Alfa galaktosidaz genlerinin insan patojeni aspergillus fumigatusdan klonlanması ve karakterizasyonu

In this study, molecular cloning studies were performed on the a-galactosidase genes of Aspergillus fumigatus IMI 385708. This organism is an opportunistic saprophytic fungus and a human pathogen, mainly affecting immunocompromised patients. A. fumigatus is a thermotolerant fungus and can efficiently produce thermostable a-galactosidase. Two different cloning strategies were undertaken in this study. A. fumigatus cDNA library, prepared previously, was screened with three different probes. No net results were obtained from these screenings. However, the DNA probes used were shown to be homologous to the a-galactosidase gene (agl1) of Trichoderma reesei. After the completion of the genome project of A. fumigatus, regions with homology to a-galactosidase genes were searched on the genome of A. fumigatus. PCR-based cloning studies were performed by designing specific primers for these regions. Two a-galactosidase genes, namely aglA and aglB were amplified with these specific primers, sequenced, and ligated to vector pUC19. The recombinant plasmid was then used to transform E. coli XL1 Blue MRF̕ cells. aglB gene consists of an open reading frame of 1684 bp containing six introns. The gene encodes a protein of 447 amino acids with a signal sequence of 22 amino acids and four N-glycosylation sites. aglA gene has an open reading frame of 1599 bp without introns. The gene encodes a protein of 532 amino acids with a putative signal sequence of 21 amino acids and four putative N-glycosylation sites. Cloning of a-galactosidase genes represents a first step towards the high level expression of these enzymes in a GRAS host.M.S. - Master of Scienc

KAPSAİSİN YÜKLÜ NANOEMULSİYONLARIN TASARLANMASI ve KARAKTERİZASYONU

TÜBİTAK TOVAG Proje01.01.2016Tıp, ilaç ve gıda endüstrisinde kullanımı artan nanoemülsiyonlar, fonksiyonel özellik gösteren bileşenleri enkapsüle ederek bu bileşenlerin etkili ve kontrollü salınımını, daha yüksek antimikrobiyal aktiviteye sahip olmalarını ve biyoyararlılıklarının artmasını sağlamaktadır. Emülsiyonlar, emülgatör molekülüyle çevrelenmiş her bir yağ damlacığının oluşturduğu yağ fazının su faz içinde dağılmasıyla oluşmaktadır. Kapsaisin, acı biberin birincil etken maddesidir ve literatürde hidrofobik kapsaisinin, hem kardiyovasküler, solunum ve sinir sistemleri üzerindeki biyoaktivitesinin hem de Staphylococcus aureus, Salmonella typhimurium, Bacillus cereus, Listeria monocytogenes, Helicobacter pylori gibi patojenler üzerinde antimikrobiyal aktivitesinin olduğu bulunmuştur. Bu çalışmanın hedefi, mikroakışkanlaştırma ve ultrasonik homojenizasyon yöntemleri ile antimikrobiyal aktiviteyi arttırmak için kapsaisin yüklü nanoemülsiyon sistemlerinin hazırlanması ve karakterizasyonunun yapılmasıdır. Projede, nanoemülsiyon oluşumunda mikroakışkanlaştırıcı ve ultrasonik homojenizasyonunun, sürfektan tipinin, pH ve sıcaklığın etkileri incelenmiştir. Nanoemülsiyonların karakterizasyonu ve fiziksel dayanıklılığın ölçülmesi için NMR Relaksometre başlıca metot olarak kullanılmış bunun yanında karakterizasyon amaçlı, parçacık boyut analizi, zeta potansiyel, transmisyon elektron mikroskopi, atomik kuvvet mikroskopisi de kullanılmıştır. Antimikrobiyal aktivite denemeleri yüzeye yayma yöntemi ile Escherichia coli için VRBA, Staphylococcus aureus için BPA ve disk difüzyonu yöntemi ile Penicillium italicum için DRBC kullanılarak yapılmıştır. %2’lik kapsaisin oleoresin içeren nanoemülsiyonların 15 dakika temas süresi sonunda E. colipopulasyonunda 3.4 log, S. aureus için ise 2 saat temas süresi sonunda 5.89 log düşüş görülürken Penicillium italicum üzerinde inhibisyon görülememiştir. %2’lik kapsaisin yüklü nanoemülsiyonların antioksidan aktivitesi, DPPH metoduna göre en yüksek 1.36 mg DPPH/ L bulunurken FRAP metoduna göre 0.14 mM Fe(II)/L olarak ultrasonikasyon tekniği ile sürfektan sükroz mono palmitat kullanılarak elde edilmiştir. Sürekli fazda gliserolün, her iki sistem ile sonuçlar üzerinde olumlu etki oluşturduğu gözlenmiştir. Mikroakışkanlaştırma yoluyla işlenmiş nanoemülsiyonlar, gelişmiş bir antimikrobiyal aktivite göstermiştir.NMRrelaksometri ile nanoemülsiyonların moleküler düzeyde yapılarında meydana gelen değişiklikler gözlenip parçacık boyutu, bulanıklık gibi diğer metotlarla ilişkilendirilebilmiştir.Böylece nanoemülsiyon sistemi ile etkisi zenginleştirilmiş gıdanın ihtiyacına yönelik kapsaisin içeren nanoemülsiyon sistemleri geliştirilebileceği gösterilmiştir.The application of nanoemulsions to food, medical and pharmaceutical industries has received great attention among the researchers especially for encapsulation of functional components for controlled release purposes and ensuring the prevention of degradation of active substances, increased rates of bioavailability and antimicrobial effect. Emulsions consist of a lipid phase dispersed in an aqueous continuous phase, with each oil droplet being surrounded by a thin interfacial layer consisting of emulsifier molecules. The primary active ingredient of chili pepper, capsaicin, is a hydrophobic substance that in the literature, it is proved to be a compound with a large bioactivity with manifested actions over the cardiovascular, respiratory and nervous systems and showing good antimicrobial activity against Staphylococcus aureus, Salmonella typhimurium, Bacillus cereus, Listeria monocytogenes, Helicobacter pylori. The aim of the proposed study is to prepare and characterize capsaicin loaded nanoemulsion systems by using microfluidization and ultrasonic homogenization techniques. In the project, effect of microfluidization, ultrasonication, emulsifier type, pH and temperature on nanoemulsion formation will be investigated. For characterization of the capsaicin-loaded nanoemulsions, the nondestructive technique NMR Relaxometry will be used. At the same time, particle size analysis, zeta potential, transmission electron and atomic force microscopy techniques will be used for characterization. Antimicrobial activity of nanoemulsions were evaluated using spread plate technique against Escherichia coli by using VRBA, Staphylococcus aureus by using BPA and disc diffusion technique against Penicillium italicum by using DRBC. In the experiments, nanoemulsion containing 2% capsaicin showed 3.4 log reductions of E. coli population after 15 min of contact time and 5.89 log reductions of S.aureuspopulation after 2 hours of contact time whereas the same concentration of nanoemulsions showed no inhibition on Penicillium italicum. Nanoemulsion containing 2% capsaicin showed highest antioxidant activity according to the DPPH method with 1.36 mg DPPH / L as well as according to FRAP method with 0.14 mM of Fe (II) / L by using surfactant sucrose monopalmitate and ultrasonication technique. Addition of glycerol to the continuous phase showed enhanced effect on the results within both systems. On the other hand, nanoemulsions processed by microfluidization were exhibited an enhanced antimicrobial activity. NMR relaxometry technique was helped to track changes in nanoemulsions at a molecular level that could be associated with the other methods such as particle size, turbidity. Therefore, nanoemulsions with an improved functionally which obtained by using essential oil capsaicin have been shown to meet the needs of food industry

Influence of mechanically alloyed fraction and hot rolling temperature in the last pass on the structure of Fe-14Mn-6Si-9Cr-5Ni (mass. %) shape memory alloys processed by powder metallurgy

Samples of Fe-14Mn-6Si-9Cr-5Ni (mass. %) shape memory alloy were sintered from two powder mixtures: (i) as blended (0_MA) and (ii) equal amounts of as blended and mechanically alloyed (MA’d) powders (50_MA). In order to increase the compactness degree, different specimens were hot rolled at 1373 K and the last rolling pass was performed at four different temperatures: (i) room temperature (RT); (ii) 873 K; (iii) 1073 K and (iv) 1373 K. In order to emphasize the influence of both MA’d fraction and rolling final temperature, the eight resulting specimens were analysed by X-ray diffraction (XRD) as well as by scanning electron microscopy (SEM). XRD results allowed the qualitative and quantitative identification of the three component phases, namely γ (face centred cubic, fcc)-parent phase, ε-(hexagonal close packed, hcp) martensite and α’- (body centred tetragonal, bct) martensite. SEM micrographs enabled to observe the influence of MA’d fraction on the relative amounts of the three phases. The results suggest that the presence of MA’d fraction contributes to destabilization of α’(bct) martensite and increase of the amount of ε(hcp) martensite, with favourable effects on shape memory behaviour.status: publishe

Structural changes caused by high-temperature holding of powder shape memory alloy 66% Fe - 14% Mn - 6% Si - 9% Cr - 5% Ni

Shape memory alloy Fe – 14Mn – 6Si – 9Cr – 5Ni (wt.%) obtained by cold pressing and sintering of a mixture of powder elements or of mechanically alloyed powder is studied. To compact the alloy additionally the specimens are subjected to hot rolling. Solution treatment of different duration (0.6 – 4.8 ksec) at 1473 K in low vacuum, nitrogen or argon is performed. The structure of the alloy after such treatments is studied by the methods of x-ray diffraction and optical and scanning electron microscopy.status: publishe