Protease Enzyme Production and Characterization from Aspergillus Niger Grown in Medium Obtained from Organic Waste

Yıl 2025, Cilt: 23 Sayı: 1, 37 - 46, 11.05.2025

Betül Tekyıldız , Özge Aslan , Arzu Çağrı Mehmetoğlu

https://doi.org/10.24323/akademik-gida.1697198

Öz

Proteases, a subgroup of proteolytic enzymes with a significant role in the commercial enzyme market, are in high demand due to their broad and diverse biotechnological applications. Fermentation using molds is a preferred method for producing these enzymes because their extracellular production in a concentrated form facilitates easier recovery, making the process more cost-effective. Therefore, this study aims to provide an alternative approach for the production of proteases from Aspergillus niger using organic waste as a substrate and to establish a basis for enzyme purification. In this study, the optimal medium for enzyme production was determined as a culture medium prepared from organic waste with a 50% protein content, inoculated with A. niger, and incubated at 30°C for 24 h at pH 5.5. The protease enzyme produced from A. niger was partially purified using the three-phase partitioning (TPP) system, which has become a versatile bio-separation tool with broad applications, high recovery rates, and high purity levels. Using the TPP system, the protease enzyme was purified 13.15-fold with a yield of 235.7% under conditions of a 1.0:1.5 ratio (crude enzyme solution: t-butanol) and 80% ammonium sulfate saturation. The molecular weight of the enzyme was found to be 15.56 kDa using the MALDI-TOF method. The optimum and stable pH values of the purified protease enzyme from A. niger were determined as 10.0 and 9.0, respectively, while the optimum and stable temperature was 60°C. The presence of oxidizing agents, surfactants, inhibitors, and substrates caused a decrease in the enzyme activity values.

Anahtar Kelimeler

Protease , Aspergillus niger , Triple phase separation system (TPP) , Taguchi

Teşekkür

Arş. Gör. Dr. Alev Akçay’a çalışmaya yardımlarından dolayı teşekkür ederiz.

Organik Atıklardan Elde Edilen Besiyerinde Üreyen Aspergillus niger’den Proteaz Enzimi Üretimi ve Karakterizasyonu

Öz

Ticari enzim pazarında önemli bir yeri olan proteolitik enzim grubundan proteazlar, geniş ve farklı biyoteknolojik uygulamaları nedeniyle yüksek talep gören bir enzim grubudur. Bu enzimlerin küfler kullanılarak fermantasyon yoluyla üretilmesi tercih edilen bir yöntemdir. Çünkü, enzimlerin konsantre halde ekstraselüler olarak üretilmesiyle geri kazanımı daha kolay ve maliyet açısından daha elverişli bir üretim yöntemi olmaktadır. Bu nedenle çalışmada, organik çöpler substrat olarak kullanılarak Aspergillus niger'den proteazların üretimine alternatif oluşturmak ve enzimin saflaştırılmasına bir temel oluşturmak amaçlanmıştır. Araştırmada, A. niger inoküle edilen %50 protein oranında organik atıklardan hazırlanan besiyerinin, 30°C’de 24 saat inkübasyon süresinde, pH 5.5’te enzim üretimi için optimum besiyeri olduğu tespit edilmiştir. A. niger’den üretilen proteaz enzimi geniş bir uygulama alanına sahip, çok yönlü bir biyo-ayırma aracı haline gelen, yüksek geri kazanım ve saflık seviyelerine sahip bir dizi enzim ve proteini saflaştırmak için kullanılan üçlü faz ayırma (TPP) sistemi ile kısmen saflaştırılmış ve biyokimyasal karakterizasyonları incelenmiştir. TPP sistemi ile proteaz enzimi 1.0:1.5 (ham enzim çözeltisi: t-bütanol oranı) ve %80 doygun amonyum sülfat konsantrasyonu ile %235.7 verimle 13.15 kat saflaştırılmıştır. Enzimin molekül ağırlığı MALDI-TOF yöntemiyle 15.56 kDa olarak belirlenmiştir. A. niger’den saflaştırılan proteaz enziminin optimum ve stabil pH’ları sırasıyla 10.0 ve 9.0 iken optimum ve stabil sıcaklığı ise 60°C olmuştur. Okside edici ajanlar, yüzey aktif maddeleri, inhibitör ve substrat varlığı proteaz enziminin aktivite değerlerinde azalmaya neden olmuştur.

Anahtar Kelimeler

Proteaz , Aspergillus niger , Üçlü faz ayırma sistemi (TPP) , Taguchi

Kaynakça

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