Purification and Characterization of Lactoperoxidase from Goat Milk, Investigation of Inhibition Effects of Cefotaxime Sodium

Metin Tansu Uğuz; Mehmet Muhsin Erol

doi:10.18016/ksutarimdoga.vi.1532133

TR EN

Laktoperoksidaz Enziminin Keçi Sütünden Saflaştırılması ve Karakterizasyonu, Cefotaxime Sodyumun İnhibisyon Özelliklerinin Araştırılması

Öz

LPO’ nun doğal bağışıklık için önem arzettiği literatürlerde gösterilmiştir. Bu çalışmanın amacı, keçi sütünden laktoperoksidaz (LPO) enzimini saflaştırmak ve sefotaksim sodyumun enzim üzerindeki inhibisyon özelliğini araştırmaktır. Bu çalışmada, keçi sütünden LPO enzimi saflaştırılmış ve sefotaksim sodyumun inhibisyon etkisi incelenmiştir. Enzim saflaştırma süreçleri çeşitli kromatografik yöntemlerle gerçekleştirilmiş ve enzim inhibitör etkisi Lineweaver-Burk grafikleri ile analiz edilmiştir. Sefotaksim sodyumun enzim üzerindeki inhibisyonu yarışmalı inhibisyon olarak belirlenmiştir. Enzimin keçi sütünden saflaştırılması için, Amberlite CG-50 H+ reçinesiyle kısmi saflaştırma yapılarak, CM-Sephadex C-50 iyon değişim kromatografisi, Sephadex G-100 jel filtrasyon kromatografisi, sırasıyla çalışıldı. ABTS (3-ethylbenzothiazoline-6-sulphonicacid) substratı için Km ve Vmax değerleri ile optimum pH, optimum sıcaklık belirlendi. Enzim saflığı SDS-PAGE elektroforezi ile tespit edildi. Enzimin bir litre keçi sütünden spesifik aktivitesi 7.21 EÜ mg-1 protein miktarı belirlendi. LPO saflaştırma işlemleri sonucunda 1 litre yağı alınmış taze sütten 13.35 kat saflaştırıldı ve 1.9 mg (Rz=0.7) elde edildi. Enzim saflaştırılması sırasında aktivite tayinleri için ABTS substratı (ε412nm=32400M-1 cm-1) kullanıldı.

Anahtar Kelimeler

Purification and Characterization of Lactoperoxidase from Goat Milk, Investigation of Inhibition Effects of Cefotaxime Sodium

Abstract

İt has been shown in the literature that LPO is important for natural immunity. The purpose of this study is to purify the lactoperoxidase (LPO) enzyme from goat milk and to investigate the inhibition property of cefotaxime sodium on the enzyme. In this study, the LPO enzyme was purified from goat milk and the inhibition effect of cefotaxime sodium was examined. Enzyme purification processes were carried out by various chromatographic methods and the enzyme inhibitory effect was analyzed with Lineweaver-Burk graphs. İnhibition of cefotaxime sodium on LPO was determined as competitive inhibition. The enzyme was purified by first partially purifying milk using Amberlite CG-50 H+ resins, CM-Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The 2.2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate was used to determine the enzyme’s Km and Vmax values. Enzyme purity was determined using SDS-PAGE electrophoresis. A specific activity of 7.21 EU mg-1 of protein per liter was detected. The LPO was purified 13.35 times from 1 liter of defatted milk, and 1.9 mg of enzyme was obtained. ABTS substrate (ε412nm=32400M-1 cm-1) was used for the enzyme-activity assays.

Keywords

Supporting Institution

This study was supported by Bingol University Scientific Research Projects Unit (Project no: BAP-14-95-2011)

Thanks

This study was supported by Bingol University Scientific Research Projects Unit (Project no: BAP-14-95-2011)

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Details

Primary Language

English

Subjects

Biochemistry and Cell Biology (Other)

Journal Section

Research Article

Authors

Metin Tansu Uğuz ^*
0000-0002-3557-1677
Türkiye

Mehmet Muhsin Erol
0009-0009-3660-0271
Türkiye

Early Pub Date

March 20, 2025

Publication Date

March 27, 2025

Submission Date

August 13, 2024

Acceptance Date

January 17, 2025

Published in Issue

Year 2025 Volume: 28 Number: 2

DOI

https://doi.org/10.18016/ksutarimdoga.vi.1532133

IZ

https://izlik.org/JA59TE38LC

Cite

RIS / Bibtex

APA

Uğuz, M. T., & Erol, M. M. (2025). Purification and Characterization of Lactoperoxidase from Goat Milk, Investigation of Inhibition Effects of Cefotaxime Sodium. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi, 28(2), 298-305. https://doi.org/10.18016/ksutarimdoga.vi.1532133