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Lipase Enzymes Purification from the Chamomile (Matricaria chamomilla L.) and Its Immobilization on Various Supports

Yıl 2018, Cilt: 8 Sayı: 1, 131 - 143, 18.06.2018
https://doi.org/10.31466/kfbd.396563

Öz

In this study, lipase enzyme was firstly purified from chamomile (Matricaria chamomilla L.) which is abundant in
Giresun (Turkey) and surroundings and the kinetic properties of the enzyme were investigated. 70 % of (NH4)2SO4
fraction of lipase esterase activity homogenate was obtained. After dialysis, this fraction was applied to DEAE-cellulose
column, and lipase was purified 26 times. In the purification process, the protein amount was determined by using
Lowry and E280/E260 Warburg methods while lipase esterase activtiy was assayed with Erlanson’s method. It was found
that the purified enzyme, which obtained with SDS-PAGE electroforesis, had a molecular weight of 30 kDa. The
optimum pH and temperature values were determined. It was found that the lipase purified from chamomile had an
optimum pH value of 9 and an optimum temperature of 50 °C. When the affinity against different substrates of the
enzyme was examined, the highest value was determined in p-nitrophenyl palmitate, with respectively Km and Vmax
values being 0.2899 mM and 144.93 Units. Also, the lipase enzyme purified from chamomile was immobilized on
various carriers by adsorption, covalent and ionic binding methods. The kinetic properties of the immobilized
chamomile lipase such as optimum pH, optimum temperature were also studied.

Kaynakça

  • Abdou, M. A. (2003). Purification and partial characterization of psychrotrophiz Serratia marcescens lipase. Journal of Dairy Science, 86, 127-132.
  • Abigor, D. R., Opute, F. I., Opoku, A. R., Osagie, A. U.(1985). Partial purification and some properties of the lipase present in oil palm (Elaeis guineensis) mesocarp. Journal of the Science of Food and Agriculture, 36, 599-606.
  • Baytop A., 1996, Farmasötik Botanik Ders Kitabı, İstanbul Üniversitesi Eczacılık Fakültesi. Üniversite Yayın No:3637, Eczacılık Fakültesi Yayın No:58, 315s.
  • Beisson, F., Gardies, A. M., Teissere, M., Ferte, N., Noat, G. (1997). An esterase neosynthesized 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.
  • Dong, H., Gao, S., Han, S., Cao, S. (1999). Purification and characterization of a Pseudomonas sp. lipase and its properties in non-aqueous media. Biotechnology and Applied Biochemistry, 30, 251-256.
  • Erlanson, C. (1970). p-Nitrophenyl acetate as a substrate for a carboxyl-ester hydrolase in pancreatic juice and intestinal content. Scandinavian Journal of Gastroenterology, 5, 333-336.
  • Fuciños, P., Abadin, C. M., Sanromán, A., Longo, M. A., Pastrana, L., Rúa, M. L. (2005). Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: Partial purification and preliminary biochemical characterization. Journal of Biotechnology, 117, 233-241.
  • Hadi, M. R. H. S., Darz, M. T., Ghandehari, Z., Riazi, G. (2011). Effects of vermicompost and amino acids on the flower yield and essential oil production from Matricaria chamomile L. Journal of Medicinal Plants Research, 5(23), 5611-5617.
  • Haas, M. J., Cichowicz, D. J., Dierov, J. K. (2001). Lipolytic activity of Californica-laurel (Umbellularia Californica) seeds. Journal of American Oil Chemist’s Society, 78, 1067-1071.
  • Hiol, A., Jonzo, M. D., Rugani, N., Druet, D., Sarda, L., Comeau, L. C. (2000). Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme and Microbial Technology, 26, 421-430.
  • Hong, M. C., Chang, M. C. (1988). Purification and characterization of an alkaline lipase from a newly isolated Acinetobacter radioresistens CMC-1. Biotechnology Letters, 20, pp. 1027-1029.
  • İlgün, R. (2016). Kestane tohumundan (Castanea sativa) lipaz enziminin saflaştırılması ve kinetik özelliklerinin incelenmesi, Yüksek Lisans Tezi, Giresun Üniversitesi, Fen Bilimleri Enstitüsü, Giresun.
  • Kılınç, A. (1990). Alginatta immobilize edilmiş lipazın bazı özelliklerinin incelenmesi, Yüksek Lisans Tezi, Ege Üniversitesi, Fen Bilimleri Enstitüsü, İzmir.
  • Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly 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.
  • Mohamed, M. A., Mohamed, T. M., Mohamed, S. A., Fahmy, A. S. (2000). Distribution of lipases in the gramineae: Partial purification and characterization of esterase from Avena fatua, Bioresource Technology, 73, 227-234.
  • 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.
  • Rúa, M. L., Díaz-Mauriño, T., Fernández, V. M., Otero, C., Ballesteros, A. (1993). Purification and characterization of two distinct lipases from Candida cylindracea. Biochimica et Biophysica Acta, 1156, 181-189.
  • Sağıroğlu, A., Telefoncu, A. (1993). Synthesis of stereospecific esters and resolution of rasemic alcohols with immobilized lipases. Indian Journal of Chemistry, 32B, 227-233.
  • 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 Jatropha curcas L. Seeds. Journal of Biotechnology, 75, 117-126.
  • Taipa, M. A., Aires-Barros, M. R., Cabral, J. M. S. (1992). Purification of lipases. Journal of Biotechnology, 26, 301-311.
  • Veeraragavan, K. (1990). A simple and sensitive method for the estimation of microbial lipase activity. Analytical Biochemistry, 186, 301-305.
  • Vorderwulbwcke, T., Kieslich, K., Erdmann, H. (1992). Comparison of lipases by different assays. Enzyme and Microbial Technology, 14, 631-639.
  • Warburg, O., Christian, W. (1941). Isolierung und 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.
  • Yağar, H. (2000). Ayva polifenol oksidazının çeşitli taşıyıcılara immobilizasyonu ve karakterizasyonları, Doktora Tezi, Trakya Üniversitesi, Fen Bilimleri Enstitüsü, Edirne.

Papatyadan (Matricaria chamomilla L.) Lipaz Enziminin Saflaştırılması ve Çeşitli Taşıyıcılara İmmobilize Edilmesi

Yıl 2018, Cilt: 8 Sayı: 1, 131 - 143, 18.06.2018
https://doi.org/10.31466/kfbd.396563

Öz



Bu çalışmada, Giresun İli ve çevresinde bol miktarda
yetişen papatyadan (Matricaria chamomilla
L.) lipaz enzimi ilk defa
saflaştırıldı ve kinetik özellikleri incelendi.
Lipaz
esteraz aktivitesi gösteren homojenizatın % 70’lik (NH4)2SO4
kesiti elde edildi. Dializden sonra bu kesit DEAE-selüloz kolonlara uygulanarak
lipaz enzimi 26 kat saflaştırıldı. Saflaştırma işlemlerinde protein miktarı
Lowry ve E280/E260 Warburg yöntemlerine göre, lipaz
esteraz aktivitesi ise Erlanson yöntemine göre tayin edildi. SDS-PAGE
elektroforezi ile elde edilen saflaştırılmış enzimin molekül ağırlığının 30 kDa
olduğu bulundu. Optimum pH ve sıcaklık değerleri belirlendi. Papatyadan
saflaştırılan lipazın optimum pH’sının 9, optimum sıcaklığının 50 °C olduğu
bulundu. Enzimin çeşitli substratlara karşı ilgisi incelendiğinde, lipazın
ilgisinin en çok p-nitrofenil palmitatta olduğu ve bu substrata karşı Km
ve Vmax değerlerinin sırasıyla 0,2899 mM ve 144,93 Ünite olduğu
saptandı.Ayrıca
, papatyadan saflaştırılan lipaz enzimi çeşitli
taşıyıcılar
 üzerine
adsorpsiyon, kovalent ve iyonik bağlama yöntemleri ile immobilize edildi.
İmmobilize
edilmiş papatya lipazının optimum pH, optimum sıcaklık gibi kinetik özellikleri
de incelendi.




Kaynakça

  • Abdou, M. A. (2003). Purification and partial characterization of psychrotrophiz Serratia marcescens lipase. Journal of Dairy Science, 86, 127-132.
  • Abigor, D. R., Opute, F. I., Opoku, A. R., Osagie, A. U.(1985). Partial purification and some properties of the lipase present in oil palm (Elaeis guineensis) mesocarp. Journal of the Science of Food and Agriculture, 36, 599-606.
  • Baytop A., 1996, Farmasötik Botanik Ders Kitabı, İstanbul Üniversitesi Eczacılık Fakültesi. Üniversite Yayın No:3637, Eczacılık Fakültesi Yayın No:58, 315s.
  • Beisson, F., Gardies, A. M., Teissere, M., Ferte, N., Noat, G. (1997). An esterase neosynthesized 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.
  • Dong, H., Gao, S., Han, S., Cao, S. (1999). Purification and characterization of a Pseudomonas sp. lipase and its properties in non-aqueous media. Biotechnology and Applied Biochemistry, 30, 251-256.
  • Erlanson, C. (1970). p-Nitrophenyl acetate as a substrate for a carboxyl-ester hydrolase in pancreatic juice and intestinal content. Scandinavian Journal of Gastroenterology, 5, 333-336.
  • Fuciños, P., Abadin, C. M., Sanromán, A., Longo, M. A., Pastrana, L., Rúa, M. L. (2005). Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: Partial purification and preliminary biochemical characterization. Journal of Biotechnology, 117, 233-241.
  • Hadi, M. R. H. S., Darz, M. T., Ghandehari, Z., Riazi, G. (2011). Effects of vermicompost and amino acids on the flower yield and essential oil production from Matricaria chamomile L. Journal of Medicinal Plants Research, 5(23), 5611-5617.
  • Haas, M. J., Cichowicz, D. J., Dierov, J. K. (2001). Lipolytic activity of Californica-laurel (Umbellularia Californica) seeds. Journal of American Oil Chemist’s Society, 78, 1067-1071.
  • Hiol, A., Jonzo, M. D., Rugani, N., Druet, D., Sarda, L., Comeau, L. C. (2000). Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme and Microbial Technology, 26, 421-430.
  • Hong, M. C., Chang, M. C. (1988). Purification and characterization of an alkaline lipase from a newly isolated Acinetobacter radioresistens CMC-1. Biotechnology Letters, 20, pp. 1027-1029.
  • İlgün, R. (2016). Kestane tohumundan (Castanea sativa) lipaz enziminin saflaştırılması ve kinetik özelliklerinin incelenmesi, Yüksek Lisans Tezi, Giresun Üniversitesi, Fen Bilimleri Enstitüsü, Giresun.
  • Kılınç, A. (1990). Alginatta immobilize edilmiş lipazın bazı özelliklerinin incelenmesi, Yüksek Lisans Tezi, Ege Üniversitesi, Fen Bilimleri Enstitüsü, İzmir.
  • Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly 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.
  • Mohamed, M. A., Mohamed, T. M., Mohamed, S. A., Fahmy, A. S. (2000). Distribution of lipases in the gramineae: Partial purification and characterization of esterase from Avena fatua, Bioresource Technology, 73, 227-234.
  • 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.
  • Rúa, M. L., Díaz-Mauriño, T., Fernández, V. M., Otero, C., Ballesteros, A. (1993). Purification and characterization of two distinct lipases from Candida cylindracea. Biochimica et Biophysica Acta, 1156, 181-189.
  • Sağıroğlu, A., Telefoncu, A. (1993). Synthesis of stereospecific esters and resolution of rasemic alcohols with immobilized lipases. Indian Journal of Chemistry, 32B, 227-233.
  • 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 Jatropha curcas L. Seeds. Journal of Biotechnology, 75, 117-126.
  • Taipa, M. A., Aires-Barros, M. R., Cabral, J. M. S. (1992). Purification of lipases. Journal of Biotechnology, 26, 301-311.
  • Veeraragavan, K. (1990). A simple and sensitive method for the estimation of microbial lipase activity. Analytical Biochemistry, 186, 301-305.
  • Vorderwulbwcke, T., Kieslich, K., Erdmann, H. (1992). Comparison of lipases by different assays. Enzyme and Microbial Technology, 14, 631-639.
  • Warburg, O., Christian, W. (1941). Isolierung und 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.
  • Yağar, H. (2000). Ayva polifenol oksidazının çeşitli taşıyıcılara immobilizasyonu ve karakterizasyonları, Doktora Tezi, Trakya Üniversitesi, Fen Bilimleri Enstitüsü, Edirne.
Toplam 30 adet kaynakça vardır.

Ayrıntılar

Birincil Dil Türkçe
Konular Mühendislik
Bölüm Makaleler
Yazarlar

Bahar Bilgin Sökmen

Burçak Sarı Bu kişi benim

Tuğba Azap Bu kişi benim

Yayımlanma Tarihi 18 Haziran 2018
Yayımlandığı Sayı Yıl 2018 Cilt: 8 Sayı: 1

Kaynak Göster

APA Bilgin Sökmen, B., Sarı, B., & Azap, T. (2018). Papatyadan (Matricaria chamomilla L.) Lipaz Enziminin Saflaştırılması ve Çeşitli Taşıyıcılara İmmobilize Edilmesi. Karadeniz Fen Bilimleri Dergisi, 8(1), 131-143. https://doi.org/10.31466/kfbd.396563