Partial purification, immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus
Author(s)
Ana Lúcia Ferrarezi*,
Daniele H. Pivetta,
Gustavo Orlando Bonilla-Rodriguez,
Roberto da Silva,
José Manuel Guisan,
Eleni Gomes,
Benevides Costa Pessela*
Affiliation(s)
Department of Biology, Laboratory of Applied Biochemistry and Microbiology, IBILCE-UNESP, S?o José do Rio Preto SP, Brazil . Department of Chemistry and Environmental Sciences, IBILCE-UNESP, S?o José do Rio Preto SP, Brazil . Department of Enzyme Biocatalysis, Institute of Catalysis and Petrochemistry, CSIC, Campus UAM, Cantoblanco, Madrid, Spain. Department of Biotechnology and Food Microbiology, Research Institute for Food Science, CIAL-CSIC, CalleNicolás Cabrera 9, Campus UAM, Madrid, Spain.
Department of Biology, Laboratory of Applied Biochemistry and Microbiology, IBILCE-UNESP, S?o José do Rio Preto SP, Brazil . Department of Chemistry and Environmental Sciences, IBILCE-UNESP, S?o José do Rio Preto SP, Brazil . Department of Enzyme Biocatalysis, Institute of Catalysis and Petrochemistry, CSIC, Campus UAM, Cantoblanco, Madrid, Spain. Department of Biotechnology and Food Microbiology, Research Institute for Food Science, CIAL-CSIC, CalleNicolás Cabrera 9, Campus UAM, Madrid, Spain.
ABSTRACT
Lipases have important applications in biotechnological processes, motivating us to produce, purify, immobilize and perform a biochemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purification by gel filtration chromatography in Se-phacryl S-100 allowed obtaining a yield of about 85% and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in its properties when compared to crude extract. The crude enzyme extract has an optimum pH and temperature of 7.5 ° C and 40 ° C, respectively. After purification, a shift of the optimum pH from 7 to 8 was observed, as well as a rise in optimumtemperature to 60 ° C and an increase in stability. The enzyme was immobilized on CNBr-Agarose and Octyl-Agarose supports, having the highest immobilization yield of 94% in the second resin. The major advantage of immobilization in hydrophobic media such as Octyl is in its hyper activation, which in this case was over 200%, a very interesting finding. Another advantage of this type of immobilization is the possibility of using the derivatives in biotechnological applications, such as in oil enriched with omega-3 as the results obtained in this study display the hydrolysis of 40% EPA and 7% DHA from sardine oil, promising results compared to the literature.
Lipases have important applications in biotechnological processes, motivating us to produce, purify, immobilize and perform a biochemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purification by gel filtration chromatography in Se-phacryl S-100 allowed obtaining a yield of about 85% and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in its properties when compared to crude extract. The crude enzyme extract has an optimum pH and temperature of 7.5 ° C and 40 ° C, respectively. After purification, a shift of the optimum pH from 7 to 8 was observed, as well as a rise in optimumtemperature to 60 ° C and an increase in stability. The enzyme was immobilized on CNBr-Agarose and Octyl-Agarose supports, having the highest immobilization yield of 94% in the second resin. The major advantage of immobilization in hydrophobic media such as Octyl is in its hyper activation, which in this case was over 200%, a very interesting finding. Another advantage of this type of immobilization is the possibility of using the derivatives in biotechnological applications, such as in oil enriched with omega-3 as the results obtained in this study display the hydrolysis of 40% EPA and 7% DHA from sardine oil, promising results compared to the literature.
Cite this paper
Ferrarezi, A. , Pivetta, D. , Bonilla-Rodriguez, G. , Silva, R. , Guisan, J. , Gomes, E. and Pessela, B. (2013) Partial purification, immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus. Advances in Enzyme Research, 1, 79-90. doi: 10.4236/aer.2013.14009.
Ferrarezi, A. , Pivetta, D. , Bonilla-Rodriguez, G. , Silva, R. , Guisan, J. , Gomes, E. and Pessela, B. (2013) Partial purification, immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus. Advances in Enzyme Research, 1, 79-90. doi: 10.4236/aer.2013.14009.
References
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Journal of Agricultural Food Chemistry, 56, 10445-10451. http://dx.doi.org/10.1021/jf801928e [5] Berger, M., Laumen, K. and Schneider, M.P. (1992) Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sndiacylglycerols. Journal of the American Oil Chemists’ Society, 69, 955- 960. [6] Chulalaksananukul, W., Condoret, J.-S. and Combes, D. (1993) Geranyl acetate synthesis by lipase-catalyzed- transesterification in supercritical carbon dioxide. Enzyme and Microbial Technology, 15, 691-698. http://dx.doi.org/10.1016/0141-0229(93)90071-9 [7] SzczesnaAntczak, M., Kubiak, A., Antczak, T. and Bielecki, S. (2009) Enzymatic biodiesel synthesis—Key factors affecting efficiency of the process. Renewable Energy, 34, 1185-1194. http://dx.doi.org/10.1016/j.renene.2008.11.013 [8] Holm, H.C. and Cowan, D. (2008) The evolution of enzymatic inter-esterification in the oils and fats industry. 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[1] Domi?nguez De Mari?a, P., Carboni-Oerlemans, C., Tuin, B., Bargeman, G., Van Der Meer, A. and Van Gemert, R. (2005) Biotechnological applications of Candida antarctica lipase A: State-of-the-art. Journal of Molecular Catalysis B: Enzymatic, 37, 36-46. [2] Jaeger, K.E. and Reetz, M.T. (1998) Microbial lipases form versatile tools for biotechnology. Trends in Biotech- nology, 16, 396-403. http://dx.doi.org/10.1016/S0167-7799(98)01195-0 [3] Anderson, E.M., Larsson, K.M. and Kirk, O. (1998) One biocatalyst—Many applications: The use of Candida antarctica Blipase in organic synthesis. Biocatalysis and Biotransformation, 16, 181-204. http://dx.doi.org/10.3109/10242429809003198 [4] Akoh, C.C., Chang, S.-W., Lee, G.-C. and Shaw, J.-F. (2008) Bio-catalysis for the production of industrial products and functional foods from rice and other agricultural produce. Journal of Agricultural Food Chemistry, 56, 10445-10451. http://dx.doi.org/10.1021/jf801928e [5] Berger, M., Laumen, K. and Schneider, M.P. (1992) Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sndiacylglycerols. Journal of the American Oil Chemists’ Society, 69, 955- 960. [6] Chulalaksananukul, W., Condoret, J.-S. and Combes, D. (1993) Geranyl acetate synthesis by lipase-catalyzed- transesterification in supercritical carbon dioxide. Enzyme and Microbial Technology, 15, 691-698. http://dx.doi.org/10.1016/0141-0229(93)90071-9 [7] SzczesnaAntczak, M., Kubiak, A., Antczak, T. and Bielecki, S. (2009) Enzymatic biodiesel synthesis—Key factors affecting efficiency of the process. Renewable Energy, 34, 1185-1194. http://dx.doi.org/10.1016/j.renene.2008.11.013 [8] Holm, H.C. and Cowan, D. (2008) The evolution of enzymatic inter-esterification in the oils and fats industry. European Journal of Lipid Science and Technology, 110, 679-691. http://dx.doi.org/10.1002/ejlt.200800100 [9] Brenna, E., Fuganti, C. and Serra, S. (2008) Applications of biocatalysis in fragrance chemistry: The enantiomers of α-, β-, and γ-irones. Chemical Society Reviews, 37, 2443-2451. http://dx.doi.org/10.1039/b801557k [10] Woodley, J.M. (2008) New opportunities for biocatalysis: Making pharmaceutical processes greener. Trends in Biotechnology, 26, 321-327. http://dx.doi.org/10.1016/j.tibtech.2008.03.004 [11] Iyer, P.V. and Ananthanarayan, L. (2008) Enzyme stability and stabili-zation—Aqueous and non-aqueous environment. Process Biochemistry, 43, 1019-1032. http://dx.doi.org/10.1016/j.procbio.2008.06.004 [12] O?’Fa?ga?in, C. (2003) Enzyme stabilization—Recent experimental progress. Enzyme and Microbial Technology, 33, 137-149. [13] Shaw, A. and Bott, R. (1996) Engineering enzymes for stability. Current Opinion in Structural Biology, 6, 546-550. http://dx.doi.org/10.1016/S0959-440X(96)80122-9 [14] Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M. and Fernandez-Lafuente, R. (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 40, 1451-1463. http://dx.doi.org/10.1016/j.enzmictec.2007.01.018 [15] Illanes, A. (1999) Stability of biocatalysts. Electronic Journal of Biotechnology, 2, 15-30. http://dx.doi.org/10.2225/vol2-issue1-fulltext-2 [16] Gianfreda, L. and Scarfi, M.R. (1991) Enzyme stabilization: State of the art. Molecular and Cellular Biochemistry, 100, 97-128. http://dx.doi.org/10.1007/BF00234161 [17] Katchalski-Katzir, E. (1993) Immobilized enzymes—Learning from past successes and failures. Trends in Biotechnology, 11, 471-478. http://dx.doi.org/10.1016/0167-7799(93)90080-S [18] Hartmeier, W. (1985) Immobilized biocatalysts—From simple to complex systems. Trends in Biotechnology, 3, 149-153. http://dx.doi.org/10.1016/0167-7799(85)90104-0 [19] Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E., Dodson, G., Tolley, S., Turkenburg, J.P., Christiansen, L., Huge-Jensen, B., Norskov, L., Thim, L. and Menge, U. (1990) A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature, 343, 767-770. http://dx.doi.org/10.1038/343767a0 [20] Brzozowski, A.M., Derewenda, U., Derewenda, Z.S., Dodson, G.G., Lawson, D.M., Turkenburg, J.P., Bjorkling, F., Huge-Jensen, B., Patkar, S.A. and Thim, L. (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature, 351, 491-494. http://dx.doi.org/10.1038/351491a0 [21] Derewenda, Z.S., Derewenda, U. and Dodson, G.G. (1992) The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 ? resolution. Journal of Molecular Biology, 227, 818-839. http://dx.doi.org/10.1016/0022-2836(92)90225-9 [22] Schmid, R.D. and Verger, R. (1998) Lipases: Interfacial enzymes with attractive applications. Angewandte Chemie International Edition, 37, 1609-1633. http://dx.doi.org/10.1002/(SICI)1521-3773(19980703)37:12<1608::AID-ANIE1608>3.0.CO;2-V [23] Verger, R. (1997) “Interfacial activation” of lipases: Facts and artifacts. Trends in Biotechnology, 15, 32-38. http://dx.doi.org/10.1016/S0167-7799(96)10064-0 [24] Pessela, B.C.C., Munilla, R., Betancor, L., Fuentes, M., Carrascosa, A.V., Vian, A., Fernandez-Lafuente, R. and Guisa?n, J.M. (2004) Ion exchange using poorly activated supports, an easy way for purification of large proteins. Journal of Chromatgraphy A, 1034, 155-159. [25] Fuentes, M., Mateo, C., Pessela, B.C.C., Batalla, P., Fernandez-Lafuente, R. and Guisa?n, J.M. (2007) Solid phase proteomics: Dramatic reinforcement of very weak protein-protein interactions. Journal of Chromatography B, 849, 243-250. [26] Kumar, A., Galaev, I.Yu. and Mattiasson, B. (2000) Polymer displacement/shielding in protein chromatography. Journal of Chromatography B, 74, 103-113. http://dx.doi.org/10.1016/S0378-4347(00)00089-X [27] Mateo, C., Abian, O., Fernandez-Lafuente, R. and Guisan, J.M. (2000) Reversible enzyme immobilization via a very strong and non-distorting ionic adsorption on support-polyethylenimine composites. Biotechnology and Bioengineering, 68, 98-105. http://dx.doi.org/10.1002/(SICI)1097-0290(20000405)68:1<98::AID-BIT12>3.0.CO;2-T [28] Bastida, A., Sabuquillo, P., Armisen, P., Ferna?ndez-Lafuente, R., Huguet, J. and Guisa?n, J.M. (1998) A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnology and Bioengineering, 58, 486- 493. [29] Fernandez-Lafuente, R., Armise?n, P., Sabuquillo, P., Ferna?ndez-Lorente, G. and Guisa?n, J.M. 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