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Kestane Tohumundan (Castanea sativa) Lipaz Enziminin Saflaştırılması ve Kinetik Özelliklerinin İncelenmesi

Year 2018, Volume: 3 Issue: 3, 101 - 106, 18.12.2018

Bahar Bilgin Sökmen Rıdvan İlgün

https://doi.org/10.35229/jaes.416800

Abstract

Bu çalışmada, ülkemizde Giresun İli ve çevresinde bol miktarda yetişen kestane (Castanea Sativa) tohumlarından lipaz enzimi ilk defa saflaştırıldı ve kinetik özellikleri incelendi. Petrol eteri ile yağı uzaklaştırılmış kestane tohumları 0,06 M sodyum fosfat tamponu (pH=7,0) ile homojenize edildi. Lipaz esteraz aktivitesi gösteren homojenizatın % 90’lık (NH4)2SO4 kesiti elde edildi. Dializden sonra bu kesit sırasıyla hidroksilapatit ve DEAE-selüloz kolonlara uygulanarak lipaz enzimi 186,87 kez saflaştırıldı. SDS-PAGE elektroforezi sonucunda saflaştırılan enzimin molekül ağırlığının 30 kDa olduğu bulundu. Kestane tohumlarından saflaştırılan lipazın optimum pH’sının 9,0; optimum sıcaklığının 30 °C olduğu bulundu. Enzimin çeşitli substratlara karşı ilgisi incelendiğinde, lipazın ilgisinin en çok p-NFM’a olduğu ve Km ve Vmax değerlerinin sırasıyla 1,017 mM ve 163,874 U olduğu saptandı. 

Keywords

Kestane (Castanea sativa) tohumu lipaz karakterizasyon saflaştırma

References

  • Abdou, M. A. 2003. Purification and partial characterization of psychrotrophiz Serratia marcescens lipase. Journal of Dairy Science, 86, 127-132.
  • Beisson, F., Gardies, A. M., Teissere, M., Ferte, N., Noat, G. 1997. An esterase neo synthesized in post-germinated sunflower seeds is related to a new family of lipolytic enzymes. Plant Physiology and Biochemistry, 35, 761-765.
  • Bhardwaj, K., Raju, A., Rajasekharan, R. 2001. Identification, purification, and characterization of a thermally stable lipase from rice bran. a new member of the (phospho) lipase family. Plant Physiology, 127, 1728-1738.
  • Bilgin Sökmen, B. 2005. Kayısı (Armeniaca vulgaris Lam.) tohumlarından lipazın saflaştırılması ve çeşitli taşıyıcılara immobilize edilmesi. Doktora Tezi, İstanbul Üniversitesi, Fen Bilimleri Enstitüsü, İstanbul, Türkiye, 137s,
  • Dharmsthiti, S., Ammaranond, P. 1997. Purification and characterization of lipase from a raw-milkyeast (Trichosporon asteroides). Applied Biochemistry and Biotechnology, 26, 111-116.
  • Erlanson, C. 1970. p-Nitrophenylacetate as a substratefor a carboxyl-ester hydrolase in pancreatic juice and ıntestinal content. Scandinavian Journal of Gastroenterology, 5, 333-336.
  • Gupta, R., Gupta, N., Rathi, P. 2004. Bacterial lipases: An overview of production, purification and biochemical properties. Applied Microbial Biotechnology, 64, 763-781.
  • Haas, M. J., Cichowicz, D. J., Dierov, J. K. 2001. lipolytic activity of californica-laurel (Umbellularia californica) seeds. Journal of American Oil Chemist’s, 78, 1067-1071.
  • Hasan, F., Aamer, A. S., Hameed, A. 2006. Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251.
  • Laemmli, U. K. 1970. Cleavage of structural proteins during the. aassembly of the head of bacteriofage T4. Nature, 227, 680-685.
  • Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
  • Namboodiri, V. M. H., Chattopadhyay, A. 2000. Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger. Lipids, 35, 495-502.
  • Paiva, A. L., Balcao, V. M., Malcata F. X. 2000. Kinetics and mechanisms of reactions catalyzed by immobilized lipases. Enzyme and Microbial Technology, 27, 187-204.
  • Prabhu, V. A.,Tambe, S. P., Gandhi, N. N., Sawant, S. B., Joshi, J. B. 1999. Rice bran lipase: extraction, activity and stability. Biotechnology Progress, 15, 1083-1089.
  • Sanz, L. C., Olais, J. M. 1990. Characterization of lupin seed lipase. Food Chemistry, 37, 221-228.
  • Sharma, R., Chisti, Y., Banerjee, U. C. (2001). Research Review Paper: Production, purification, characterization, and applications of lipases. Biotechnology Advances, 19, 627-662.
  • Staubmann, R., Ncube, I., Gubitz, G. M, Steiner, W., Read, J. S. 1999. Esterase and lipase activity in Jatrophacurcas L. seeds. Journal of Biotechnology, 75, 117-126.
  • Subaşı, B. 2004. Kestane Sektör Profili, İstanbul Ticaret Odası Etüt ve Araştırma Şubesi, İstanbul.
  • Şengel, B. 2007. Deterjan katki maddesi olarak mikrobiyal kaynakli lipaz üretim koşullari. Yüksek Lisans Tezi, Ankara Üniversitesi Fen Bilimleri Enstitüsü, Ankara, Türkiye, 174s.
  • Ünlü, A. E. 2004. Candida Rugosa Lipazının özellikleri ve enantiyo seçimliliğinin arttırılması. Seminer. Ankara Üniversitesi, Kimya Mühendisliği Anabilim Dalı, Ankara. Türkiye.
  • Vorderwulbwcke, T., Kieslich, K., Erdmann, H., 1992. Comparison of lipases by different assays. Enzyme and Microbial Technology, 14, 631-639.
  • Yeşiloğlu, Y., Başkurt, L. 2008. Partial purifacation and characterization of almond seed lipase. Preparative Biochemistry Biotechnology, 38, 397-410.
  • Warburg, O., Christian, W. 1941. Isolierungund kristallinsation des garungsferments enolase. Biochemische Zeitschrift, 310, 384-421.
  • Winkler, U. K., Stuckmann, M., 1979. Glycogen, hyaluronate, and some other polysaccaharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138, 663-670.

Lipase Purification from the Seeds of chestnut (Castanea sativa) and Its Investigation of Some Kinetic Properties

Year 2018, Volume: 3 Issue: 3, 101 - 106, 18.12.2018

Bahar Bilgin Sökmen Rıdvan İlgün

https://doi.org/10.35229/jaes.416800

Abstract



In this study, lipase was firstly purified from chestnut (Castanea sativa) seeds, being abundant in Giresun (Turkey) and surroundings and the kinetic properties of the enzyme were investigated. Chestnut seeds, being fat-free with petroleum ether extraction, were homogenized with 0.06 M sodium phosphate buffer (pH=7.0) 90% of (NH4)2SO4 fraction of lipase esterase activity homogenate was obtained. After dialysis, this fraction was applied respectively to hydroxyapatite and DEAE-cellulose columns, and lipase was purified 186.87 times. The
purified enzyme, which showed SDS-PAGE, had a molecular weight of 30 kDa. It was found that the lipase purified from chestnut seeds had an optimum pH value of 9.0 and an optimum temperature of 30C. When affinity against various substrates of the enzyme is investigated, lipase affinite was found that have to p-NPM and its Km and Vmax values were dedected 1.017 mM and 163.874 U, respectively.

Keywords

Kestane (Castanea sativa) tohumu lipaz karakterizasyon saflaştırma

References

  • Abdou, M. A. 2003. Purification and partial characterization of psychrotrophiz Serratia marcescens lipase. Journal of Dairy Science, 86, 127-132.
  • Beisson, F., Gardies, A. M., Teissere, M., Ferte, N., Noat, G. 1997. An esterase neo synthesized in post-germinated sunflower seeds is related to a new family of lipolytic enzymes. Plant Physiology and Biochemistry, 35, 761-765.
  • Bhardwaj, K., Raju, A., Rajasekharan, R. 2001. Identification, purification, and characterization of a thermally stable lipase from rice bran. a new member of the (phospho) lipase family. Plant Physiology, 127, 1728-1738.
  • Bilgin Sökmen, B. 2005. Kayısı (Armeniaca vulgaris Lam.) tohumlarından lipazın saflaştırılması ve çeşitli taşıyıcılara immobilize edilmesi. Doktora Tezi, İstanbul Üniversitesi, Fen Bilimleri Enstitüsü, İstanbul, Türkiye, 137s,
  • Dharmsthiti, S., Ammaranond, P. 1997. Purification and characterization of lipase from a raw-milkyeast (Trichosporon asteroides). Applied Biochemistry and Biotechnology, 26, 111-116.
  • Erlanson, C. 1970. p-Nitrophenylacetate as a substratefor a carboxyl-ester hydrolase in pancreatic juice and ıntestinal content. Scandinavian Journal of Gastroenterology, 5, 333-336.
  • Gupta, R., Gupta, N., Rathi, P. 2004. Bacterial lipases: An overview of production, purification and biochemical properties. Applied Microbial Biotechnology, 64, 763-781.
  • Haas, M. J., Cichowicz, D. J., Dierov, J. K. 2001. lipolytic activity of californica-laurel (Umbellularia californica) seeds. Journal of American Oil Chemist’s, 78, 1067-1071.
  • Hasan, F., Aamer, A. S., Hameed, A. 2006. Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251.
  • Laemmli, U. K. 1970. Cleavage of structural proteins during the. aassembly of the head of bacteriofage T4. Nature, 227, 680-685.
  • Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
  • Namboodiri, V. M. H., Chattopadhyay, A. 2000. Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger. Lipids, 35, 495-502.
  • Paiva, A. L., Balcao, V. M., Malcata F. X. 2000. Kinetics and mechanisms of reactions catalyzed by immobilized lipases. Enzyme and Microbial Technology, 27, 187-204.
  • Prabhu, V. A.,Tambe, S. P., Gandhi, N. N., Sawant, S. B., Joshi, J. B. 1999. Rice bran lipase: extraction, activity and stability. Biotechnology Progress, 15, 1083-1089.
  • Sanz, L. C., Olais, J. M. 1990. Characterization of lupin seed lipase. Food Chemistry, 37, 221-228.
  • Sharma, R., Chisti, Y., Banerjee, U. C. (2001). Research Review Paper: Production, purification, characterization, and applications of lipases. Biotechnology Advances, 19, 627-662.
  • Staubmann, R., Ncube, I., Gubitz, G. M, Steiner, W., Read, J. S. 1999. Esterase and lipase activity in Jatrophacurcas L. seeds. Journal of Biotechnology, 75, 117-126.
  • Subaşı, B. 2004. Kestane Sektör Profili, İstanbul Ticaret Odası Etüt ve Araştırma Şubesi, İstanbul.
  • Şengel, B. 2007. Deterjan katki maddesi olarak mikrobiyal kaynakli lipaz üretim koşullari. Yüksek Lisans Tezi, Ankara Üniversitesi Fen Bilimleri Enstitüsü, Ankara, Türkiye, 174s.
  • Ünlü, A. E. 2004. Candida Rugosa Lipazının özellikleri ve enantiyo seçimliliğinin arttırılması. Seminer. Ankara Üniversitesi, Kimya Mühendisliği Anabilim Dalı, Ankara. Türkiye.
  • Vorderwulbwcke, T., Kieslich, K., Erdmann, H., 1992. Comparison of lipases by different assays. Enzyme and Microbial Technology, 14, 631-639.
  • Yeşiloğlu, Y., Başkurt, L. 2008. Partial purifacation and characterization of almond seed lipase. Preparative Biochemistry Biotechnology, 38, 397-410.
  • Warburg, O., Christian, W. 1941. Isolierungund kristallinsation des garungsferments enolase. Biochemische Zeitschrift, 310, 384-421.
  • Winkler, U. K., Stuckmann, M., 1979. Glycogen, hyaluronate, and some other polysaccaharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138, 663-670.
There are 24 citations in total.

Details

Primary Language Turkish
Journal Section Articles
Authors

Bahar Bilgin Sökmen 0000-0003-3904-8178

Rıdvan İlgün This is me 0000-0002-8613-0353

Publication Date December 18, 2018
Submission Date April 19, 2018
Acceptance Date June 12, 2018
Published in Issue Year 2018 Volume: 3 Issue: 3

Cite

APA Bilgin Sökmen, B., & İlgün, R. (2018). Kestane Tohumundan (Castanea sativa) Lipaz Enziminin Saflaştırılması ve Kinetik Özelliklerinin İncelenmesi. Journal of Anatolian Environmental and Animal Sciences, 3(3), 101-106. https://doi.org/10.35229/jaes.416800
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