Yeni izolat bacillus subtilis e6-5 suşundan proteaz: üretim koşullarının optimizasyonu, kısmi saflaştırılması, karakterizasyonu ve deri endüstrisindeki potansiyel uygulamaları
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Date
2020-08-18
Authors
Zeren, Behice
Journal Title
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Publisher
Bursa Uludağ Üniversitesi
Abstract
Bu çalışmada, Bacillus subtilis E6-5 suşu proteaz kaynağı olarak kullanılmış ve farklı besi yerlerinde proteaz üretim kapasitesi açısından test edilmiştir. Proteaz üretimi için ortamın optimizasyonu besinsel ve fiziksel koşullar değiştirilerek elde edilmiştir. Maksimum proteaz üretimi, besi yeri bileşiminde, sükroz (karbon kaynağı), tripton (azot kaynağı) BaCl2 + CaCl2 (metal iyonları) kullanılarak elde edilmiştir. Maksimum proteaz üretimi için optimal fiziksel parametreler 35°C, pH 8.0, 150 rpm çalkalanma hızı, %4 inokülasyon miktarı, 72 saat inokülasyon yaşı olarak belirlenmiştir. Optimal besinsel ve fiziksel parametreler kombine edilerek elde edilen yeni modifiye ortamda proteaz üretiminde 5 kat bir artış sağlanmıştır. Saflaştırılmış proteazın optimal sıcaklık ve pH değeri sırasıyla 60°C ve 7.0 olarak belirlenmiştir. Termostabilite çalışmaları, enzimin termostabil olduğunu göstermiştir. Saflaştırılmış enzim Ca varlığında aktiftir. Enzim bir metalloproteinazdır. Enzimin Vmax ve Km kinetik değerleri sırasıyla 3333 U/mL ve 1 mM olarak saptanmıştır. Moleküler ağırlığı yaklaşık 51.5 kDa olarak belirlenmiştir. Enzimin -20°C'de 45 güne kadar stabilitesini korunduğu belirlenmiştir. Deriden kıl gidermede enzimatik işlem kimyasal işlemden daha efektif bulunmuştur. Histolojik analizler, proteazın derilerin kalitesini arttırdığını göstermiştir. Bacillus subtilis E6-5 suşu proteazı, deri endüstrisinde kullanım potansiyeline sahip olabilir.
In this study, Bacillus subtilis E6-5 strain was used as a protease source and tested for protease production capacity in different media. Optimization of the medium for protease production was obtained by modifying nutritional and physical conditions. Maximum protease production has achieved using sucrose (carbon source), tryptone (nitrogen source) and BaCl2 + CaCl2 (metal ions) in medium composition. The optimal physical parameters for maximum protease production were determined as 35°C, pH 8.0, 150 rpm agitation rate, 4% inoculum size, 72 hours inoculum age. In the new modified medium obtained by combining optimal nutritional and physical parameters, a 5-fold increase in protease production was achieved. The optimal temperature and pH of the purified protease were determined as 60°C and 7.0, respectively. Thermostability studies have shown that the enzyme is thermostable. The purified enzyme was active in the presence of Ca 2+, 2+ . The enzyme was a metalloprotease. Vmax and Km kinetic values of the enzyme were determined as 3333 U/mL and 1 mM, respectively. The molecular weight was determined about 51.5 kDa. The activity of the enzyme was found to be stable up to 45 days at -20ºC. The enzymatic treatment was found to be more effective than the chemical treatment for hair removal from the skin. Histological analyses revealed that the protease has enhanced the quality of skins. The protease of Bacillus subtilis E6-5 strain may have the potential to be used in the leather industry.
In this study, Bacillus subtilis E6-5 strain was used as a protease source and tested for protease production capacity in different media. Optimization of the medium for protease production was obtained by modifying nutritional and physical conditions. Maximum protease production has achieved using sucrose (carbon source), tryptone (nitrogen source) and BaCl2 + CaCl2 (metal ions) in medium composition. The optimal physical parameters for maximum protease production were determined as 35°C, pH 8.0, 150 rpm agitation rate, 4% inoculum size, 72 hours inoculum age. In the new modified medium obtained by combining optimal nutritional and physical parameters, a 5-fold increase in protease production was achieved. The optimal temperature and pH of the purified protease were determined as 60°C and 7.0, respectively. Thermostability studies have shown that the enzyme is thermostable. The purified enzyme was active in the presence of Ca 2+, 2+ . The enzyme was a metalloprotease. Vmax and Km kinetic values of the enzyme were determined as 3333 U/mL and 1 mM, respectively. The molecular weight was determined about 51.5 kDa. The activity of the enzyme was found to be stable up to 45 days at -20ºC. The enzymatic treatment was found to be more effective than the chemical treatment for hair removal from the skin. Histological analyses revealed that the protease has enhanced the quality of skins. The protease of Bacillus subtilis E6-5 strain may have the potential to be used in the leather industry.
Description
Keywords
Protease, Bacillus, Proteaz, Optimizasyon, Saflaştırma, Karakterizasyon, Deri, Kıl giderme, Optimization, Purification, Characterization, Skin, Dehairing
Citation
Zeren, B. (2020). Yeni izolat bacillus subtilis e6-5 suşundan proteaz: Üretim koşullarının optimizasyonu, kısmi saflaştırılması, karakterizasyonu ve deri endüstrisindeki potansiyel uygulamaları. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Fen Bilimleri Enstitüsü.