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Received August 23, 2023
Accepted August 23, 2023
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Immobilization and stabilization of Pseudomonas aeruginosa SRT9 lipase on tri(4-formyl phenoxy) cyanurate
School of Life Sciences, Biotechnology Research Laboratory, Swami Ramanand Teerth Marathwada University, Nanded 431 606, India 1Department of Chemistry, University College of Science, Osmania University Campus, Hyderabad 500 007, India 2Department of Chemistry, N.S.B. College, Nanded 431 601, India
Korean Journal of Chemical Engineering, March 2011, 28(3), 867-874(8), 10.1007/s11814-010-0431-0
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Abstract
Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 ℃ to 70 ℃. The immobilized lipase showed 20-25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ~60% when incubated with 30% butanol. The kinetic studies revealed increase in KM value from 0.043mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the Vmax of immobilized enzyme (142.8 μmol min.1 mg.1), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 ℃ in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.
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Stocklein W, Sjtajer H, Menge U, Schmid RD, Biochim. Biophys Acta., 1168, 181 (1993)
Jaeger KE, Liebeton K, Zonta A, Schimossek K, Reetz MT, Appl. Microbiol. Biotechnol., 46(2), 99 (1996)
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Claon PA, Akoh CC, Enzyme Microb. Technol., 16(10), 835 (1994)
Bertinotti A, Carrea G, Ottolina G, Riva S, Tetrahedron., 50, 13165 (1994)
Frykman H, Ohrner N, Norin T, Hult K, Tetrahedron. Lett., 34, 1367 (1993)
Yahya ARM, Anderson WA, Moo-Young M, Enzyme Microb. Technol., 23(7-8), 438 (1998)
Margolin AL, Enzyme Microb. Technol., 15, 266 (1993)
Azerad R, Bull. Soc. Chim. Fr., 132, 17 (1995)
Colowick SP, Kaplan NO, Mosbach K, Eds., Methods in Enzymology vol 137, Academic Press Inc. (1988)
Yamane T, Funada T, Ishida S, J. Ferment. Technol., 60, 517 (1982)
Lavayre J, Baratti J, Biotechnol. Bioeng., 24, 1007 (1982)
Otero C, Guisan JM, Ballesteros A, Appl. Biochem. Biotechnol., 19, 163 (1988)
Shaw JF, Chang RC, Wang FF, Wang YJ, Biotechnol. Bioeng., 35, 132 (1990)
Goldman R, Kadam O, Silman IH, Caplan SR, Katchalski E, J. Biochemistry., 64, 486 (1968)
Moreno JM, Sinisterra JV, J. Mol. Catal., 93, 357 (1994)
Stark MB, Holmberg K, Biotechnol. Bioeng., 34, 942 (1989)
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Deutscher MP, Guide to protein purification: Method Enzymol, Academic Press, New York, 182 (1990)
Rao RS, Borkar PS, Khobragade CN, Sagar AD, Enzyme Microb. Technol., 39(4), 958 (2006)
Kavitake BP, Patil VP, Salunkhe MM, Bull. Soc. Chim.Belg., 104, 675 (1995)
Stahl E and Kalten BU, Thinlayer chromatography a laboratory hand book, Academic Press, NewYork (1995)
Gacche RN, Ghole VS, Khobragade CN, Sagar AD, J. Sci. Ind. Res., 61, 621 (2002)
Soares CMF, de Castro HF, Moraes FF, Zanin GM, Appl. Biochem. Biotechnol., 79, 745 (1999)
Kordel M, Hofmann B, Schomburg D, J. Bacteriol., 173, 4836 (1991)
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, Nucleic Acids Research., 25, 3389 (2007)
Copeland RA, Enzymes: a practical introduction to structure, mechanism and data analysis, New York, VCH Publisher INC, New York (1996)
Yang BK, Chen JP, J. Food Sci., 2, 424 (1994)
Gupta MN, Thermostability of enzymes. New Delhi, India: Narosa Publishing House (1993)
Martinek K, Klibanov AM, Goldmacher VS, Berezen IV, BiochimBiophys., 485, 1 (1997)
Klibanov AM, Science., 219, 722 (1983)
Bastida A, Sabuquillo P, Armisen P, Fernandez-Lafuente R, Huguet J, Guisan JM, Biotechnol. Bioeng., 58(5), 486 (1998)
Knezevic Z, Mojovic L, Adnadjevic B, Enzyme Microb. Technol., 22(4), 275 (1998)
Laane C, Boeren S, Vos K, Veeger C, Biotechnol. Bioeng., 30, 81 (1986)
Gorman LA, Dordick JS, Biotechnol. Bioeng., 39, 392 (1992)
Wu JC, Zhang GF, He ZM, Biotechnol. Lett., 23(3), 211 (2001)
Yang D, Rhee SS, Biotechnol. Bioeng., 40, 478 (1992)
