ÇİĞDEM (CROCUS BİFLORUS) YUMRULARINDAN PROTEAZ ENZİMİNİN SAFLAŞTIRILMASI VE SAFLAŞTIRILAN ENZİMİN KAZEİNİN KOAGÜLASYONUNDA KULLANILABİLİRLİĞİNİN ARAŞTIRILMASI

Year 2018, Volume: 43 Issue: 2, 231 - 239, 20.02.2018

Safinur Yıldırım Çelik

https://doi.org/10.15237/gida.GD17095

Cited By: 3

Abstract

Son yıllarda, hayvanlardan insanlara geçen bazı
hastalıklarda (SARS, deli dana, domuz ve kuş gribi) artış olduğu bilinmektedir.
Tüketicilerde, hayvansal proteazların gıda maddesi yapımında kullanımı bu
hastalıkların kendilerine bulaşacağına dair endişelere yol açmaktadır. Bu
nedenlerden dolayı bitkisel kaynaklı ürünlere bir talep artışı olmuştur.  Ticari olarak satılan enzimlerin ise sadece
az bir kısmının bitkisel kaynaklı olması, araştırmacıları ticari olarak da
kullanılabilecek yeni bitkisel proteaz kaynakları aramaya sevk etmiştir. Bu
çalışmada, ilk kez çiğdem (Crocus biflorus) yumrularından proteaz enzimi
saflaştırılmış ve bazı karakteristik özellikleri belirlenmiştir. Enzim, amonyum
sülfat çöktürmesi ve iyon değişim kromatografisi ile 86 kat saflaştırılmıştır.
Proteaz enziminin optimum pH’sı 5.5 ve optimum sıcaklığı 50^oC olarak
bulunmuştur. V_max ve K_M değerleri ise sırasıyla 47
mg/L.dak ve 0.9 g/L olarak hesaplanmıştır. Enzimin molekül ağırlığı SDS-PAGE
ile 35 kDa olarak hesaplanmıştır. Saflaştırılan enzimin, peynir yapımının ilk
basamağı olan kazeinin koagülasyonu işlemlerinde kullanılabileceği belirlenmiştir. 

Keywords

Çiğdem (Crocus biflorus), proteaz, koagülasyon, kazein

PURIFICATION OF PROTEASE ENZYME FROM ÇİĞDEM (CROCUS BIFLORUS) TUBERS AND INVESTIGATION OF USABILITY OF THE PURIFIED PROTEASE ENZYME IN COAGULATION OF CASEIN

Abstract

It is known that in some diseases
(SARS, mad cow, pig and bird flu) that have passed from animals to humans have
increased in recent years. In consumers, the use of animal proteases in the
production of food products leads to concerns about the spread of these
diseases. For these reasons, it has been an increase in demand for the product
obtained from plant sources. The fact that only a small proportion of
commercially available enzymes are herbaceous, have encouraged researchers to
search for new herbal protease sources that can be used commercially. In this
study, for the first time protease enzyme was purified from the tubers of a Crocus
biflorus and characterized. The protease enzyme was purified 86 fold by
ammonium sulfate precipitation and ion exchange chromatography. Optimum pH and
temperature was found as 5.5 and 50 °C. The values of V_max and K_M
were calculated as 47 mg/mL.min and 0.9 g/L, respectively. The molecular weight
of the enzyme was calculated to be 35 kDa by using SDS-PAGE. It was determined
that the purified enzyme can be used in the process of casein coagulation, the
first step of cheese making. 

Keywords

Çiğdem (Crocus biflorus), protease, coagulation, casein

References

• Referans1 Akyol, Y., Yetişen, K., Özdemir, C., Bozdağ, B., & Kocabaş, O. (2012). Türkiye’deki Crocus biflorus Miller subsp. tauri (Maw) Mathew (Iridaceae) Üzerine Morfolojik ve Anatomik Bir Çalışma. Iğdır Univ. J. Inst. Sci. Tech., 2:15–20.
• Referans2 Antão, C. M., & Malcata, F. X. (2005). Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol. Biochem. PPB, 43(7):637–50. https://doi.org/10.1016/j.plaphy.2005.05.001
• Referans3 Berridge, N. J. (1945). The purification and crystallization of rennin. Biochem. J., 39(2):179–86.
• Referans4 Beynon, R., & Bond, J. S. (2000). Proteolytic Enzymes. Biochem. Educ., 18(1):359. https://doi.org/10.1016/0307-4412(90)90038-P
• Referans5 Bradford, M. M. (1976). A rapid and sensitive method for the for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem., 72:248–254.
• Referans6 Cavalcanti, M. T. H., Teixeira, M. F. S., Lima Filho, J. L., & Porto, A. L. F. (2004). Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp. Bioresour. Technol., 93(1):29–35. https://doi.org/10.1016/j.biortech.2003.10.003
• Referans7 Demir, Y., Alayli, A., Yildirim, S., & Demir, N. (2005). Identification of protease from Euphorbia amygdaloides latex and its use in cheese production. Prep. Biochem. Biotechnol., 35(4):291–299. https://doi.org/10.1080/10826060500218107
• Referans8 Fadýloǧlu, S. (2001). Immobilization and characterization of ficin. Nahrung - Food, 45(2):143–146. https://doi.org/10.1002/1521-3803
• Referans9 Kumar Dubey, V., & Jagannadham, M. V. (2003). Procerain, a stable cysteine protease from the latex of Calotropis procera. Phytochemistry, 62(7):1057–1071. https://doi.org/10.1016/S0031-9422(02)00676-3
• Referans10 Menon, M., Vithayathil, P. J., Raju, S. M., & Ramadoss, C. S. (2002). Isolation and characterization of proteolytic enzymes from the latex of Synadenium grantii Hook, “f.” Plant Sci., 163(1):131–139. https://doi.org/10.1016/S0168-9452(02)00085-7
• Referans11 Modler, H. W. (1985). Functional Properties of Nonfat Dairy Ingredients - A Review. Modification of Products Containing Casein. J. Dairy Sci., 68(9):2195–2205. https://doi.org/10.3168/jds.S0022-0302(85)81091-2
• Referans12 Sumantha, A., Sandhya, C., Szakacs, G., Soccol, C. R., & Pandey, A. (2005). Production and partial purification of a neutral metalloprotease by fungal mixed substrate fermentation. Food Technol. Biotechnol., 43(4):313–319.