Cloning and Determination of Kinetic Activity of Thermophilic GCH-I from Anoxybacillus flavithermus DSM 2641T

Yıl 2022, , 178 - 183, 30.06.2022

Fatih Şaban Beriş , Özlem Hızal Hakan Karaoğlu

https://doi.org/10.35229/jaes.1068313

Öz

Phenylketonuria (PKU) is a disease caused by phenylalanine hydroxylase enzyme deficiency in newborn infants and is the most important cause of treatable mental retardation. One of the causes of the disease comes from the defects of the PTPS in the metabolic pathway of phenilalanine metabolisms. Treatment of the disease is not feasible, and life-time tetrahydrobiopterin loading is performed in chronic patients. Today, tetrahydrobiopterin is chemically synthesized. Biological production is a different point of view due to the long duration of chemical synthesis, costs, and exposure to chemical pollutants. For this reason, the gch-I gene from the thermophilic A. flavithermus DSM 2641T was identified by PCR method. We cloned the gchI gene that was 603 bp and its open reading frame has 200 amino acids. The gene was cloned into pET28a(+) expression vector with 6xHis tags and transform in E. coli BL21(DE3)pLys host cells to express with 1 mM IPTG induction. After purification with Ni-NTA resin, we determined that GCH-I is 24 kDa, its optimum pH is 8.0 and temperature is 65C.Under optimal conditions, GCH-I exhibited enzymatic activity with Km- and Vmax- values of 243 ± 23,25 μM and 100,93 ± 3,5 nM/min/mg protein, respectively.

Anahtar Kelimeler

A. flavithermus DSM 2641T, BH4, GCHI, PKU, Thermophilic bacterium

Destekleyen Kurum

Recep Tayyip Erdogan University Research Foundation

Proje Numarası

2011.102.03.4

Anoxybacillus flavithermus DSM 2641T Bakterisinin Termofilik GCH-I Enziminin Klonlanması ve Kinetik Aktivitesinin Belirlenmesi

Öz

Fenilketonüri (FKU) yenidoğan bebeklerde fenilalanin hidroksilaz enzim eksikliğinin neden olduğu bir hastalıktır ve tedavi edilebilir zihinsel geriliğin en önemli nedenidir. Hastalığın nedenlerinden biri, fenilalanin metabolizmasının metabolik yolundaki PTPS'nin kusurlarından kaynaklanmaktadır. Hastalığın tedavisi mümkün değildir ve kronik hastalarda yaşam boyu tetrahidrobiyopterin yüklemesi yapılır. Bugün, tetrahidrobiyopterin kimyasal olarak sentezlenir. Biyolojik üretim, uzun süre kimyasal sentez, maliyetler ve kimyasal kirleticilere maruz kalma nedeniyle farklı bir bakış açısıdır. Bu nedenle, termofilik A. flavithermus DSM 2641T'den gchI geni PCR yöntemi ile tanımlandı. 603 bp olan gchl genini klonlandı ve açık okuma çerçevesi 200 amino aside sahip olduğu tespit edildi. AfgchI geni, 6xHis etiketleri ile pET28a(+) ekspresyon vektörüne klonlandı ve 1 mM IPTG indüksiyonu ile eksprese etmek için E. coli BL21 (DE3)pLys konakçı hücrelerine transforme edildi. Ni-NTA afinite kromatografisi ile saflaştırıldıktan sonra GCH-I’in 24 kDa, optimum pH’sının 8,0 ve optimum sıcaklık isteminin 65C olduğu belirlendi. Optimal koşullar altında GCH-I, Km- ve Vmax- değerleri sırasıyla 243 ± 23,25 μM ve 100,93 ± 3,5 nM/dak/mg protein olarak tespit edildi.

Anahtar Kelimeler

A. flavithermus DSM 2641T, BH4, GCHI, FKU, Termofilik bakteri

Proje Numarası

2011.102.03.4

Kaynakça

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