Hakan Karaoglu

In this study, the cloning, expression, and biochemical characterization of GTP cyclohydrolase I, the first enzyme involved in phenylketonuria metabolism, from the thermophilic bacterium Anoxybacteroides amylolyticum was investigated. The gchI gene, identified by PCR from A. amylolyticum, is a single open reading frame encoding a total of 186 amino acids with a size of 555 bp. The expression of the enzyme, which catalyzes the conversion of GTP to dihydroneopterin triphosphate, was induced in E. coli BL21(DE3) cells using 1 mM IPTG cloned into pET28a(+). A Ni-NTA affinity column was used for purification of the enzyme. The characterization studies revealed that the enzyme's Vmax was 6.9x10-5 ± 2.3x10-4 μM/min/mg protein and its Km was 7.0 ± 1.4 mM. The enzyme showed the best activity at a temperature of 70°C and an optimal pH of 7.0. It was demonstrated that thermophilic A. amylolyticum GCHI could be used in the first step of the conversion of GTP under in vitro conditions as an alternative to the chemical synthesis of BH4, which is used in the treatment of phenylketonuria.

Ksilanazlar [endo-1,4-β-D-ksilanaz (E.C. 3.2.1.8)] ksilanın çevre dostu bir yöntemle işlenmesine ihtiyaç duyulan endüstriyel birçok uygulamada kullanılan son derece önemli enzimlerdir. Bu çalışmada Anoxybacillus amyloliticus bakterisine ait intraselüler ksilanaz geni pET28a+ vektörüne klonlanarak üretilmiştir. Daha sonra enzim (AnaXYN329) saflaştırılarak enzimin sahip olduğu biyokimyasal ve kinetik özellikler açığa çıkarılmıştır. Elde edilen sonuçlara göre, AnaXYN329 en yüksek aktiviteyi pH 6.5’da; 60 ve 65 °C’de, göstermiştir. AnaXYN329 enziminin ısıl kararlılık deneyleri yaklaşık 120 dakika sürmüştür ve enzim yarılanma ömrünü 40. dakikada tamamlamıştır. Ayrıca enzimin Km değeri 3.631 ± 0.162 µg/µL, Vmax değeri 100.05 ± 0.00292 µmoL/dk/mg protein, kcat değeri 128,978 1/sn, kcat/Km değeri ise 35.514 olarak hesaplandı. Sonuç olarak AnaXYN329, endüstriyel uygulamalarda kullanılma potansiyeline sahiptir.

Fructose is the most preferred sugar to provide benefits for sweetening and health. As many industrial enzymes are used to produce High Fructose Syrup (HFS), it is vital to explore alternative enzymes for fructose production. Oligo-α-1,6-glucosidase (O-1-6-glucosidase) hydrolyzes non-reducing ends of isomaltooligosaccharides, panose, palatinose, and an a-limit dextrin by breaking α-1,6-glucoside bonds, although it generally has no activity on α-1,4-glucoside bonds of maltooligosaccharides. In this study, sucrose-hydrolyzing activity of O-1-6-glucosidase of thermophilic A. gonensis was evaluated. For this purpose, O-1-6-glucosidase gene region of A. gonensis was cloned in the pET28(a)+ expression vector, the expression product was purified, modeled, and biochemically characterized. The optimal activity of the enzyme was to be at pH 7.0 and 60 °C. The enzyme activity was halved at the end of the 276^th h at 60 °C. The enzyme maintained its activity even after 300 h at pH 6.0-10.0. The values of K_m, V_max, k_cat ,and k_cat/K_m were determined as 44.69 ± 1.27 mM, 6.28 ± 0.05 µmoL/min/mg protein, 6.70 1/s and 0.15 1/mMs^-1, respectively. While Zn²⁺, Cu²⁺, Pb²⁺, Ag²⁺, Fe³⁺, Hg²⁺, and Al²⁺ metal ions inhibited O-1-6-glucosidase, Mn²⁺, Fe²⁺, and Mg²⁺ ions activated the enzyme. Consequently, A. gonensis O-1-6-glucosidase (rAgoSuc2) has interesting properties, especially for HFS production.

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.