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Objectives: Unlike higher organisms such as domestic animals and cultivated plants, which display a robust reproductive isolation and limited dispersal ability, microbes exhibit an extremely promiscuous gene flow and can rapidly disperse across the planet by multiple ways. Thus, microbial plasmids, including synthetic replicons, containing antibiotic resistance genes are a serious risk to public health. In this short communication, we explored the presence of synthetic elements in alfalfa symbionts (Ensifer meliloti strains) from agricultural soils. Methods: A total of 148 E. meliloti isolates from alfalfa plants growing under field conditions were collected from January 2015 to June 2019. Antimicrobial susceptibility testing was performed under laboratory conditions. We identified five kanamycin-resistant E. meliloti strains (named K1-K5). Whole genome sequencing analysis and conjugations were used to identify and study the plasmids of K strains. Results: We found that the genomes of K strains contain ampicillin, kanamycin and tetracycline resistance genes, the reporter gene lacZ from Escherichia coli and multiple cloning sites. These sequences were found within

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. &beta, Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding &beta, galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with &beta, galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-&beta, galactoside and lactose. The activity levels of one of these novel &beta, galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic &beta, galactosidases from diary stabilization ponds&rsquo, metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries&rsquo, by-products.

The conformational itineraries taken by carbohydrate residues in the catalytic subsite of retaining glycoside hydrolases (GHs), harness the link between substrate conformation and reactivity. GHs’ active sites may be described as a combination of subsites dedicated to the binding of individual sugar residues and to catalysis. The three-dimensional structure of GH:carbohydrate complexes has demonstrated that carbohydrate ring conformation changes in an ordered manner during catalysis. Here we demonstrate in silico that a link exists between subsite binding dynamics and substrate specificity for β-galactosidases from clan GH-A families GH1, GH2, GH35, GH42 and GH59. Different oligosaccharides were docked in the active site of reference β-galactosidase structures using Vina-Carb. Subsequent molecular dynamics (MD) simulations revealed that these enzymes favor a high degree of flexibility and ring distortion of the substrate the lytic subsite −1. Although the β-galactosidase families examined are structurally and mechanistically related, distinct patterns of ring distortion were unveiled for the different families. For β-galactosidases, three different family-dependent reaction itineraries (1S3 → 4H3‡ → 4C1, 1,4B → 4H3/ 4E‡ → 4C1, and 1S5 → 4E/ 4H5‡ → 4C1) were identified, all compatible with the antiperiplanar lone pair hypothesis (ALPH) for the hydrolysis of β-glycosides. This comparative study reveals the fuzzy character of the changes in carbohydrate ring geometry prior to carbohydrate hydrolysis.

Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. Recent studies have shown, neurogenesis continues into adulthood but is known to be decreasing during advancing age and these changes may contribute to cognitive alterations. Advances, which aim to promote better aging are of paramount importance. Dietary restriction (DR) is the only non-genetic intervention that reliably extends life and health-span. Mechanism's of how and why DR and age affect neurogenesis are not well-understood, and have not been utilized much in the zebrafish, which has become a popular model to study brain aging and neurodegenerative disease due to widely available genetic tools. In this study we used young (8-8.5 months) and old (26-32.5 months) zebrafish as the model to investigate the effects of a short-term DR on actively proliferating cells. We successfully applied a 10-week DR to young and old fish, which resulted in a significant loss of body weight in both groups with no effect on normal age-related changes in body growth. We found that age decreased cell proliferation and increased senescence associated beta-galactosidase, as well as shortened telomere lengths. In contrast, DR shortened telomere lengths only in young animals. Neither age nor DR changed the differentiation patterns of glial cells. Our results suggest that the potential effects of DR could be mediated by telomere regulation and whether these are beneficial or negative remains to be determined. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (&beta, gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with &beta, gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.

Ethnopharmacological relevance: Ganoderma lucidum (Lingzhi or Reishi) is a medicinal mushroom historically used in Asian countries to treat a wide variety of diseases and prolong life. In the last years, G. lucidum has been internationally recognized as an effective adjuvant in cancer treatment. Among active components, the most recent research indicates that polysaccharides modulate the immune response favoring the recovery from toxicity of chemo and radiotherapy while triterpenes are cytotoxic to tumoral cells mainly by altering gene expression. Beyond this body of evidence on the efficacy of G. lucidum in cancer treatment, it is not yet understood whether these extracts exert the same mechanisms of action than current antitumoral drugs. Aim of the study: In this study, we tested the DNA damaging potential of G. lucidum extracts by the β-galactosidase biochemical prophage induction assay (BIA) using doxorubicin, a DNA intercalating agent, as a positive control. This assay was traditionally used to screen microbial metabolites towards antitumoral agents. Here, we used this bacterial assay for the first time to assess DNA damage of herbal drugs. Results: After a bioguided assay, only a purified fraction of G. lucidum containing a mixture of C16 and C18:1 fatty acids exerted weak activity which could not be attributed to direct interaction with DNA. At the same concentrations, the induction observed for doxorubicin was clearly contrasting. Conclusions: The micro BIA assay could be successfully used to demonstrate differences in cellular effects between G. lucidum extracts and doxorubicin. These results showed that G. lucidum extracts display weak DNA damaging potential. Since DNA injury promotes aging and cancer, our results substantiate the traditional use of this mushroom to prolong life. Fil: Vela Gurovic, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Viceconte, Fátima Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Pereyra, Marcelo Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentina Fil: Bidegain, Maximiliano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Cubitto, María Amelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina

Çalışmamızda Bingöl Binkap sıcak su kaplıcasından izole edilen Bacillus subtilis 4NK ve Bacillus paralicheniformis 5NK’nın intrasellüler β-Galaktosidaz gen ve enzimi üzerine çalışmalar yapılmıştır. β-Galaktosidaz geni için tasarlanan bazı spesifik primerler kullanılarak PCR yardımıyla bu genin amplifikasyonu gerçekleştirilmeye çalışılmıştır. Her üç bakterinin β-galaktosidaz gen varlığının tayini için farklı primerler kullanarak yapılan PCR ürünleri incelendiğinde üç genin ürününün de ~2.4 kb olduğu tespit edildi. Enzim üzerine yapılan sıcaklık ve pH’nın etkisi 25-65˚C ve pH 4.0-11.0 aralığında incelendi. Bacillus subtilis 4NK ve Bacillus paralicheniformis 5NK için optimum sıcaklık ve pH değerleri sırasıyla 45˚C, pH 6.0 ve 55˚C, pH 6.0 olarak bulundu. Bacillus subtilis 4NK ve Bacillus paralicheniformis 5NK enzimi için termal stabiliteler sırasıyla 40-50˚C ve 45-60˚C aralıklarında 30-120dk. da incelendi. Bacillus subtilis 4NK’nın termal stabiliteye sahip olduğu bulundu. Bacillus paralicheniformis 5NK termal stabiliteye sahip olduğu ancak 55˚C de aktiviteyi kısmen koruduğu 60˚C de ise koruyamadığı bulundu. Bacillus subtilis 4NK ve Bacillus paralicheniformis 5NK için amonyum sülfat çöktürmesi ve diyaliz yoluyla kısmi olarak saflaştırma işlemi uygulandı. Bu saflaştırma sonucunda Bacillus subtilis 4NK diyaliz sonrasında enzimin verimi %85.2, saflaştırma katsayısı da 2.8 olarak bulundu. Bacillus paralicheniformis 5NK verimi %76.8, saflaştırma katsayısı 2.0 olarak bulundu. Enzim üzerine bazı kimyasal, metal ve şelatörlerin etkisi denendi. Bacillus subtilis 4NK β-galaktosidazı için bu kimyasallardan PCMB (4-kloro civa benzoik asid)’in çok yüksek oranda, DTT, PMSF ve NEM’in düşük oranda inhibisyona yol açtığı, iodoasetamid’ in ise enzim üzerine ciddi etkisi olmadığı bulundu. Bacillus paralicheniformis 5NK β-galaktosidazı için bu kimyasallardan iodoasetamid ve PCMB’in yüksek oranda inhibisyona neden olduğu, PMSF’nin 10 mM da yüksek düzeyde inhibisyonuna neden olduğu ve NEM’in 5 ve 10 mM’ da kısmı inhibisyonuna neden olduğu bulundu. DTT’nin ise enzim üzerine önemli oranda etkisi olmadı. Bacillus subtilis 4NK β-galaktosidazı için yapılan incelemeler sonucunda düşük konsantrasyonlarda CoCl2 ve MnCl2‘ün (1ve 2.5 mM) yüksek oranlarda enzimi aktive ettiği bulundu. CuCl2 ve CdCl2 yüksek konsantrasyonlarda (10 ve 20 mM) inhibisyona neden oldu. EDTA’nın ise tüm konsantrasyonlarda enzimi aktive ettiği bulundu. Bacillus paralicheniformis 5NK βgalaktosidazı için yapılan incelemeler sonucunda birçok konsantrasyonda CuCl2 ve CdCl2’ün yüksek oranlarda enzimi inhibe ettiği bulundu. EDTA’nın ise enzimi aktivasyonunda ciddi anlamda etki etmediği bulundu.NB besiyerinde %1 laktozlu ve laktozsuz besiyerleri kullanılarak 6-48 saat aralığında kültüre alınan Bacillus subtilis 4NK ve Bacillus paralicheniformis 5NK bakterilerinin çoğalması ve enzim üretimi üzerine laktozun etkisi incelendi. 4NK bakteri varyetesinde laktozlu ortamda 24. saatten sonra bakteri çoğalmasında artış olduğu bulundu. Fakat 5NK bakteri çoğalması açısından laktozlu ortamda laktozsuz ortama göre değişiklik olmadı. Her iki bakteri için de laktozlu ortam enzim üretimini arttırdığı bulundu. Enzimin Km ve Vmax değerlerini bulmak için farklı oNPG konsantrasyonları kullanıldı. Km ve Vmax değerleri 4NK ve 5NK için sırasıyla; 23.80 mM, 1.978 abs/dk. ve 5.61 mM, 1.869 abs/dk olarak bulundu. Anahtar Kelimeler: Bacillus subtilis 4NK, Bacillus paralicheniformis 5NK, Anoxybacillus ayderensis FMB1, β-galaktosidaz karakterizasyonu, PCR In our study, intracellular β-Galactosidase gene and enzyme of Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK isolated from Bingol Binkap hot spring water, as well as the enzyme gene of Anoxybacillus ayderensis FMB1 have been studied. In addition, PCR amplification of β-galactosidase gene was performed by using some specific primers of the gene. β-Galactosidase gene presence of all three bacteria were detected by using using different primers and PCR products obtained were ~2.4kb. The effect of temperature and pH on the enzyme was investigated at temperatures of 25-65˚C and at pH 4.0-11.0. The optimal temperature and pH values for Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK were 45˚C, pH 6.0 and 55˚C, pH 6.0, respectively. Thermal stability for enzymes of Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK was examined at 40-50˚C and 45-60˚C for 30-120 min, respectively. β-Galactosidase of Bacillus subtilis 4NK was found to posses thermal stability. The enzyme of Bacillus paralicheniformis was found to have thermal stability at lower temperature, partially at 55˚C activity but the activity was not protected at 60˚C. Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK were partially purified by ammonium sulfate precipitation and dialysis. As a result of this partial purification, the yield of enzyme was 85.2% and the purification fold was 2.8 for enzyme of Bacillus subtilis 4NK. The yield of Bacillus paralicheniformis 5NK enzyme was 76.8% and the purification fold was 2.0. Some chemicals, metals and chelators were tested on the enzyme activity. It was found that PCMB (4-chloro mercuric benzoic acid) caused very high inhibition on Bacillus subtilis 4NK β-galactosidase, while inhibition was very low by DTT, PMSF and NEM, but iodoacetamide did not seriously affect enzyme activity. For Bacillus paralicheniformis 5NK β-galactosidase, it was found that iodoacetamide and PCMB caused high inhibition, PMSF caused high inhibition at 10 mM, and partial inhibition was observed by NEM at 5 and 10 mM. DTT had no significant effect on the enzyme. Studies on Bacillus subtilis 4NK β-galactosidase revealed that CoCl2 and MnCl2 highly activated the enzyme at low concentrations (1, 2.5 mM). CuCl2 and CdCl2 caused inhibition at high concentrations (10 and 20 mM). Whereas EDTA was found to activate the enzyme at all concentrations. Studies on Bacillus paralicheniformis 5NK β- galactosidase revealed that CuCl2 and CdCl2 inhibited the enzyme at high ratios at several concentrations. However, EDTA had no significant effect on enzyme activation. Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK bacteria cultured on a NB medium with lactose and lactose-free media for 6-48 h were examined for bacterial growth and enzyme production. The strain 4NK showed an increase in bacterial growth after 24 hours in the medium with lactose. However, bacterial growth in the lactose medium for the strain 5NK did not change compared to lactose free medium. It was found that lactose increased enzyme production for both bacteria. Different oNPG concentrations were used to find the Km and Vmax values of the enzyme. Km and Vmax values for the strains 4NK and 5NK were 23.80 mM, 1.978 abs / min. and 5.61 mM, 1.869 abs / min, respectively.

Çalışmamızın amacı ağız kokusu (AK) olan kronik periodontitisli, ağız kokusu olmayan kronik periodontitisli ve periodontal sağlıklı bireylerde plak indeksi (PI), gingival indeks (GI), cep derinliği (CD), klinik ataşman seviyesi (KAS), halimeter değerleri (HMD), organoleptik ölçüm skorları (OLS), Winkel dil kaplama indeksi (WDKI) ve tükürük β – galaktozidaz düzeylerinin başlangıçta ve faz I periodontal tedaviden 1 ay sonra değerlendirilmesi ve faz I periodontal tedavinin parametreler üzerindeki etkinliğinin araştırılmasıdır. Çalışmada yer alan ağız kokusu olan kronik periodontitisli (Grup1-AK(+); 25 hasta), ağız kokusu olmayan kronik periodontitisli (Grup2-AK(-); 25 hasta) ve periodontal olarak sağlıklı (Grup3-Kontrol; 25 hasta) bireylerde PI, GI, CD, KAS, HMD, OLS, WDKI ve tükürük β – galaktozidaz düzeyleri ölçümleri başlangıçta ve faz I periodontal tedaviden 1 ay sonra gerçekleştirildi. Kronik periodontitisli hastalar dil yüzeği temizliği konusunda bilgilendirildi. Sonuçlar istatistiksel olarak değerlendirildi (p

Bu çalışmada, Türkiye'nin Güneydoğu Bölgesi'nde yer alan Batman ilindeki birakaryakıt istasyonundan izole edilen mezofilik Enterobacter sp. 3TP2A bakterisinin yüksekmiktarda mezofilik β-galaktosidaz enzimi ürettiği görülmüştür. Ayrıca, laktozun β-galaktosidaz üretimini önemli ölçüde arttırdığı görülmüş olup bu bulgu, bu enzimin endükeedilebilir bir enzim olduğunu göstermektedir. Enterobacter sp. 3TP2A bakterisinden izoleedilen intraselüler β-galaktosidaz saflaştırılıp karakterize edildi. Saflaştırma işlemi %70amonyum sülfat çöktürmesi, diyaliz, ultrafiltrasyon ve son olarak da Sephadex G-75kromatografisi yöntemi kullanılarak gerçekleştirilmiştir. Enzim, jel geçirgenlikkromatografisi kullanılarak yaklaşık %11 verimle 17.3 kat saflaştırıldı. Saflaştırılanenzimin pH 8.0 ve 35 oC'de stabil olduğu ve moleküler ağırlığının ise SDS-PAGE venative-PAGE metodlarına göre yaklaşık 60 kDa olduğu tespit edilmiştir.Çeşitli metal iyonların farklı CaCl2, MgCl2, ZnCl2, CuCl2 ve EDTA (1, 2, 5, 10 ve20 mM) konsantrasyonlardaki etkileri incelenmiştir. EDTA ve Cu2+'nın Enterobacter sp.3TP2A'dan saflaştırılan β-galaktosidaz üzerinde inhibe edici bir etkiye sahip olduklarıbelirlenmiştir. EDTA'nın enzim aktivitesini %76'ya kadar inhibe ettiği ve Cu2+'nın β-galaktosidaz üzerindeki inhibe edici etkisinin düşük konsantrasyonlarda bile güçlü olduğugörülmüştür (96.9%). Bununla birlikte, Mg2+'nın saflaştırılan enzimin aktivasyonuna sebepolduğu görülmüştür. Ca2+ ise enzim aktivitesini önemli ölçüde etkilememiş olup 20 mM (yalnızca 16%)'de enzimin deaktivasyonuna sebep olmuştur. Ancak, Zn2+ 1, 2 ve 5 mM'deenzimin aktivasyonuna sebep olmuştur (sırasıyla, %32, %27, %8). Mg2+konsantrasyonundaki artışın 47%'ye kadar enzim aktivasyonuna sebep olması ve EDTAtarafından inhibe edilmesi, bu enzimin metal-bağımlı veya metaloenzim olduğunugöstermektedir. Buna ilaveten, farklı konsantrasyonlardaki inhibitörlerin saflaştırılan enzimüzerindeki etkileri şu konsantrasyonlarda incelenmiştir: PCMB (0,2, 0,4, 1, 2 mM) , Iodo,DTT, β-mer, N-etil, (1, 2, 4, 8 mM). Enzim, N-etil tarafından tamamen inhibe edilmesinekarşın (%100), DTT tarafından etkilenmemiştir. Enzim, β-galaktosidaz aktivitesini 8mM'de %14 oranında arttıran β-mer tarafından hafifçe etkilenmiştir. Iodo da β-galaktosidazaktivitesini hafifçe etkilemiştir (%13'e kadar). PCMB ise enzimatik aktiviteyi %87'lerevaran inhibisyonla büyük oranlarda etkilemiştir.Lineweaver-Burk grafiği lineer olarak belirlenmiş olup bu sonuç basit birMichealis-Menten kinetiğine işaret etmektedir. Vmax 0.701 (μmol/ dak mg) ve Km 0.104mM olarak tespit edilmiştir. Ayrıca, laktoz hidrolizi süresinin, saflaştırılan β-galaktosidaztarafından katalize edilen reaksiyondan dolayı, 10 saate kadar çıktığı gözlemlenmiştir.Bu çalışmanın amacı Enterobacter sp. 3TP2A'dan mezofilik β-galaktozidaz'ısaflaştırmak, karakterize etmek ve laktoz hidrolizi gibi biyoteknoloji alanında kullanımınıtest etmekti. Elde edilen bulgular bu enzimin iyi bir aday olabileceğini göstermiştir. In this study, a mesophilic Enterobacter sp. 3TP2A isolated from petroleum stationin Batman in the southeast of Turkey was identified and found to produce a high amount ofmesophilic β-galactosidase. The lactose was found to increase the β-galactosidaseproduction to a great extent, meaning that this enzyme is inducible. An intracellular β-galactosidase from Enterobacter sp. 3TP2A was purified and characterized. The enzymewas purified by ammonium sulphate precipitation 70%, dialysis, ultrafiltration and finallyusing Sephadex G-75 chromatography method. The enzyme was purified to 17.3-fold afterGel permeation chromatography with a yield of approximately 11%. The purified enzymewas found to be stable in pH 8.0 and temperature of 35 oC. The Molecular weight of thepurified enzyme was found to be about 60 kDa by both SDS-PAGE and native-PAGE.The effects of various metal ions at different concentrations of CaCl2, MgCl2,ZnCl2, CuCl2, and EDTA (1, 2, 5, 10 and 20 mM) were tested. EDTA and Cu2+ had aninhibitory effect on the β-galactosidase purified from Enterobacter sp. 3TP2A. EDTAinhibited the enzyme activity (upto 76%) and Cu2+ had strong inhibitory effect on β-galactosidase even at low concentrations (96.9%). However, Mg2+ caused activation of thepurified enzyme. Ca2+ did not effect enzyme activity to a great extent, causing deactivationof the enzyme at 20 mM (only 16%), while Zn2+ at 1, 2 and 5 mM inhibited enzymeactivity (32, 27, 8%, respectively). Increase in the concentration of Mg2+ causing activation upto 47% and also inhibition by EDTA show that the enzyme is metal-dependent or ametalloenzyme. Also determining the effect of different concentration of inhibitors onpurified enzyme; PCMB (0.2, 0.4,1, 2 mM) , Iodo, DTT, β-mer, N-ethyl, (1, 2, 4, 8 mM).The enzyme was completely inhibited by N-ethyl (100%), but not affected by DTT. Theenzyme was slightly affected by β-mer enhancing β-galactosidase activity at 8mM with14% . The Iodo had a slight effect on β-galactosidase activity (upto 13%). PCMB inhibitedthe enzymatic activity to a great extent upto approx. 87%.The Lineweaver-Burk plot was linear, suggesting a simple Michealis-Mentenkinetics. The Vmax was found as 0.701 (μmol/ min mg) and Km was found as 0.104 mM. Itwas also found that the time for lactose hydrolysis continues up to 10 h with the reactioncatalyzed by purified β-galactosidase.The aim of this study was to purify and characterize the mesophilic β-galactosidasefrom Enterobacter sp. 3TP2A and then to test for use in biotechnology such as lactosehydrolysis. The results obtained indicated that this species may well be a good candidate. 121

Re-expression of E2 in human papillomavirus (HPV) transformed tumour cells can induce apoptosis; however, some evidences also attribute an important role to E2 in sustaining tumorigenesis. In the present paper, we studied the effects of tumour necrosis factor (TNF)-α-mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) activation on E2-induced senescence in HPV16-integrated SiHa cells. The results show that E2 inhibits endogenous E6 gene expression and sensitizes SiHa cells to TNF-α-induced NF-κB activation. Under this condition there was an increase in the expression of senescent proteins p53, p21, p27 and p16 and senescence-associated (SA)-β-galactosidase activity indicating that TNF-α augments E2-mediated senescence. Re-expression of E2 expression with TNF-α treatment resulted in an increase in the expression of anti-apoptotic Bcl2 (B-cell lymphoma 2) protein and other pro-survival genes like cyclin D1 (cyc D1), survivin and hTERT (human telomerase reverse transcriptase). Concomitantly, E2 + TNF-α combination increased the survival of SiHa cells by positive changes in viability, proliferation and colony formation. E2-induced apoptotic tendency shifted towards senescence in presence of TNF-α by arresting the cells at both G0/G1 and G2/M phases, thus enhancing cell survival. Another observation in the present study is the significant up-regulation of key senescence messaging factors regulated by NF-κB namely interleukin (IL)-6, IL-8, high-mobility group protein A (HMGA)1 and B (HMGB)1 in E2-transfected cells treated with TNF-α. Our data provide a mechanistic basis and a new insight for the role of TNF-α and E2 in linking cellular senescence, tumorigenesis and HPV re-infection., Human papillomavirus (HPV)16 E2 potentiates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) activation induced by tumour necrosis factor (TNF)-α in SiHa (human cervical cancer) cells and significantly influences cell viability, apoptosis and expression of pro-survival genes regulated by NF-κB.

A novel packed-bed bioreactor, operating under isothermal and non-isothermal conditions, has been constructed. The core of the apparatus consisted in a polypropylene ring filled with beta-galactosidase immobilized on beads of polyacrylic acid, grafted with dimethylaminoethyl methacrylate. Phenylendiamine and glutaraldehyde were used as spacer and coupling agent, respectively. Two lateral nylon membranes held the enzyme beads into the ring and allowed the occurrence of the process of thermodialysis when the bioreactor was operating under non-isothermal conditions. Comparison of the enzyme activity under isothermal and non-isothermal conditions has shown that in the presence of temperature gradients the rate of lactose hydrolysis was increased, with a reduction of the apparent Km value. Under non-isothermal conditions the percentage increases of enzyme activity were found to decrease with the increase of the substrate concentration. The results have been explained within the frame of reference of the process of thermodialysis.

A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the λgt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. β-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S -transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.

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The aim of this study was to characterize the probiotic characteristics of Lactobacillus fermentum strains isolated from Tulum cheese. Seven L.fermentum strains were selected among the isolated and identified lactobacillus strains due to their abundance. When the gastric condition was considered, L.fermentum LP3 and LP4 were able to tolerate pH 2.5 and 1% bile salt. All L.fermentum strains had similar enzymatic activity and antibiotic resistance pattern but the highest antagonistic effect was detected within LP3, LP4 and LP6. Cholesterol assimilation amount of L.fermentum strains ranged between 12.1 and 45.3% in MRS and 20.7-71.1% in MRS with bile. The highest cholesterol assimilation in MRS and MRS with bile was occurred by LP3 and LP4, respectively. L.fermentum LP2 adhered to caco-2 cells more than Lactobacillus rhamnosus LGG where LP3, LP4 and LP5 adhered at similar level. In conclusion, L.fermentum LP3 and LP4 fulfilled sufficient criteria to be probiotics for use as a starter culture in the production of tulum cheese or other dairy products. Also this study indicated that some food-associated Lactobacillus strains non-predominant for gut biota have significant probiotic potential. © 2014 Elsevier Ltd.

β-D-Galaktosidaz (EC 3.2.1.23, laktaz) laktozu, glikoz ve galaktoza hidrolizini katalizleyen enzimdir. Doğada yaygın olarak bulunur ve bitkisel, hayvansal ve mikrobiyal kaynaklardan izole edilir. Bu çalışmada, nohuttan β-galaktosidaz saflaştırılması üç fazlı ayırma (TPP) yöntemi kullanılarak ilk defa uygulandı. pH 6,8'de, 1:0,5 ham enzim çözeltisi:t-bütanol oranı ve %60 doygun amonyum sülfat kullanarak uygulanan TPP yöntemi sonucunda enzim, %133 verim ile 10,1 kat saflaştırıldı. Saflaştırılan enzimin, SDS-PAGE analizi ile molekül ağırlıkları 48 ve 38 kDa olan iki alt birim içerdiğini gösterdi. Native-PAGE ve zimogram analizi ile enzimin molekül ağırlığı 85 kDa olarak bulundu. Saflaştırılan enzimin optimal pH ve sıcaklığı, sırasıyla 2,8 ve 50oC olarak bulundu. Arrheius aktivasyon enerjisi (Ea) 28,63 kJ/mol olarak hesaplandı. Saflaştırılan enzim, 45oC'de 30 dakika inkübasyon sonunda aktivitesinin %60'ını korurken, bununla birlikte, 60oC'de 30 dakika inkübasyon sonunda aktivitesinin %80'ini kaybettiği gözlemlendi. 37oC'de pH 2,8'de enzimin, Michaelis-Menten sabiti (Km) ve maksimum hızı sırasıyla 1,1 mM ONPG ve 0,90 U/ml/dk olarak bulundu. Saflaştırılan enzimin turnover sayısı (kcat) ve katalitik performansı (kcat/Km) sırasıyla 212,26 dk-1 ve 193 dk-1mM-1 olarak bulundu. Enzimin termodinamik parametreleri; ΔG#, 62,21 kJ/mol; Δ#E-T, -13,56 kJ/mol; Δ#E-S, 0,25 kJ/mol; ΔH#, 26,06 kJ/mol; ΔS#, -0,12 kJ/mol.K olarak hesaplandı. β-Galactosidase (Lactase, E:C 3.2.1.23) is an enzyme which catalyze the hydrolysis of lactose to glucose and galactose. The enzyme is widespread in nature and can be isolated from herbal, animal and microbial sources. In this study, the purification of β-galactosidase from chick pea (Cicer arietinum) was carried out first time by three phase partitioning method (TPP). Enzyme was 10.1 fold purified with 133% activity recovery by the method of TPP obtained by using 60% saturated ammonium sulphate and crude enzyme extract:t-butanol in 1:0.5 ratio at pH 6.8. SDS-PAGE analysis of purified enzyme showed that enzyme has consisted two subunits in 48 and 38 kDa molecular weight. The molecular weight of native enzyme was found to be 85 kDa as results of Native-PAGE and zymogram analysis. Optimal pH and temperature of purified enzyme were found to be 2.8 and 50oC, respectively. Arrhenius activation energy (Ea) was determined as 28.63 kJ/mol. Purified enzyme is retained the 60% of its activity after 30 minutes incubation at 45oC, however, 80% activity loss was observed at 60oC. Michaelis-Menten constant (Km) and maximal velocity of enzyme were found to be 1.1 mM ONPG and 0.90 U/ml/min at 37oC and pH 2.8. Turnover coefficient (kcat) and catalytic efficiency (kcat/Km) of purified enzyme were found to be 212.26 min-1 and 193 min-1mM-1 in order. Thermodynamic parameters of enzyme were calculated as ΔG#, 62.21 kJ/mol; Δ#E-T, -13.56 kJ/mol; Δ#E-S, 0.25 kj/mol; ΔH#, 26.06 kj/mol; ΔS#, -0.12 kJ/mol K. 57

Apoptosis or programmed cell death plays a pivotal role in embryonic development and maintenance of homeostasis. It is also involved in the etiology of pathophysiological conditions such as cancer, neurodegenerative, autoimmune, infectious, and heart diseases. Consequently, the study of apoptosis is now at center of both basic and clinical research applications. Therefore, sensitive and simple apoptosis detection techniques are required. Here we describe a monoclonal antibody–defined novel antigen, namely NAPO (negative in apoptosis), which is specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle, including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive, and easy method for differential identification of apoptotic and nonapoptotic cells.

International audience; Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45°C) or bacteriostatic (30°C) incubation in egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated through the release of a specific intracellular enzyme (β-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45°C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was disrupted. In addition, the cytoplasmic β-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30°C, biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.

Introduction Salmonella enterica Typhimurium (ST) is a phagosomal pathogen that can infect placental trophoblast cells leading to abortion and severe maternal illness. It is unclear how the trophoblast cells promote profound bacterial proliferation. Methods The mechanism of internalization, intracellular growth and phagosomal biogenesis in ST-infected human epithelial (HeLa), macrophage (THP-1) and trophoblast-derived cell lines (JEG-3, BeWo and HTR-8) was studied. Specific inhibitors were used to block bacterial internalization. Phagosomal maturation was determined by confocal microscopy, Western-blotting and release of lysosomal β-galactosidase by infected cells. Bacterial colony forming units were determined by plating infected cell lysates on agar plates. Results ST proliferated minimally in macrophages but replicated profoundly within trophoblast cells. The ST-ΔinvA (a mutant of Salmonella pathogenicity island-1 gene effector proteins) was unable to infect epithelial cells, but was internalized by scavenger receptors on trophoblasts and macrophages. However, ST was contrastingly localized in early (Rab5+) or late (LAMP1+) phagosomes within trophoblast cells and macrophages respectively. Furthermore trophoblast cells (unlike macrophages) did not exhibit phagoso-lysosomal fusion. ST-infected macrophages produced IL-6 whereas trophoblast cells produced IL-10. Neutralizing IL-10 in JEG-3 cells accelerated phagolysomal fusion and reduced proliferation of ST. Placental bacterial burden was curtailed in vivo in anti-IL-10 antibody treated and IL-10-deficient mice. Discussion Macrophages phagocytose but curtail intracellular replication of ST in late phagosomes. In contrast, phagocytosis by trophoblast cells results in an inappropriate cytokine response and proliferation of ST in early phagosomes. Conclusion IL-10 production by trophoblast cells that delays phagosomal maturation may facilitate proliferation of pathogens in placental cells. © 2013 Elsevier Ltd. All rights reserved.

Bu çalışmada, Gazi Üniversitesi Fen Fakültesi Biyoteknoloji Laboratuvarı kültür koleksiyonunda bulunan insan, gıda ve hayvan kaynaklı olmak üzere 39 adet Lactobacilllus cinsine ait ve yenidoğan gaitasından izole edilmiş olan 3 adet Bifidobacterium cinsine ait toplam 42 adet bakteri kullanılmıştır. O-nitrofenil-beta-D-galaktosid (o-NPG) substrat olarak kullanılarak, kültürlerin ß-galaktosidaz enzim aktiviteleri belirlenmiştir. Suşların enzim aktiviteleri değerlendirildiğinde, Lactobacillus cinsine ait kültürlerden L. fermentum ZYN17 (2,468±0,000 U/mg), L. acidophilus BAZ36 (0,947±0,052 U/mg), L. rhamnosus GD11 (1,034±0,027 U/mg) ve L. casei LB65 (1,116±0,036 U/mg) suşlarının, Bifidobacterium cinsine ait kültürlerden de Bifidobacterium breve A26 (0,726±0,032 U/mg) suşunun en yüksek spesifik enzim aktivitesi yeteneğine sahip oldukları belirlenmiştir. Yüksek spesifik ß-galaktozidaz enzim aktivitesi gösterdiği belirlenen kültürlerde farklı pH, sıcaklık ve tamponların enzim aktivitesi üzerinde etkisinin belirlenmesi ile, optimizasyon çalışmaları yapılmıştır. Kültürlerin pH 6,8, 37ºC ve potasyum fosfat tamponunda en yüksek spesifik enzim aktivitesi yeteneğine sahip oldukları gözlenmiştir. Suşların farklı konsantrasyonlarda substrat (laktoz) içeren besiortamlarında geliştirilmesi durumunda, en yüksek enzim aktivitesi %6 laktoz içeren besiortamında belirlenmiştir. Yapay mide suyunda, pH 7,0'da Lactobacillus fermentum ZYN17 suşu 2,262±0,000 U/mg ile en yüksek spesifik enzim aktivitesine sahipken, pH 7,5'te 1,686±0,000 U/mg ile L. fermentum ZYN17 suşu yapay bağırsak sıvısında en yüksek ß-galaktozidaz spesifik enzim aktivitesi göstermiştir. ß-galaktozidaz spesifik aktivitesi üzerine, enzim ekstraksiyon yöntemlerinden kimyasal yöntemlerden Triton X-100'ün en etkili yöntem olduğu belirlenmiştir.Anahtar Kelimeler : Lactobacillus, Bifidobacterium, ß-galaktosidaz In this study, human-being, nutritional and animal originated 39 Lactobacillus species and 3 Bifidobacterium isolated from new-born faeces were used. Total 42 bacteria were all from the culture collection of Biotechnology Laboratory of Faculty of Science in Gazi University. ß-galactosidase enzyme activities of the cultures were identified by using o-nitrophenyl-b-D-galactopyranoside (o-NPG) as a substrate. L. fermentum ZYN17 (2.468±0.000 U/mg), L. acidophilus BAZ36 (0.947±0.052 U/mg), L. rhamnosus GD11 (1.034±0.027 U/mg) and L. casei LB65 (1.116±0.036 U/mg) strains, from Lactobacillus family and Bifidobacterium breve A26 (0.726±0.032 U/mg) strain from Bifidobacterium family had the highest spesific enzyme activity. Optimization studies were carried out with these cultures with high ß-galactosidase enzyme activity. Different pH, temperature, buffer and nutritional environment conditions had an effect on the enzyme activity. It is observed that, cultures had the highest spesific enzyme activity at pH 6.8, at 37°C and in potassium phosphate buffer. As the strains were grown up in different concentrations of substrate (lactose) containing nutritional environment, 6 % lactose served for the highest enzyme activity. In the artificial gastric juice, at pH 7.0, Lactobacillus fermentum ZYN17 strain (2.262±0.000 U/mg) showed the highest spesific enzyme activity, while in the artificial intestinal fluid, at pH 7.5, L. fermentum ZYN17 strain (1.686±0.000 U/mg) showed the highest spesific ß-galactosidase enzyme activity. Among the chemical extraction methods, Triton X-100 was determined as the most effective ß-galactosidase enzyme extraction method.Key Words : Lactobacillus, Bifidobacterium, ß-galactosidase 119

On démontre que la synthèse simultanée des octylglycosides correspondant aux deux hexoses de lactose est possible. A partir de lactose et d'octanol, en présence de deux biocatalyseurs spécifiques à chacun des deux hexoses, la bêta-galactosidase et une farine d'amande avec une bêta-glucosidase, on obtient l'octylglucoside et l'octylgalactoside. Les conditions optimales de cette réaction sont étudiées afin d'obtenir un rendement maximal

Recent findings concerning Drosophila melanogaster intestinal pathology suggest that this model is well suited for the study of intestinal stem cell physiology during aging, stress and infection. Despite the physiological divergence between vertebrates and insects, the modeling of human intestinal diseases is possible in Drosophila because of the high degree of conservation between Drosophila and mammals with respect to the signaling pathways that control intestinal development, regeneration and disease. Furthermore, the genetic amenability of Drosophila makes it an advantageous model species. The well-studied intestinal stem cell lineage, as well as the tools available for its manipulation in vivo, provide a promising framework that can be used to elucidate many aspects of human intestinal pathology. In this Perspective, we discuss recent advances in the study of Drosophila intestinal infection and pathology, and briefly review the parallels and differences between human and Drosophila intestinal regeneration and disease. 4 21 30 Cited By :95

Bu tezde, epoksi grubu içeren farklı polimerler kullanılarak Kluyveromyces fragilis kökenli ticari ß- galaktosidaz enziminin immobilizasyonu araştırılmıştır. Ticari ß- galaktosidaz enzimi Lactozym, Sepabeads EC-HFA, Sepabeads EC-EP, Dilbeads ve Immobeads epoksi polimerleri üzerine immobilize edilmiş ve immobilizasyon etkinliğinin en yüksek olduğu Sepabeads EC-HFA polimeri çalışmanın geri kalan kısmında kullanılmıştır.Sepabeads EC-HFA polimeri kullanılarak yapılan immobilizasyon işleminde immobilizasyon etkinliğine, immobilizasyon süresi (24 ? 72 saat), immobilizasyon materyali miktarı ( 0.1 ? 2.5 g), tampon konsantrasyonu (0.025 ? 0.4 M) ve pH'nın (5 ? 8 ) etkileri incelenmiştir. En yüksek immobilizasyon etkinliği (%97), 24 saat, 0.6 g immobilizasyon materyali miktarı, 0.1 M tampon konsantrasyonu ve pH 6.5'da elde edilmiştir.İmmobilizasyon etkinliği üzerine etkili olan ve immobilizasyon işleminin optimizasyonunda kullanılmak üzere seçilen parametreler, immobilizasyon materyali miktarı, pH ve tampon konsantrasyonu olarak belirlenmiştir. Bu parametrelerin immobilizasyon işlemine olan etkilerini ve birbirileri arasındaki interaksiyon etkilerini incelemek için Cevap Yüzey Yöntemi kullanılmıştır.İmmobilizasyon işlemi üzerine immobilizasyon materyali miktarının negatif, pH ve tampon konsantrasyonunun ise pozitif doğrusal etkiye sahip olduğu görülmüştür. Belirlenen parametrelerin optimum seviyelerinde (0.87 g immobilizasyon materyali, pH 6.61, 0.13 M tampon konsantrasyonu, 24 saat) maksimum immobilizasyon etkinliği (%100) elde edilmiştir. Model denklemi ile hesaplanan immobilizasyon etkinliği ise % 99.8 olarak belirlenmiştir.İmmobilizasyon işleminin enzimin optimum pH ve sıcaklık noktalarını değiştirmediği belirlenmiştir. pH ve sıcaklık stabiliteleri incelendiğinde immobilize enzimin stabilitesinin serbest enzime kıyasla daha iyi olduğu sonucuna varılmıştır. Serbest enzimin kinetik sabitleri Km 1.273 mM ve Vmax 909.09 µmol/dk/mg protein, immobilize enzimin kinetik sabitleri Km 3.023 mM ve Vmax 57.47 µmol/dk/mg protein olarak hesaplanmıştır. Optimize edilen immobilize enzimin tekrar kullanılabilirliği incelenmiş, ve 10 kez önemli bir aktivite kaybı gözlenmeden tekrar kullanılmıştır.Anahtar Sözcükler: ß- galaktosidaz, epoksi, immobilizasyon, cevap yüzey yöntemi In this thesis, the immobilization of commercial ß- galactosidase of Kluyveromyces lactis on different epoxy polymers was investigated. Commercial ß- galactosidase Lactozym, immobilised on Sepabeads EC-HFA, Sepabeads EC-EP, Dilbeads and Immobeads epoxy polymers. The highest immobilisation efficiency was obtained with Sepabeads EC-HFA and this polymer was used in the rest of this study.The effects of, immobilization time (24-72 h), amount of support material (0.1 ? 2.5 g), buffer concentration (0.025 ? 0.4 M) and pH (5 ? 8) parameters on immobilization efficiency were investigated, during the immobilization process of ß- galactosidase on Sepabeads EC-HFA. The highest immobilization efficiency (%97) was obtained at 24 hour, 0.6 g support material, 0.1 M buffer concentration, pH 6.5.The effects of parameters (amount of support material, pH, buffer concentration) on immobilization process and the interactions between each parameter was investigated with Responce Surface Methodology.Amount of support material had a negative linear effect, pH and buffer concentration had a positive linear effect on the immobilization process. The maximum immobilization efficiency of % 100 was obtained at the optimum levels of immobilization parameters (0.87 gsupport material, pH 6.61, 0.13 M buffer concentration, 24 hour). The immobilization efficiency calculated by the model equation was % 99.8.It is found that optimum pH and temperature levels of immobilised enzyme did not change. pH and temperature stability of enzymes were examined and improved pH and temperature stability were found for immobilised enzyme. Kinetic constants of free and immobilised were found as, Km 1.273 mM, Vmax 909.09 µmol/dk/mg protein; Km 3.023 mM and Vmaz 57.47 µmol/dk/mg protein, respectively. Reusability of the optimised and immobilised enzyme was investigated, and immobilised enzyme used for 10 times without a necessary activity loss.Keywords: ß- galactosidase, epoxy, immobilization, respunce surface method 88

La actividad de la -galactosidasa refleja la tasa de envejecimiento celular in vitro. Mediante quimioluminiscencia se cuantificó dicha actividad a pH 6 en células epiteliales ováricas provenientes de 28 donantes sin antecedentes de cáncer. Las células fueron cultivadas en forma seriada hasta alcanzar el estado de detención permanente de crecimiento. Durante la fase de crecimiento exponencial, todos los cultivos mostraron un patrón semejante de crecimiento y una baja actividad -galactosidasa. Sin embargo, el inicio de la disminución de la capacidad replicativa que caracteriza el final de dicha fase, así como el inicio de la fase estacionaria o senescente presentaron un aumento significativo en la actividad enzimática. Nuestros resultados mostraron que la actividad -galactosidasa puede ser considerada como marcador de senescencia replicativa del epitelio superficial del ovario a pH 6. ?-galactosidase activity reflects the rate of cellular aging in vitro. Such activity was quantified at pH 6 in ovarian epithelial cells from 28 donors without a history of cancer, by the chemoluminiscent method. The cells were serially cultured until they achieved the state of permanent growth arrest. During the exponential growth phase, all cultures showed a similar pattern of growth and low ?-galactosidase activity. However, both in the onset of decrease replicative activity, as well as in the onset of the stationary phase, there was a significant rise in the enzyme activity. Our results showed that ?-galactosidase activity can be considered as a replicative senescence marker of the ovarian surface epithelium at pH 6.

Karbonhidratlar insan diyetinin önemli bir kısmını teşkil eder. Karbonhidratlarınsindirim bozukluklarından en sık rastlananı laktoz intoleransıdır.Primer erişkin tiphipolaktazya yani laktoz intoleransı erişkin populasyonunun %75'ten fazlasınıetkilemektedir. Hastalığın insidansı ırklar arasında önemli farklılıklar gösterir. Bufarklılık laktoz intoleransının yalnızca çevresel faktörlerden değil genetik bir temeledayalı olduğunu göstermiştir. Enattah ve arkadaşları bu yüzyılın başında laktozintoleransı ile C/T-13910 tek nükleotit polimorfizmini tanımlamışlardır. Aynıçalışmada laktoz intoleransı ile daha zayıf ilişkili bir mutasyon tespit edilmiştir(G/A-22018).Farklı populasyonlarda yapılan birçok çalışma bu iki gen polimorfizmi ile laktozintoleransı arasında C/C-13910 genotipi ile ilişkili olduğunu ve C/T ve T/Tgenotiplerinin persistansı (laktoz toleransı) ile ilişkili olduğu gösterilmiştir. C/T-13910polimorfizminin özellikle Kuzey Avrupa populasyonunda laktaz aktivitesi ile önemliderecede ilişkili olduğu tespit edilmiştir. Literatürde Türk toplumunda laktoz intoleransıile ilişkili polimorfizmlerin sıklığı konusunda yapılmış bir çalışma bulunmamaktadır.Bu amaçla sistemik bir hastalığı olmayan 20 laktoz intoleransı tanısı konmuş ve 17gönüllü grupta genetik polimorfizim analizi yapıldı. Polimorfizimlerin analizi içinEDTA'lı hemogram tüplerine alınan venöz kan örneklerinde DNA izolasyonu yapıldı,DNA örneklerine PCR işlemi uygulandı. Daha sonra hibridizasyon ve renklendirmeişlemleri ile oluşturulan renkli striplerde mutasyonlar tespit edildi.Sonuç olarak hasta grubunda 18 C/C (%90), 2 C/T (%10,) 12 G/G (%60), 7 G/A(%35), Grup 2'de 13 C/C (%76), 4 C/T (%23), 7 G/G (%41), 7 G/A (%41), 3 A/A(%17) genotiplerine sahiptiler.Bu bulguların ışığında Türk toplumunda C/C genotipinin laktoz intoleransı iledirekt ilişkili olduğunu tespit ettik.Anahtar Kelimeler: Laktoz İntoleransı, Polimorfizm, C/T -13910, G/A -22018 Carbohydrates constitute an important part of human diet. Lactose intolerance isthe most common disorder of digestion of carbohydrates. Primary adult typehypolactasia, namely lactose intolerance, affects over 75% of the adult population. Theincidence of the disease varies among the races. This variation showed that lactoseintolerance depends on not only environmental factors but also a genetic basis. At thebeginning of this century Enattah et al described C/T-13910 single nucleotidepolymorphism in lactose intolerance. In the same study a mutation (G/A-22018) whichhas lower relation with lactose intolerance has been determined. In many studies ondifferent populations it has been shown that among these two gene polymorphisms C/C-13910 genotype is related with lactose intolerance and also C/T and T/T genotypes arerelated to persistancy (lactose intolerance). Especially in the North Europe population,C/T-13910 polymorphism has been determined to have significant relation with lactoseactivity.In the literature, there is no study about the incidence of lactose intolerancerelated polymorphisms in Turkish population. Therefore in this study, geneticpolymorphism analyses performed in 20 healthy volunteers with no systemic disordersand 17 volunteers with lactose intolerance diagnosis. To analyse polymorphisms, DNAisolation was performed in venous blood samples taken into vaccium tubes with EDTA,and PCR was applied on DNA samples. Then, mutations were determined in stainedstrips prepared by hybridization and staining procedures.As a result, the patient group had the 18 C/C (90%), 2 C/T (10%), 12 G/G (60%)and 7 G/A (35%) genotypes while the healty control group had 13 C/C (76%), 4 C/T(23%), 7 G/G (41%), 7 G/A (41%) and 3 A/A (17%) genotypes.In the light of these findings, we determined that C/C genotype is directly relatedto lactos intolerance in Turkish population.Key Words : Lactose İntolerance, Polymorphism, C/A-13910, G/A-22018 103

Kluyveromyces lactis önemli bir endüstriyel maya türüdür. K. lactis'in farklı suşları laktaz enzimi ve bazı rekombinant proteinlerin üretiminde kullanılmaktadır. K. lactis çeşitli habitatlarda bulunabilir. K. lactis çoğunlukla süt ve peynir örneklerinden kolaylıkla saf kültür olarak izole edilmektedir. Bu araştırmada Bursa ilindeki yerel üreticilerden sağlanan süt ve peynir örneklerinden 6 farklı K. lactis suşu izole edildi. İzole edilen bu K. lactis suşlarında glikojen miktarı, laktaz ve invertaz aktiviteleri belirlendi. Saf kültür olarak izole edilip tanımlanan K. lactis suşlarının standart K. lactis suşuna göre laktaz ve invertaz aktivitelerinde önemli farklılıklar belirlendi. Bu K. lactis suşlarının LAC4 ve LAC9 genlerinin bazı yapısal özellikleri de incelendi. Elde edilen sonuçlar Bursa ilindeki yerel üreticilerden elde edilen süt ve peynir örneklerinden izole edilen K. lactis örneklerinin farklı metabolik özellikleri olan yeni K. lactis suşları olduğunu göstermektedir. Kluyveromyces lactis is an important industrial yeast species. Different strains of K. lactis are widely used in the production of lactase and also certain recombinant proteins. K. lactis can be found in a variety of habitats. K. lactis species are easily isolated from milk and cheese samples as a pure culture. In this research 6 different K. lactis strains from milk and cheese samples, obtained from local producers in Bursa province, are isolated. In these isolated K. lactis strains their glycogen amounts, lactase and invertase activities are determined. Important differences in lactase and invertase activities of K. lactis strains which are isolated and described as pure culture, are found as compaired with standard K. lactis strain. Some of the structural features of LAC4 and LAC9 genes of these K. lactis strains are also examined. The results show that K. lactis strains isolated from milk and cheese samples obtained from local producers in Bursa province are the new K. lactis strains with their different metabolic properties. 103

Bu tezde Kluyveromyces türü mayalar ile ß-galaktosidaz enzim üretiminde bazı proses değişkenleri incelenmiş, çalkalamalı kültürde ve karıştırmalı tank tipi biyoreaktörde Cevap Yüzey Yöntemi kullanılarak ß-galaktosidaz enzim üretimi optimize edilmiştir.Kluyveromyces lactis NRRL Y-8279 ile sentetik ortam kullanılarak çalkalamalı kültürde ß-galaktosidaz enzim üretimine etki eden faktörler araştırılmıştır. ß-galaktosidaz enzim üretiminde önemli fermentasyon parametreleri olan ortam pH'sı (6-8), karıştırma hızı (150-250 rpm), başlangıç substrat konsantrasyonu (20-40 g/l) ve fermentasyon süresi (36-60 saat) optimize edilmiştir. Maksimum spesifik enzim aktivitesi, proses değişkenlerinin optimum seviyelerinde (başlangıç ortam pH 7.35, karıştırma hızı 179.2 rpm, başlangıç substrat konsantrasyonu 24.9 g/l, fermentasyon zamanı 50.9 saat) 4218.4 U/g olarak tespit edilmiştir. Karıştırmalı tank tipi biyoreaktörde ß-galaktosidaz enzim üretiminin optimizasyonu Kluyveromyces lactis NRRL Y-8279 ile sentetik ortam kullanılarak gerçekleştirilmiştir. ß-galaktosidaz enzim üretimi üzerine başlangıç substrat konsantrasyonu (15-35 g/l), karıştırma hızı (100-300 rpm), havalandırma hızı (1-3 vvm) ve fermentasyon süresinin (12-32 s) etkileri incelenmiş ve optimum seviyeleri belirlenmiştir. Maksimum spesifik enzim aktivitesi olan 4622.7 U/g, proses değişkenlerinin optimum seviyeleri olan 33.8 g/l başlangıç substrat konsantrasyonu, 2.21 vvm havalandırma hızı, 24 saat fermentasyon süresi ve 173.4 rpm karıştırma hızında elde edilmiştir. CYY'nin, çalkalamalı kültürde ve karıştırmalı tank tipi biyoreaktörde ß-galaktosidaz enzim üretiminin optimizasyonunda ve proses parametreleri arasındaki interaksiyon etkilerinin belirlenmesinde başarı ile kullanılabileceği bulunmuştur.ß-galaktosidaz enziminin saflaştırılması, Sephadex A50 iyon değiştirici jel kullanılarak gerçekleştirilmiş ve enzim yaklaşık 3 kat saflaştırılmış, saflaştırma verimi %74.41 olarak bulunmuştur. Kluyveromyces lactis NRRL 8279'dan elde edilen saflaştırılmış ß-galaktosidaz enzimine ait SDS PAGE görüntülerinde enzime ait dört adet alt birim (133.77, 58.07, 49.81, 24.71 kDa) tespit edilmiştir Üretilen ß-galaktosidaz enziminin optimum sıcaklık ve pH değerleri 37°C ve pH 7.0 olarak bulunmuştur. Enzimin pH 6.0-7.5 ve 25-37°C sıcaklık aralığında stabil olduğu belirlenmiştir. Enzimin Km ve Vmax değerleri sırasıyla 1.10 mM ve 1000 µmol/min mg protein olarak belirlenmiştir. In this thesis, process variables in ß-galactosidase production by Kluyveromyces were investigated and ß-galactosidase production was optimized by using Response Surface Methodology. In the first part of the thesis, production and optimization of ß-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in shake flask cultures were investigated. Among the different cell disintegration methods used, the highest specific activity was obtained when the cells were permeabilized using isoamyl alcohol. Response surface methodology was used to investigate the effects of four fermentation parameters (agitation speed, pH, initial substrate concentration and incubation time) on ß-galactosidase enzyme production. Results of the statistical analysis showed that the fit of the model was good in all cases. Maximum specific enzyme activity of 4218.4 U/g was obtained at the optimum levels of process variables (pH 7.35, agitation speed 179.2 rpm, initial sugar concentration 24.9 g/l and incubation time 50.9 h). In the second part of the thesis, production and optimization of ß-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in stirred tank bioreactor were investigated. Response surface methodology was used to investigate the effects of four fermentation parameters (aeration rate, agitation speed, initial sugar concentration and incubation time) on ß-galactosidase enzyme production. The fermentation parameters mentioned above had strong linear and negative quadratic effects on specific enzyme activity. Maximum specific enzyme activity of 4622.7 U/g was obtained at the optimum levels of process variables (aeration rate 2.21 vvm, agitation speed 173.4 rpm, initial sugar concentration 33.8 g/l, incubation time 24.0 h). The purification of the enzyme from a crude protein extract of K. lactis by ion exchange chromatography resulted in a purification factor of 3-fold over the crude extract having an overall yield based on total enzyme units of 74.41%. Enzyme is consist of four subnits (133.77, 58.07, 49.81, 24.71 kDa) The optimum temperature and pH of the ß-galactosidase enzyme produced under optimized conditions were 37°C and pH 7.0, respectively. The enzyme was stable over a pH range of 6.0-7.5 and a temperature range of 25-37°C. The Km and Vmax values for ?-nitrophenol-ß-D-galactopyranoside (ONPG) were 1.10 mM and 1000 µmol/min mg protein, respectively. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in ß-galactosidase enzyme production. Hence, this study fulfills the lack of using mathematical and statistical techniques in optimizing the ß-galactosidase enzyme production in stirred tank bioreactor. 216

Thesis (Master)--Izmir Institute of Technology, Food Engineering, Izmir, 2008, Includes bibliographical references (leaves: 66-72), Text in English; Abstract: Turkish and English, xiii, 75 106 leaves, B-galactosidase (lactase) enzyme is of great industrial interest, since it can be used to solve problems associated with whey disposal and lactose crystallization in sweetened and frozen dairy products such as ice cream. All over the world, many people suffer from lactose intolerance and lactase preparations are used as supplements for these persons. B-galactosidase is also used to produce prebiotics since this enzyme hydrolyses lactose into galactooligosaccharides. Immobilized B-galactosidase preparations are preferred in many processes because they can be recycled and maintain their activities for a long time without loosing their chemical stability.Novel cross-linked chitosan-hydroxyapatite composite support has beenprepared, lactase from Kluyveromyces lactis was immobilized onto these beads. Lactase immobilization mechanism and effect of factors such as initial glutaraldehyde concentration, temperature, pH, initial lactase concentration, solid-liquid ratio on immobilzation mechanism were studied.Optimum cross-linking was obtained at the glutaraldehyde concentration of 100 mg/L. The optimum values of temperature, pH and solid/liquid ratio on lactase/HAChitosan were found to be 200C, pH 7.5 and 0.3g/ml Vg/Vl, respectively. The pH and thermal stabilities of free and immobilized enzymes were also investigated and it was observed that the relative activity remained above 83.2% within pH 6-7.5 and maximum activity yield was obtained at 370C for free and all immobilized enzymes. The Michaelis constant Km and Vmax of immobilized and free enzyme on chitosanhydroxyapatite composite beads were found to be 9.5 mM and Vm 454.5 .mol ONP min.1 mg.1 protein and 1.011 mM and 1098.9 .mol ONP min.1 mg.1 protein, respectively.