely used in food industry, detergent, paper, textile, leather and pharmaceutical industries because of their stability, selectively and substrate specificity for wider industrial applications.

Keywords:  Microbial enzymes, Bacterial lipase, Thermoalkaliphilic enzymes, Bio-detergent





Abol FDM, Bayoumi RA, Mohamed A (2016): Production of thermoalkaliphilic lipase from Geobacillusthermoleovorans DA2 and application in leather industry. Accepted for publication in 3 Dec.2015. in Enzyme Research Journal. Enzyme Res.2016.9034364. Published on line 2016 Jan 3.doi:10.1155/2016/9034364.

Aisaka K, Terada O (1980): Purification and properties of lipoprotein lipase from Rhizopusjaponiclls. Agricultural and Biological Chemistry 44, 799-805.

Al-Dhumri SA, Bayoumi RA (2019): Bacterial hyperthermostable alkaline lipase production by B. stearothermophilus isolated from oil polluted soil. Int. J. Adv. Res. Biol. Sci. (2019). 6(2): 166-184.

Ammar MS, Shash SM, El-Said TI, Abd-El Monem MO (1999): Environmental andnutritional parameterscontrolling the biosynthesis of thermostable alkaline protease (TAP) from Bacillus stearothermophilus, S-­WNl616 1B isolated from Wadi EI-Natroun Lakes in Egypt. (3rd Intern.) Sci. Confr. Fac. of Sci. Al-Azhar Univ., Cairo, Egypt. 22-25 March, (1999).

Annamalai N, Elayaraja S, Vijayalakshmi S, Balasubramanian T (2011): Thermostable alkaline tolerant lipase from Bacilluslicheniformis using peanut oil cake as a substrate. Afr. J. Biochem. Res. 5:176–181.

Barrow GI, Feltham RK (Eds.) (1993): Cowan & steel’s: Manual for the Identification of Medical Bacteria. 2nd ed. Cambridge Univ. Press. London.

Bayoumi RA, Atta HM,  El-Sehrawy MH (2012): Bioremediation of Khormah Slaughter House Wastes by Production of Thermoalkalistable Lipase for Application in Leather Industries. Life Science J.  9(4): 1324-1335.

Castro-Ochoa LD, Rodriguez-Gomez C, Valerio-Alfaro G, Ros RO (2005): Screening, purification and characterization of the thermoalkalophilic lipase produced by Bacillusthermoleovorans CCR (1998):Selective growth of a mutant in continuous culture of   B. caldolyticus   for production of α-amylase.Appl. Microbiol. Biotechnol. 30,125-132.

Chisti Y (1998): Strategies in downstream processing. In:Bio-separation and bioprocessing:a handbook, Vol. 2.  Subramanian G. (editor). pp. 3-30. New York: Wiley-VCH.

Choo DW, Kurihara T, Suzuki T, Soda K, Esaki N (1998): A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1 gene cloning and enzyme purification and characterization. Appl. Environ. Microbiol.64:486-491.PMCID: PMC106070.

Das A, Shivakumar S, Bhattachaya Shakaya S, Swathi S (2016): Purification and characterization of a surfactant-compatible lipase from Aspergillus tamari JG1F06 exhibiting energy-efficient removal of oil stains from polycotton fabric. 3 Biotech.6.131.

Dharmsthiti S, Luchai S (1999): Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027. FEMS Microbiology Letters, 179, 241-246. doi:10.1111/j.1574-6968. 1999.tb08734.x

Dixon M, Webb E (1964): Enzymes, 2nd Edition. Academic Press Inc. New York.

Dong H, Gao S, Han S, Cao S (1990): Purification and characterization of a Pseudomonas sp. Lipase and its properties in non-aqueous media. BiotechnologyApplicationBiochemistry. 30, 251.

El-Kasaby AH, Bayoumi RA, Sidkey NM, Soliman AM (2018): Production, Purification and Applications of Thermostable Slaughterhouse (SH), Fish (FW) and Poultry (PW) Wastes Protease(s) Under Solid State Fermentation (SSF) Conditions. Int. J. Adv. Res. Biol. Sci. (2018). 5(2): 108-132.

Ferreira-Dias Sandoval G, Francisco P, Valero F (2015): The potential use of lipases in the production of fatty acid derivatives for the food and nutraceutical industries. Electron. J. Biotechnol.16(2015).

Franken LPG, Marcon NS, Treichel H, Oliveira D, Freire DMG, Dariva C (2009): Effect of treatment with compressed propane on lipases hydrolytic activity. Food and Bioprocess Technology; 10:0087-0095.

Godfrey T, West S (1996): Introduction to industrial enzymology. In: Industrial enzymology. 2nd ed. Godfrey, T.; West, S. editors., pp. 1-8.  New York: Stockton Press.

Gokbulut AA, Alper (2013): Purification and biochemical characterization of an extracellular lipase from psychrotolerant Pseudomonasfluorescens KE38. Turkish J. Biol. 37, 538.

Gomori G (1955): Preparation of buffers for use in enzyme active studies. In: Methods in Enzymol. Vol 1. Colwick, S. P.; and Kaplan, N. O.(Eds.). Academicpress Inc. Pub. New York.

Grbavcic SZ, Dimitrijevic-Brankovic SI, Bezbradica DI, Siler-Marinkovic SS, Knezevi ZD (2007): Effect of fermentation conditions on lipase production by Candidautilis. J, the Serbian Chem. Society; 72(8-9), 757.

Gupta M, Mehra G, Gupta R (2004): A glycerol inducible thermostable lipase from Bacillus sp., Medium optimization by a placket Burman design and by responses surface methodology. Can.J. Microbiol. 50(5): 361-368.

Gupta N, Shai V, Gupta R (2007): Alkaline lipase from a novel strain Burkholderiamultivorans: Statistical medium optimization and production in a bioreactor. Process Biochemistry; 42(2), 518.

Gupta R, Rathi P, Gupta N. and Bradoo S (2003): Lipase assays for conventional and molecular screening; an overview. Biotechnol. Appl. Biochem 37(1): 63-71.

Gurung N, Ray S, Bose S, Rai V (2013): A broader view: Microbial enzymes and their relevance in industries, medicine, and beyond. Bio. Med. Res. Int., 329121.

Gururaj P, Ramalingam S, Devi GN, Gautam P (2016): Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07.Braz. J. Microbiol. 47(20):647-657.

Haalck I, Hedrich H, Hassink J (1992): Prog. Biotech 8:505-572.

Haalck L, Kallabis B, Schoemaker M, Cammann K, Spener F (1991): Lipases for   biosensors.Fat.Sci.Tech 93:415-416.

Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O (2005): Appl. Microbiol. Biotechnol. 68, 588.

Handelsman T, Shoham Y (1994): Production and characterization of an extracellular thermostable lipase from a thermophilic Bacillus sp. J. Gen. Appl. Microbiol., 40: 435-443.

Haq IU, Ashraf H, Iqbal J, Qadeer M (2003): Production of α-amylase by Bacillus licheniformis using an economical medium. Bioresource Technology. 87, 57-61.

Hasan F, Shah AA, Hameed A (2006): Enzyme Microb. Technol. 39, 235.

Hasan F, Shah AA, Hameed A (2014): Industrial applications of microbial lipases. Enzym. and Microb. Technol.39:235-251.

Hass MJ, Cichowicz OJ, Bailey DG (1992): Purification and characterization of an extracellular lipase from the fungus Rhizopusdelemar. Lipids 27, 571-576.

Hensyl WR (Ed) (1994): Bergy’s Manual of Determinative Bacteriology 9th edition, Williams  and Wilkins, Baltimore.

Hiol A, Jonzo M, Rugani N, Druct D, Sanla L (2000): Purification and characterization  of an extracellular lipase from a thermophilic Rhizopusoryza strain isolated from palm fruit. Enzyme Micrab. Technol. 26:421.

Houde A, Kademi A, Leblanc D (2004): Lipases and their industrial applications. Appl Biochem and Biotechnol., 118: 155-170.

Jaegar KE, Eggert T (2002): Lipases for biotechnology. Curr. Opin. Biotech. 13(4):390-397.

Jaeger KE, Dijkstra BW, Reetz MT (1999): Bacterial biocatalysts molecular biology, Three-Dimensional Structures, and Biotechnological applications of Lipases. Annual Review of Microbiology. 53, 315. 

Jaeger KE, Rettz TM (1998): Microbial lipases from versatile tools for biotechnology. Trends Biotechnol.16, 396-403.

Jansen AEM, Vaidya AM, Halling P (1996): Eng. Microb Tech 18:340-346.

Kademi A, Ait-Abdelkader N, Fakhreddine L, Baratti J (2000): Purification and characterization of a thermostable esterase from the moderate thennophile Bacillus circulans. Appl. Microbiol. Biotechnol. 54, 173-179.

Kambourova M, Kirilova N, Mandeva R, Derekova A (2003): Purification and properties of thermostable lipase from a Bacillus stearothermophilus MC 7. J. Molecular Catalysis B: Enzymatic 22, 307-313.

Kaur G, Singh A, Sharma R, Sharma V, Verma S, Sharma PK (2016): Cloning expression, purification and characterization of lipase from Bacilluslicheniformis, isolated from hot spring of Himachal Pradesh, India. 3 Biotech.6-49.

Kim HK, Park SY, Lee JK, Oh TK (1995): Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Bioscienc., Biotechnol.  Biochem. 62, 66-71.

Kumar A, Dhar K (2016): Lipase Catalysis in Organic Solvents: Advantages and Applications. Biol. Proced. Online 2016, 18(2). Available at: 459

Laemmli UK (1970):Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227,180-185.

Lawrence RC, Fryer TF, Reiter B (1967): A rapid method for the quantitative estimation of microbial lipases. Nature 21 (1967)1264.

Lee DW, Kim HW, Lee KW, Kim BC, Choe EA, Lee HS, Kim DS, Pyun YR (2001): Purification and characterization of two distinct thermostable lipases from the Gram-positive thermophilic bacterium Bacillusthermoleovorans ID-1. Enzyme and Microbial Technology. 29, 363-371.

Lee SY, Rhee JS (1993): Production and partial purification of a lipase from Pseudomonas putida 3SK. Enzyme Microb. Technol. 15, 617-24.

Leisola M, Jokela J, Pastinen O, Turunen O, Schoemaker H (2017): Encyclopedia of Life Support Systems (EOLSS). Physiology and maintenance. Vol. II. Industrial use of enzymes (UNESCO-EOLSS), available from: http://www.eolss. net/sample-chapters/ (Accessed: 4th February 2017).

Lima V, Krieger N, Mitchell D, Baratti J, De Filippis I, Fontana J (2004): Evaluation of the potential for use in bio-catalysis of a lipase from a wild strain of Bacillusmegaterium. J. Mol. Catal. and Biolo. Enzy.; 31: 5361.

Lin SF, Chiou CM, Yeh C, Tsai YC (1996): Purification and partial characterization of an alkaline lipase from Pseudomonaspseudoalcaligenes F-111. Appl. Environ. Microbiol; 62:1093–1095.

Mahmoud AI (2004): Biotechnological recycling of infectious hospital wastes for the sake of safe environment. M.Sc. Thesis. Bot.& Microbiol. Dept., Fac. of Sci. Al- AzharUniversity, Cairo, Egypt

Moreira KA, Albuquerque BF, Teixeira MF, Porto AL, Filho JL (2002): Application of protease from Nocardiopsis sp as a laundry detergent additive. W. J. Microbiol. Biotechnol. 18, 307-312.

Muthusamy A, Beslin LG (2018): Extracellular lipase production, purification and characterization using Bacillussubtilis in submerged state fermentation. Biomed. J. Sci, &Tech.Res.7641-2645.

Nahas E(1988): Control of lipase production by Rhizopusoligosporus under various growth conditions, J. Gen. Microbiol. 134(1):227-237.

Nawani N, Kaur I (2000): Purification, characterization and thermostability of a lipase from a thermophilic Bacillus sp.J33. Mol. Cell Bioch.206, 91-96.

Park H, Lee K, Chi Y, Jeong S (2005): Effects of methanol on the catalytic properties of porcine pancreatic lipase. Journal of Microbiology and Biotechnology; 15(2), 296.

Patil KJ, Chopda MZ, Mahajan RT (2011): Lipase biodiversity. Indian J Sci. Technol., 4(8): 971-982.

Pellet PI, Young VR (1980): Nutritional Evaluation of Protein Foods. The United Nation’s University Hunger Program. Food and Nutrition Bulletin, Suppl. 4, The United University, Tokyo.

Prakash D, Nawani N, Prakash M, Bodas M, Mandal A, Khetmalas M, Kapadnis B (2013): Biomed Res. Int. 2013, 264020.

Qamsari EM, Kermanshahi RK, Nejad ZM (2011): Isolation and identification of a novel, lipase producing bacterium, Pseudomonasaeruginosa KM110. Iranian Journal of Microbiology. 3, 92.

Rapp P, Backhaus S (1992): Formation of extracellular lipases by filamentous fungi, yeast and bacteria. Journal of Enzyme Microbiology and Technology. 14, 938.

Roushdy MM (2001): Application of modern fermentation biotechnology in the field of thermostable protease production. M.Sc. Thesis, Botany & Microbiology Dept. Faculty of Science, Al -Azhar University, Cairo, Egypt.

Saxena R, Davidson W, Sheoran A, Giri B (2003): Purification and characterization of an alkaline thermostable lipasefrom Aspergillus carneus. ProcessBiochemistry.39, 239-247.

Schmidt-Dannert C1, Sztajer HStöcklein W,  Menge U, Schmid RD (1994): Screening, purification and properties of a thermophilic lipase from Bacillusthermocatenulatus. Biochim  Biophys  Acta. 1994 Aug 25;1214(1):43-53.

Shaini S, Jayasree S (2016): Isolation and characterization of lipase producing bacteria from windrow compost. Int. J. Current. Microbiol. Appl. Sci. 5:926-933.

Sharma P, Sharma N, Pathania S, Handa S (2017): Purification and characterization of lipase by Bacillusmethylotrophicus PS3 under submerged fermentation and its application in detergent industry. Acad. Sienti. Res. & Technol, Egypt. J. Genet. Eng. & Biotechnol. (15): 369-377.

Sharma R, Chisti Y, Banerjee UC (2001): Production, purification, characterization, and applications of lipases.  Biotechnol. Advances.19, 627-662.

Sharma R, Soni SK, Vohra RM, Gupta LK, Gupta JK (2002): Purification and characterization of a thermostable alkaline lipase from a new thermophilic Bacillus sp. RSJ-1. ProcessBiochemistry. 37, 1075-1084.

Showell MS (1999): Enzymes, detergent. In: Encyclopedia of bioprocess technology: fermentation, bio-catalysis and bio-separation, vol 2. Flickinger, M.C.; Drew, S.W. (eds.) 958-971.Wiley, New York.

Singh J, Vollra RM, Sahoo DK (1999): Alkaline protease from a new obligate alkalophilic isolate of Bacillus sphaericus. Biotech. Lett. 21, 921­-924.

Singh R, Kumar M, Anshumali Mittal A,  Mehta PK (2016): Microbial enzymes: industrial progress in 21st centur (Review Article).3 Biotech 6:174.

Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds., 1986): Bergey’s Manual of Systematic Bacteriology, 1st ed., vol. 2, Williams & Wilkins, Baltimore.

Veerapagu M, Narayanan AS, Ponmurugan K, Jeya KR (2013): Screening, selection , identification , production and optimization of bacteria lipase from oil spilled. Asian J Pharm Clin Res, Vol 6, Suppl 3, 2013, 62-67.

Wang Y, Srivastava K, Shen G, Wang H (1995): Thermostable alkaline lipase from a newly isolated thermophilic Bacillus. Strain A30-1 (ATCC 53841). J. Ferment. Bioeng. 79, 433-438.

Wilson K, Walker J (1994): Practical biochemistry, Principles and Techniques. Fourth Edition, Cambridge University Press. pp. 182-191.

Winkler UK, Stuckmann M (1979): Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratiamarcescens. J. Bacteriol., 138, 663-670.

Yamada K, Ota U, Machida H (1962): Studies on the production of lipase by microorganisms II, Quantitative determination of lipase. Agric. Biol. Chem.26(69), http//,10,127.

Zhu K, Jutila A, Tuominen E, Patkar S, Svendsen A, Kinnunen P (2001): Impact of the tryptophan residues of Himicola lanuginosa lipase on its thermal stability. J. Biochim. Biophys. Acta. 1547:329-338

Zouaoui B, Bouziane A (2012): Production, optimization and characterization of the lipase from Pseudomonasaeruginosa. Romanian Biotechnological Letters. 17, 71-87.




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