Smf (submerged fermentatıon) yöntemi ile topraktan izole edilerek tanımlanan Bacillus subtilis'ten α-amilaz üretimi, saflaştırılması ve karakterizasyonu
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Tarih
2022
Yazarlar
Dergi Başlığı
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Cilt Başlığı
Yayıncı
Dicle Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Çalışmamızda kullanılmak üzere Ergani'de bulunan Makam Dağı'ndan toprak örneği alınmıştır. Alınan toprak örneğinden bakteri izolasyonu gerçekleştirilmiştir.İzole edilmiş mikroorganizma üzerinde bazı biyokimyasal analizleri uygulanmış ve 16S rRNA tanımlanması yapılmıştır. Elde edilen veriler sonucunda, izole edilmiş olan bakterinin Bacillus subtilis olduğu belirlenmiştir. SmF tekniği ile üretilen Bacillus subtilis izolatına ait ?-amilaz üretimi üzerine çeşitli parametrelerin etkisi araştırılarak optimum koşulları belirlenmiştir. Bakteri çoğalmasına ait optimum koşulların süre bakımından 32. saat, sıcaklık bakımından 37°C ve pH bakımından da pH 7.0 olduğu belirlenmiştir. Enzimin maksimum düzeyde üretildiği süre olarak belirlenen 48. saatte bakteri çoğaltma işlemi gerçekleştirilmiştir. Kültüre edilmiş olan bakteriden üst sıvı alınarak ?-amilaz aktivite çalışması yapılmıştır. Enzimin aktivite gösterdiği optimum pH'sı 7.0 ve optimum sıcaklık da 37°C olarak belirlenmiştir. Bakteri tarafından kullanılacak azot ve karbon kaynaklarının enzim üretimindeki etkisi incelenmiştir. Elde edilen verilere göre azot kaynaklarını kullanan bakteriden kontrole kıyaslandığında daha düşük amilaz aktivitesi belirlenmiştir. Azot kaynakları arasında amonyum nitrat ve amonyum sulfat eklenmesiyle en yüksek spesifik aktivite gözlenmiştir. Enzim üretimi üzerine karbon kaynaklarının etkisine bakıldığında ise galaktoz değerinin kontrole yakın bir değer gösterdiği ve diğer karbon kaynaklarında ise enzim üretiminin azaldığı görülmüştür. Enzimin optimum koşulları belirlendikten sonra saflaştırma işlemine geçildi. Saflaştırılan amilazın molekül ağırlığı SDS-PAGE ile ~73 kDa olarak tespit edildi. Deterjanların bazılarının, saflaştırılan enzim aktivitesi üzerine etkisini saptamak için 0.005'lik Tween-40, SDS, TritonX-100, Tween-8 ve brij kullanılmıştır. Kullanılan deterjanlardan SDS'nin önemli ölçüde amilaz enzimini inhibe ettiği görüldü. Bazı metallerin, saflaştırılan enzim aktivitesi üzerindeki etkisi incelendiğinde CaCl2'nin aktiviteyi arttırdığı, MnCl2 ve MgCl2 metallerinin eklenmesi sonucunda herhangi bir değişiklik gözlenmediği, FeCl2, CuCl2, ZnCl2 ve HgCl2 metallerinin eklenmesiyle de enzim aktivitesinin önemli ölçüde inhibe edildiği belirlenmiştir.
Soil samples were taken from Makam Mountain in Ergani to be used in our study. Bacteria isolation from the soil sample taken from Makam Mountain was made in the laboratory under sterile conditions. Some biochemical analyzes were performed on the isolated microorganism and 16S rRNA was identified. As a result of the data obtained, it was determined that the isolated bacterium was Bacillus subtilis. Optimum conditions were determined by investigating the effect of various parameters on ?-amylase production of Bacillus subtilis isolate produced by SmF technique. It was determined that the optimum conditions for bacterial growth were 32 hours in terms of time, 37°C in terms of temperature and pH 7.0 in terms of pH. Bacterial replication was carried out at the 48th hour, which was determined as the time during which the enzyme was produced at the maximum level. ?-amylase activity study was performed by taking the supernatant from the cultured bacteria. The optimum pH at which the enzyme was active was determined as 7.0 and the optimum temperature was 37°C. The effect of nitrogen and carbon sources to be used by bacteria on enzyme production was investigated. According to the data obtained, lower amylase activity was determined from the bacteria using nitrogen sources compared to the control. Among the nitrogen sources, the highest specific activity was observed with the addition of ammonium nitrate and ammonium sulfate. When the effect of carbon sources on enzyme production was examined, it was seen that the galactose value showed a value close to the control and the enzyme activity decreased in other carbon sources. After the optimum conditions of the enzyme were determined, the purification process was started. The molecular weight of the purified amylase was determined as ~73 kDa by SDS-PAGE.To determine the effect of some detergents on the purified enzyme activity, 0.005 Tween-40, SDS, TritonX-100, Tween-8 and brige were used. It was observed that SDS, one of the detergents used, inhibited the amylase enzyme significantly. When the effect of some metals on the purified enzyme activity was examined, it was determined that CaCl2 increased the activity, no change was observed as a result of the addition of MnCl2 and MgCl2 metals, and the enzyme activity was significantly inhibited by the addition of FeCl2, CuCl2, ZnCl2 and HgCl2 metals.
Soil samples were taken from Makam Mountain in Ergani to be used in our study. Bacteria isolation from the soil sample taken from Makam Mountain was made in the laboratory under sterile conditions. Some biochemical analyzes were performed on the isolated microorganism and 16S rRNA was identified. As a result of the data obtained, it was determined that the isolated bacterium was Bacillus subtilis. Optimum conditions were determined by investigating the effect of various parameters on ?-amylase production of Bacillus subtilis isolate produced by SmF technique. It was determined that the optimum conditions for bacterial growth were 32 hours in terms of time, 37°C in terms of temperature and pH 7.0 in terms of pH. Bacterial replication was carried out at the 48th hour, which was determined as the time during which the enzyme was produced at the maximum level. ?-amylase activity study was performed by taking the supernatant from the cultured bacteria. The optimum pH at which the enzyme was active was determined as 7.0 and the optimum temperature was 37°C. The effect of nitrogen and carbon sources to be used by bacteria on enzyme production was investigated. According to the data obtained, lower amylase activity was determined from the bacteria using nitrogen sources compared to the control. Among the nitrogen sources, the highest specific activity was observed with the addition of ammonium nitrate and ammonium sulfate. When the effect of carbon sources on enzyme production was examined, it was seen that the galactose value showed a value close to the control and the enzyme activity decreased in other carbon sources. After the optimum conditions of the enzyme were determined, the purification process was started. The molecular weight of the purified amylase was determined as ~73 kDa by SDS-PAGE.To determine the effect of some detergents on the purified enzyme activity, 0.005 Tween-40, SDS, TritonX-100, Tween-8 and brige were used. It was observed that SDS, one of the detergents used, inhibited the amylase enzyme significantly. When the effect of some metals on the purified enzyme activity was examined, it was determined that CaCl2 increased the activity, no change was observed as a result of the addition of MnCl2 and MgCl2 metals, and the enzyme activity was significantly inhibited by the addition of FeCl2, CuCl2, ZnCl2 and HgCl2 metals.
Açıklama
Anahtar Kelimeler
Bacillus subtilis, İzolasyon, α-amilaz, Saflaştırma, Submerged fermantasyon (SmF), Isolation, α-amylase, Purification, Submerged fermentation (SmF)
Kaynak
WoS Q Değeri
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Sayı
Künye
Ortakaya, V. (2022). Smf (submerged fermentatıon) yöntemi ile topraktan izole edilerek tanımlanan Bacillus subtilis'ten α-amilaz üretimi, saflaştırılması ve karakterizasyonu. Yayımlanmamış doktora tezi, Dicle Üniversitesi, Diyarbakır.
