Proteazın fazla üretimi için UV mutasyon yoluyla Bacillus subtilis strain 168 E6-5'de suş geliştirme çalışmaları ve üreme ortamının optimizasyonu
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Date
2016-10-06
Authors
Peker, Meltem
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Publisher
Uludağ Üniversitesi
Abstract
Bu çalışmada, UV ile muamele edilen Bacillus subtilis 168 E6-5 suşundan klasik mutasyon işlemleri ile proteaz üreten 369 adet mutant elde edilmiştir. Bu mutantlar arasında en geniş proteaz zonuna (12 mm) sahip mutan suş seçilmiş ve bu suş Bacillus subtilis MET39 olarak isimlendirilmiştir. Yeni mutantın sıvı ortamda enzim üretim kapasitesi test edilmiş ve mutant suş ana şuştan 1.2 kat daha fazla enzim üretimi göstermiştir. Mutant suşun 28.saatte maksimum seviyede proteaz ürettiği bulunmuştur. Ayrıca besinsel faktörlerin enzim üretimi üzerindeki etkileri incelenmiş ve bu amaçla, enzim üretimi üzerine farklı karbon, nitrojen kaynakları ve metal iyonları gibi temel ortam maddelerinin etkileri araştırılmıştır. En iyi karbon kaynağı olarak gliserol (114 IU/mL) bulunmuştur. Kullanılan inorganik azot kaynakları arasında, tripton (348 IU/mL) enzim üretimi üzerinde çok yüksek bir potansiyel göstermiştir. İnorganik azot kaynaklarının organik kaynakları kadar etkilli olduğu belirlenmiş. Ancak sadece NH4NO3 varlığında enzim üretimi saptanmamıştır. Kullanılan metal iyonlarının enzim üretimi üzerinde fazla bir etkisi olmamıştır. Proteaz üretimini artırmak amacıyla, yeni ortam en iyi karbon, azot kaynakları ve metal iyonlarının birleştirilmesi ile elde edilmiştir. Bu ortamda, enzim verimi (150 IU/mL), bazal ortamına (80 IU/mL) oranla % 88 artırılmıştır. Elde edilen mutant suşun endüstriyel ölçekte proteaz üretimi, büyük bir potansiyele sahip olabilir.
In this study, the parent strain Bacillus subtilis 168 E6-5 was exposed to UV irradiation by classical mutation procedure, and 369 mutants were obtained. Among of these mutants, a mutant strain was selected based on its ability to produce highest zone formation, and strain is named as Bacillus subtilis 168 MET39. New mutant was tested for enzyme production capacity in the liquid media, the mutant MET39 showed 1.2-fold increase in protease production over the parent strain B.subtilis 168 E6-5. It was found that mutant strain produced protease on maximum level at 28th hour. The effects of nutritional factors on the production of enzymes were studied, and for this purpose, the effects of major medium ingredients, such as different carbon, nitrogen sources and metal ions, on the production of the enzyme were used. The best carbon source was found to be gliserol (114 IU/mL). Among the inorganic nitrogen sources, the highest enzyme production was obtained in the presence of tripton (348 IU/mL). Inorganic nitrogen sources were detected as effective as organic sources. But enzyme production only wasn't determined in the presence of NH4NO3. The metal ions were not much effect on enzyme production. In order to enhance the production of protease, a new medium was obtained by combining the best carbon and nitrogen sources, and metal ions. In this medium, enzyme yield (150 IU/mL) was enhanced 88% compared to basal medium. This obtained mutant strain may be great potential for protease production at industrial scale
In this study, the parent strain Bacillus subtilis 168 E6-5 was exposed to UV irradiation by classical mutation procedure, and 369 mutants were obtained. Among of these mutants, a mutant strain was selected based on its ability to produce highest zone formation, and strain is named as Bacillus subtilis 168 MET39. New mutant was tested for enzyme production capacity in the liquid media, the mutant MET39 showed 1.2-fold increase in protease production over the parent strain B.subtilis 168 E6-5. It was found that mutant strain produced protease on maximum level at 28th hour. The effects of nutritional factors on the production of enzymes were studied, and for this purpose, the effects of major medium ingredients, such as different carbon, nitrogen sources and metal ions, on the production of the enzyme were used. The best carbon source was found to be gliserol (114 IU/mL). Among the inorganic nitrogen sources, the highest enzyme production was obtained in the presence of tripton (348 IU/mL). Inorganic nitrogen sources were detected as effective as organic sources. But enzyme production only wasn't determined in the presence of NH4NO3. The metal ions were not much effect on enzyme production. In order to enhance the production of protease, a new medium was obtained by combining the best carbon and nitrogen sources, and metal ions. In this medium, enzyme yield (150 IU/mL) was enhanced 88% compared to basal medium. This obtained mutant strain may be great potential for protease production at industrial scale
Description
Keywords
Proteaz, Ultraviole, Besinsel faktörler, Bacillus sp, Protease, Nutritional factors
Citation
Peker, M. (2016). Proteazın fazla üretimi için UV mutasyon yoluyla Bacillus subtilis strain 168 E6-5'de suş geliştirme çalışmaları ve üreme ortamının optimizasyonu. Yayınlanmamış yüksek lisans tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.