Anoxybacillus flavithermus DSM 2641T bakterisinin termofilik GCH-I enziminin klonlanması ve kinetik aktivitesinin belirlenmesi

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 phenylalanine 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. NH2TP is a difficult substance to obtain in studies that are difficult to synthesize and need to be used as a substrate. In this study, it was aimed to clone the gch-I gene from the thermophilic A. flavithermus bacterium and to investigate the activity of the enzyme for the enzymatic conversion of NH2TP, which is the substrate of many enzymes and is difficult to synthesize chemically. Fort his purpose, it was aimed to clone the gch-I gene from a thermophilic bacteria, Anoxybacillus flavithermus, and investigate the kinetic activity for enzymatic conversion from GTP as an alternative to the production of NH2TP. 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 65C. Under optimal conditions, GCH-I exhibited enzymatic activity with Kmand Vmax- values of 243 ± 23.25 μM and 100.93 ± 3.5 nM/min/mg protein, respectively.: 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, kimyasal sentezin zaman alıcı olması, maliyetler ve kimyasal kirleticilere maruz kalma nedeniyle farklı bir bakış açısıdır. NH2TP, kimyasal sentezi güç olan ve substrat olarak kullanılması gereken çalışmalarda temini zor bir maddedir. Bu çalışmada, bir çok enzimin substratı olan ve kimyasal yolla sentezlenmesi güç olan NH2TP’nin eldesinde alternatif olarak GTP’den enzimatik dönüşümü için termofilik A. flavithermus bakterisinden gch-I geninin klonlanması ve enzimin aktivitesinin araştırılması amaçlanmıştır. Bu amaç doğrultusunda, 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 65C 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

Antimicrobial and antiurease activities of newly synthesized morpholine derivatives containing an azole nucleus

2-[6-(Morpholin-4-yl)pyridin-3-ylamino]acetohydrazide (4) was obtained starting from 6-morpholin-4-ylpyridin-3-amine (2) via the formation of ester (3) and then converted to the corresponding Schiff bases (5, 6) with the reaction with aromatic aldehydes. The carbothioamide (9), obtained from the reaction of hydrazide with phenylisothiocyanate, was converted to the corresponding 1,2,4-triazole (11) and 1,3,4-thiadiazole (12) derivatives by the treatment with NaOH or H2SO4, respectively. The cyclocondenzation of 9 with 4-chlorophenacyl bromide or ethyl bromoacetate produced the corresponding 1,3-thiazole (10) or 1,3-thiazolidine derivatives (13), respectively. Antimicrobial and antiurease activities of newly synthesized compounds were investigated. Some of them were found to be active on M. smegmatis, and they displayed activity toward C. albicans and S. cerevisiae in high concentration. Compound 10 proved to be the most potent showing an enzyme inhibition activity with an IC50 = 2.37 +/- A 0.19 mu M

Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2

The cytidine-5'-triphosphate (CTP) synthase (EC 6.4.3.2) gene (pyrG) was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 (Ago). The gene is 1590 bp in length and encodes a protein of 530 amino acids, with a molecular mass of 59.5 kDa. The amino acid sequence of CTP synthase shares approximately 90%–94% similarity to Bacillus sp., and it belongs to the triad glutamine amidotransferases, which utilize a Cys–His–Glu triad for activity. Multiple sequence alignments revealed that the enzyme includes conserved amino acids responsible for catalytic activity and the binding of a divalent metal ion (Mg+2). AgoCTP synthase (AgoG2CTPs) was overproduced in Escherichia coli BL21 (DE3) pLysS as recombinant and purified by nickel affinity chromatography. Its biochemical characterization showed that the enzyme had maximal activity at pH 9.0–10.0 and 65 ºC. Km, Vmax, and kcat were found to be approximately 12.415 mM, 0.381 U/L, and 0.762 s–1 at 65 ºC, respectively. CTP synthase promotes the formation of CTP in dividing cells and is a recognized target for anticancer and antibacterial drugs. The results obtained from this study can be improved upon with the use of different species and substrates

Magnesite Enrichment with Pseudomonas oryzihabitans Isolated from Magnesite Ore

gurkok, sumeyra/0000-0002-2707-4371WOS: 000367816900005Magnesite is a primary source of magnesium and its compounds. The major problem in its practical use are the impurities such as silicon, iron and calcium carbonate. Some magnesite ores in Turkey cannot be used due to a high amount of CaCO3 (3%). In this study, bacterial isolates from magnesite quarries in Mersin were tested by plate assay for their ability to decalcify magnesite. A bacterial strain producing the largest clear zones in the plate assay was identified as Pseudomonas oryzihabitans by 16S rDNA-PCR and applied to magnesite ore. It was found to be effective in decalcifying magnesite ore without significant concurrent dissolution of the magnesium carbonate

Synthesis and antimicrobial activities of some new biheterocyclic compounds containing 1,2,4-triazol-3-one and 1,3,4-thiadiazole moieties

WOS: 0002807370000042-(4-Amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-N'-[(2,6-dihalogenophenyl)methylene]acetohydrazides (3a, b) was obtained via the formation of 2-(4-amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl) acetohydrazide (2), which was obtained starting from 4-amino-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (1) in 2 steps. 2-{[ 4-amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl] acetyl}-N-phenylhydra-zine carbothioamide (4), which was prepared starting from 2, was converted to the corresponding 1,3,4-thiadiazole derivative (5) in acidic media. Moreover, the basic treatment of 4 resulted in the formation of 4-amino-5-(4-chlorophenyl)-2-[(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl) methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (7). The reactions of compounds 5 and 7 with methyl iodide in the presence of sodium ethoxide afforded the corresponding N-methyl (6) and S-methyl (8) derivatives, respectively. The synthesis of Mannich bases (10a and 10b) was performed from the reaction of 7 with morpholine or piperazine in the presence of formaldehyde. All the newly synthesized compounds were screened for their antimicrobial activity. The antimicrobial activity study revealed that compounds 3a, 3b, and 5 showed good antimicrobial activities against the test microorganisms as compared with ampicillin.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [107T333]This work supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project no: 107T333)

Synthesis and antimicrobial activities of 2-(5-mercapto)- 1,3-oxadiazol-2-ylmethyl-1,2,4-triazol-3-one derivatives

The synthesis of 4-amino-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-5-(4-methylphenyl)-2,4-dihydro-3H - 1,2,4-triazol-3-one (2) and 4-amino-2-[(4-amino-5-mercapto-4H -1,2,4-triazol-3-yl)methyl]-5-(4-methylphenyl)- 2,4-dihydro-3H -1,2,4-triazol-3-one (5) was performed starting from 2-[4-amino-3-(4-methylphenyl)-5-oxo- 4,5-dihydro-1H -1,2,4-triazol-1-yl]acetohydrazide (1). The treatment of 2 and 5 with 4-fluorobenzaldehyde (for 8) or salicylaldehyde (for 6) afforded the corresponding Schiff bases (6 and 8). The condensation of 5 with phenacyl bromide produced 4-amino-5-(4-methylphenyl)-2-[(6-phenyl-7H-[1,2,4]triazolo[3,4- b][1,3,4]thiadiazin-3-yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (7). The alkylation of 2 and 8 was carried out by using methyl iodide in basic media. Compounds 3 and 10a,b were prepared by aminoalkylation of 2 and 8 with 4-fluorophenyl piperazine, morpholine, or 4-methyl piperazine in the presence of formaldehyde. The synthesized compounds were screened for their antimicrobial activities and, with the exception of 2, 4, 7, and 9, were found to possess good or moderate activities against the screened bacterial strains except Candida tropicalis and Candida albicans

Design, synthesis and antimicrobial activities of some azole derivatives

WOS: 000316432100007PubMed: 23614278Three new 1,3,4-oxadiazole, 1,3-thiazolidine and 1,2,4-triazole derivatives were obtained starting from furan-2-carbohydrazide. Then, 1,2,4-triazole compound was converted to the corresponding Mannich bases using several secondary amines including piperidine, piperazine, morpholine or thiomorpholine moiety. The synthesis of 5-(furan-2-yl)-4-{[(4-methoxyphenyl)methylidene]amino}-4H-1,2,4-triazole-3-thiol (XIII) was performed starting from furan-2-carbohydrazide by three steps. The structures of the synthesized compounds were well characterized by elemental analyses, IR, H-1 NMR, C-13 NMR and mass spectral studies. Newly synthesized compounds were screened for their antimicrobial activities and some of them displayed activity against the tested microorganisms.Karadeniz Technical University, BAP, TurkeyKaradeniz Teknik University [2007.111.002.5]The support provided by Karadeniz Technical University, BAP, Turkey (Ref. No. 2007.111.002.5) is gratefully acknowledged

Preparation and antimicrobial activity evaluation of some quinoline derivatives containing an azole nucleus

WOS: 000302661200003Quinoline-2-carbohydrazide (2) obtained from quinaldic acid (1) was converted to the corresponding carbothioamide 3 and carboxamide 6 by treatment with benzyliso(thio) cyanate. The basic treatment of 3 and 6 yielded the corresponding 1,2,4-triazole derivatives 4 and 7. The synthesis of 5-(quinolin-2-yl)-1,3,4-oxadiazol-2-thiol (9) was performed from the reaction of 1 with CS2 in basic media. The Mannich reaction of compounds 4, 7, and 9 resulted in the formation of aminoalkylated derivatives 5a-c, 8, and 10a,b. The condensation of 1 with thiosemicarbazide, carbohydrazide, or thiocarbohydrazide gave the corresponding 1,2,4-triazole derivatives (11-13). The treatment of 4-amino-5-(quinolin-2-yl)-4H-1,2,4-triazole-3-thiol (13) with 4-chlorophenacyl bromide caused the formation of fused triazolothiadiazine 14. The condensation of 13 with 4-methoxybenzaldehyde generated the corresponding Schiff base 15. The newly synthesized compounds were characterized by elemental analyses, IR, H-1-NMR, C-13-NMR, and mass spectra. The antimicrobial activity study revealed that some of the newly synthesized compounds showed good to moderate activity against a variety of microorganisms.Karadeniz Technical University, BAP, TurkeyKaradeniz Teknik University [2007.111.002.5]Support was provided by Karadeniz Technical University, BAP, Turkey (Ref. No. 2007.111.002.5)

Synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives

PubMed: 20428053Some novel 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one (3, 6, 8, 9) derivatives and or 3-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole (5) were synthesized from the reaction of various ester ethoxycarbonylhydrazones (1a-e) with several primary amines. The synthesis of 4-amino-5-(4-chlorophenyl) -2-[(5-mercapto1,3,4-oxadiazol-2-yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (13) was performed starting from 4-Amino-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2, 4-triazol-3-one (2) by four steps; then 13 was converted to the corresponding Schiff base (14) by using 4methoxybenzaldehyde. Finally, two Mannich base derivatives of 14 were obtained by using morpholine or methyl piperazine as amine component. All newly synthesized compounds were screened for their antimicrobial activities and some of which were found to possess good or moderate activities against the test microorganisms. Copyright © 2010 by the authors

Reduction, mannich reaction, and antimicrobial activity evaluation of some new 1,2,4-triazol-3-one derivatives

WOS: 000305556200008Ethyl[4-arylmethyleneamino-3-(4-metylphenyl)-5-oxo-4,5-dihydro-1 H -1,2,4-triazole-1-yl] acetates (3a-e and 10a-d) were obtained starting from 4-amino-2,4-dihydro-3H -1,2,4-triazol-3-ones (1 and 9) in 2 steps. The treatment of 3a-e with NaBH4 resulted in the formation of 3 kinds of product incorporating carboxcilic acid (4a-c) or alcohol (5a-e) functionality. [4-{[(4-Methoxyphenyl) methylene] amino}- and 4-{[pyridin-4ylmethylene] amino}-3-(4-methylphenyl)-5-oxo-4,5-dihydro-1 H -1,2,4-triazole-1-yl] acetic acids (6a, 6e) were obtained by the hydrolysis of the corresponding esters (5a-5e). The treatment of 6a with NaBH4 caused the reduction of only the imine bond; the carboxyl group remained unchanged. Then this carboxylic acid was converted to the corresponding hydrazide (8) in 2 steps by reaction with ethanol and hydrazine hydrate, respectively. The synthesis of Mannich bases was performed by the reaction of the corresponding 1,2,4-triazoles containing an imine group (10a-d) with several primary or secondary amines including morpholine or piperazine nucleus. All newly synthesized compounds were screened for their antimicrobial activity. The antimicrobial activity study revealed that the Mannich bases (11-16) showed good activity against the test microorganisms as compared with ampicillin.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [107T333]; Karadeniz Technical University, BAP, TurkeyKaradeniz Teknik University [2007.111.002.6]The authors are grateful for the support provided by the Scientific and Technological Research Council of Turkey (TUBITAK, Project no: 107T333) and Karadeniz Technical University, BAP, Turkey (Ref. No. 2007.111.002.6)