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

Fatih Şaban Beriş; Özlem Hızal; Hakan Karaoğlu

doi:10.35229/jaes.1068313

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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

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

Abstract

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.

Keywords

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

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Details

Primary Language

English

Subjects

-

Journal Section

Research Article

Authors

Fatih Şaban Beriş ^*
0000-0002-0535-943X
Türkiye

Özlem Hızal This is me
0000-0002-0248-6652
Türkiye

Hakan Karaoğlu
0000-0003-4615-1157
Türkiye

Publication Date

June 30, 2022

Submission Date

February 5, 2022

Acceptance Date

April 21, 2022

Published in Issue

Year 2022 Volume: 7 Number: 2

DOI

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

IZ

https://izlik.org/JA39KC77FL

APA

Beriş, F. Ş., Hızal, Ö., & Karaoğlu, H. (2022). Cloning and Determination of Kinetic Activity of Thermophilic GCH-I from Anoxybacillus flavithermus DSM 2641T. Journal of Anatolian Environmental and Animal Sciences, 7(2), 178-183. https://doi.org/10.35229/jaes.1068313

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Journal of Anatolian Environmental and Animal Sciences

https://doi.org/10.35229/jaes.1808116

EBSCOHost Scilit CABI

JAES/AAS-Journal of Anatolian Environmental and Animal Sciences/Anatolian Academic Sciences&Anadolu Çevre ve Hayvancılık Dergisi/Anadolu Akademik Bilimler-AÇEH/AAS