Production, Purification of Aspergillus niger Catalase by Three-Phase Partitioning and Its Biochemical Characterization

Öz

In this study, it was aimed to produce, purify and characterize catalase enzyme from Aspergillus niger. For that purpose, the cells of A. niger were grown at 37°C and 155 rpm in 1 liter YpSs broth media and on the 7th day of cultivation the crude enzyme extract was obtained from the growth media. Catalase enzyme was purified by three-phase partitioning method. Accordingly, the system prepared by 80% (w/w) ammonium sulfate saturation at pH 7.0 with 1:1.5 (v/v) ratio of crude extract to t-butanol gave 7.9-fold purification with 263% activity recovery. The Km value (21.4 mM), the optimum reaction temperature (50°C) and the optimum reaction pH (6.0) were determined. Stability tests indicated that the enzyme can remain its activity in a broad pH (4.0-9.0) range. Additionally, it was observed that about 77% of initial catalase activity was maintained in the presence of 7.5% (v/v) ethanol. Besides its main function, it can also oxidase some phenolic compounds like 4-methyl catechol and pyrocatechol in a peroxide-independent way. As a consequence, it was seen that the catalase enzyme can be purified from A. niger by a three-phase partitioning system known as a time-effective and inexpensive system and very easy to use rather than a conventional chromatography. Its biochemical properties (pH and ethanol stabilities and presence of a secondary oxidase activity) can provide advantages in various industrial applications.

Anahtar Kelimeler

• Aspergillus niger,Catalase,Three-phase partitioning,Oxidase,Peroxidase

Aspergillus niger Katalazının Üretimi, Üçlü-Faz Ayırma ile Saflaştırılması ve Biyokimyasal Karakterizasyonu

Öz

Bu çalışmada Aspergillus niger katalazının üretimi, saflaştırılması ve karakterize edilmesi amaçlanmıştır. Buna göre 1 litrelik YpSs sıvı büyüme ortamında 37°C ve 155 rpm çalkalama hızında büyütülen A. niger’den 7. günde ham enzim ekstraktı elde edilmiştir. Katalaz enzimi üçlü-faz ayırma (TPP) tekniği ile saflaştırılmıştır. Bunun için %80 (w/w) amonyum sülfat içeren ve ham ekstrakt:t-butanol oranı 1:1.5 olacak şekilde pH 7.0’da hazırlanan sistemden enzim %263 verim ile 7.9 kat saflaştırılmıştır. Km değeri (21.4 mM), optimum reaksiyon sıcaklığı (50°C) ve optimum reaksiyon pH’sı (6.0) belirlenmiştir. Kararlılık testleri, enzimin geniş pH (4.0-9.0) aralığında dayanıklı kalabildiğini göstermiştir. Ayrıca katalaz aktivitesinin %7.5’lik (v/v) etanol varlığında yaklaşık %77’sinin korunduğu gözlenmiştir. Bununla birlikte, esas fonksiyonunun yanında 4-metil katekol ve katekol gibi fenolik bileşikleri peroksitten bağımsız olarak okside edebilmiştir. Sonuç olarak, A. niger’den katalaz enziminin geleneksel kromatografi yöntemi yerine zamandan tasarruf sağlayan, maliyeti ucuz ve kullanımı oldukça kolay olan üçlü faz sistemleri ile saflaştırılabildiği görülmektedir. Enzimin sahip olduğu biyokimyasal özellikleri (pH ve etanol kararlılığı ve ikincil oksidaz aktivite varlığı), çeşitli endüstriyel uygulama alanlarında avantaj sağlayabilir.

Anahtar Kelimeler

• Aspergillus niger,Katalaz,Üçlü-faz ayırma sistemi,Oksidaz,Peroksidaz

Kaynakça

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