Yazar "Önal, Cansu" seçeneğine göre listele
Listeleniyor 1 - 10 / 10
Sayfa Başına Sonuç
Sıralama seçenekleri
Öğe Asparagine 79 is an important amino acid for catalytic activity and substrate specificity of bile salt hydrolase (BSH)(Springer, 2019) Öztürk, Mehmet; Önal, CansuMicrobial bile salt hydrolases (BSHs), a member of cholylglycine hydrolase (CGH) family, catalyze the hydrolysis of glycine and taurine-linked bile salts in the small intestine of human. BSH is evolutionarily related to penicillin V acylase (PVA) which hydrolyses a penicillin V and is also a member of CGH family. Although, five of the six amino acids, C2, R16, D19, N170, N79 and R223, supposed to be responsible for catalytic activity of BSH enzyme, are strictly conserved in all CGH family members, N79 is partially conserved in this family. In this study, in order to analyze the correlation between N79 and catalytic activity or substrate specificity of BSH, the polar and acidic N79 was substituted for the aliphatic and hydrophobic V79 by PCR-based site directed mutagenesis and mutant recombinant BSH was expressed in E. coli BLR(DE3). While the effects of the mutation on catalytic activity and substrate specificity of BSH were detected by ninhydrin assay. The effect of this mutation on the stability of the BSH was observed by SDS-PAGE analysis. Although V79 mutation resulted in stable BSH, it reduced the catalytic activity and altered substrate specificity of BSH. The results suggested that N79 might be important for substrate binding and catalytic turnover of BSH.Öğe Cloning of bile salt hydrolase gene (BSH) ) from human originated Lactobacillus plantarum and characterization of BSH enzyme by site directed mutagenesis(Bolu Abant İzzet Baysal Üniversitesi, 2019) Önal, Cansu; Öztürk, MehmetGastrointestinal mikrobiyota insan fizyolojisinde önemli bir rol oynar ve konağın beslenme, fizyolojik ve immünolojik fonksiyonlarından sorumludur. Gastrointestinal sistemin mikrobiyal kolonizasyonu, kolorektal kanser etiyolojisinin genetik faktörlerini önemli ölçüde etkiler. Litokolik asit ve deoksikolik asit gibi toksik sekonder safra asitleri, inflamasyon, DNA hasarı ve apoptoz indüksiyonunu etkileyerek kolon kanserine neden olmaktadır. Glisin ya da taurin ile konjuge safra asitlerinin hidrolizi, sekonder safra asitlerinin ortaya çıkması için bir geçiş reaksiyonudur. Çeşitli kaynaklardan elde edilen STH enzimlerinin (EC 3.5.1.24) karakterizasyonu, substrat tercihi ve spesifisiteleri farklılık göstermektedir. STH enzimi ve toksik sekonder metabolitler arasındaki ilişkiyi anlamak için, STH'nin yapısı daha iyi anlaşılmalıdır. Ancak STH enziminin yapısı ve çalışma mekanizması çok iyi bilinmemektedir. Enzimin yapısını ve işlevini anlamak için substrat özgüllüğünden sorumlu olduğu düşünülen amino asitleri kodlayan bölgeye yönelik yönlendirilmiş mutagenez kullanılır. Bu çalışmada, 324-amino asitten oluşan Lactobacillus plantarum B14 STH geni klonlanmış ve kısmen korunmuş ve substrat özgüllüğünden sorumlu olduğu düşünülen Phe-18, Tyr-24, Asn-79, Leu-138 ve Asn-180 amino asitleri, yönlendirilmiş mutagenez ile değiştirilerek, enzim aktivitelerine bakılmıştır. Tüm STH 'ler E. coli BLDRE3-pET22b ekspresyon sistemi kullanılarak saflaştırılmıştır. Mutant enzimlerin ve her biri aynı molekül ağırlığı (37 kDa) ve stabilitesi SDS-PAGE ile doğrulanmıştır. Lb. plantarum B14'ün, STH aktivitesi, nicel olarak Direkt Plate Testi ve nitel olarak Ninhidrin Testi ile belirlenmiştir. Mutasyonların substrat özgüllüğü üzerindeki etkileri, altı farklı safra tuzları ile mutant STH enzimlerinin kısmen saflaştırılmasıyla araştırılmıştır. Araştırmada, Lb. plantarum B14 STH'ın tauro-konjuge safra tuzlarına kıyasla glisin ile konjuge safra tuzlarına daha fazla hidroliz sergilediği ve mutant enzimlerin STH aktivitelerinin, farklı safra tuzlarına farklı ölçüde azalan değişimler gösterdiği tespit edilmiştir.Öğe Construction of R16F and D19L mutations in the loop I of bile salt hydrolase (BSH) enzyme from Lactobacillus plantarum B14 and structural and functional analysis of the mutant BSHs(Taylor & Francis Inc, 2019) Öztürk, Mehmet; Hacıbeyoğlu, Kübra; Önal, Cansu; Kılıçsaymaz, ZekiyeBile salt hydrolase (BSH), found commonly in intestinal species of Lactobacillus and Bifidobacterium, catalyzes the hydrolysis of glycine or taurine-conjugated bile acids into the amino acids and free bile acids. Deconjugated bile acids potentially play an important role in the reduction of blood cholesterol level and formation of some gastrointestinal diseases such as cholestasis, gallstone formation, and colon cancer. Although the crystal and three-dimensional structures of BSH enzyme are known, the working mechanism of catalytic activity of such an important BSH enzyme is not known very well. Previous in silico analysis of multiple BSH has identified that Arginine-16 (R16) and Aspartate-19 (D19) were catalytically important residues in the active site of BSH. To confirm the function of these amino acids, in this study, BSH enzyme from Lactobacillus plantarum B14 strain was cloned into Escherichia coli and strictly conserved polar R16 and D19 amino acids of BSH enzyme were substituted for hydrophobic Phenylalanine-16 (F16) and Leucine-19 (L19) amino acids, respectively, by polymerase chain reaction (PCR)-based site-directed mutagenesis. The effects of the mutations on catalytic activity and structure of the BSH enzymes were detected by ninhidrin assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Research results showed that although F16 mutation led to loss of enzyme activity completely, L19 mutation led to abolishment of the synthesis of BSH enzyme. These results indicated that R16 and D19 amino acids located in loop I of the BSH enzyme might be critical for catalytic activity and assembly of the BSH enzyme respectively.Öğe Critical F129 and L138 in loop III of bile salt hydrolase (BSH) in Lactobacillus plantarum B14 are essential for the catalytic activity and substrate specificity(Taylor & Francis Inc, 2019) Öztürk, Mehmet; Önal, Cansu; Ba, Ndeye MaremeBile salt hydrolase (BSH) is a gut-bacterial enzyme that influences human health by altering the host fat digestion and cellular energy generation. BSH is essential for deconjugation of the glycine or taurine-conjugated bile salts in the small intestine of humans. Therefore, BSH may be a key microbiome target for the designing of new measures to control some diseases in humans. BSHs, a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, exhibit higher variation in substrate specificity. The phenylalanine-129 (F129) and leucine-138 (L138) in loop III of BSH, thought to be responsible for substrate specificity, are partially conserved in this superfamily. In this study, the aromatic-hydrophobic F129 and aliphatic-hydrophobic L138 of C-terminally His-tagged BSH from Lactobacillus plantarum B14 (LbBSH) was substituted for aliphatic-hydrophobic isoleucine (I) and negatively charged polar glutamate (E) amino acid, respectively, by site-directed mutagenesis and characterized using an Escherichia coli BLR(DE3) expression system. Although both mutations resulted in an assembled and stable recombinant BSHs (rBSHs), they altered the catalytic activity and substrate specificity of rBSH. This is the first experimental finding which confirmed that F129 and L138 were critical amino acids for the catalytic activity and substrate specificity turnover of BSH.Öğe Effects of structural changes in bile salt hydrolase enzyme on biocatalytic efficiency and activation energy at working pH and temperature conditions(Hrvatsko Drustvo Kemjijskih Inzenjera I Tehnologa, 2022) Ermurat, Yakup; Öztürk, Mehmet; Önal, Cansu; Kılıçsaymaz, ZekiyeMicrobial bile salt hydrolases (BSHs) catalyse the hydrolysis of glycine and taurine-linked bile salts in the small intestine of humans. Achieving the effects of structural changes in BSH molecules on biocatalytic efficiency (kcat/Km) and activation energy (Ea) is necessary to determine biocatalytic performances of the enzymes. Amino acids responsible for biocatalytic activity or substrate specificity in BSH molecules were modified to determine the effects of structural changes on kcat/Km values and Ea values of the bioconversion reactions. Purified wild type positive control enzyme (pCON2) and mutant recombinant target enzymes (F18L and Y24L) reacted with six conjugated pure bile salt substrates at working temperature and pH conditions. The results of the hydrolysis conversion analysis conducted at various pH conditions were used to estimate kcat/Km, and the assays conducted at various temperature conditions were used to approximate Ea of the biocatalytic reactions. The quantified kcat/ Km value was found remarkably highest with mutant recombinant enzymes (Y24L), while the efficiency value with wild type (pCON2) was determined as lowest, indicating that the structural modifications in BSH molecules showed higher values. The alterations with the mutant-type enzymes F18L and Y24L resulted in decreasing kcat/Km and increasing Ea estimations of the hydrolysis conversion reactions.Öğe Functional characterization of Val58Met and Phe129Ile mutants of bile salt hydrolase from Lactobacillus plantarum in E. coli BLR(DE3) strain(Wiley-Blackwell, 2016) Öztürk, Mehmet; Önal, Cansu; Kılıçsaymaz, Zekiye; Ba, Ndeye Mareme[No Abstract Available]Öğe Investigation of neuron loss in the brain by gene expression in the neonatal period in rats exposed to hyperoxia and treated with PTZ epilepsy model(Wiley, 2022) Önal, CansuInvestigation of Neuron Loss in the Brain by Gene Expression in the Neonatal Period in Rats Exposed to Hyperoxia and treated with PTZ Epilepsy ModelÖğe Molecular cloning, characterization, and comparison of four bile salt hydrolase-related enzymes from lactobacillus plantarum gd2 of human origin(Taylor & Francis Inc, 2018) Öztürk, Mehmet; Aydın, Yasin; Kılıçsaymaz, Zekiye; Önal, Cansu; Ba, NdeyeA bile salt hydrolase (bsh) enzyme deconjugate taurine and glycine-linked bile salts. Because of the strong implications between deconjugation of tauro- or gluco-conjugated salts and its positive or negative health consequences, the characterization of the bsh enzymes is important. bshs from different lactobacilli species, even strains, exhibit higher variation in sequence, kinetic properties, and substrate specificity. In the present study four bsh related genes from Lactobacillus plantarum GD2 strain were cloned and expressed in Escherichia coli BLR (DE3) strain. Amino acid residues of recombinant bshs were analyzed and their deconjugation abilities were tested with six human conjugated bile salts. Results indicated that the genetic distance among four related bsh genes in Lb. plantarum is far from each other and bsh2-4 enzymes share significant sequence homology specifically with penicillin V acylase (PVA) family members. Biochemical and the in silico analysis suggest that the bsh1 enzyme is a member of the bsh family while bsh2-4 enzymes are members of the PVA family.Öğe Penicillin-induced epileptiform ECoG activity in gerbils: effects of physical exercise and a Diospyros kaki extract(Springer, 2016) Kayacan, Yıldırım; Bahadır, Anzel; Çetinkaya, Ayhan; Soytürk Orallar, Hayriye; Çakır, Serkan; Beyazçiçek, Ersin; Önal, Cansu; Yıldırım, ArzuMongolian gerbils (28 males) were divided into four groups, control (C), treadmill-exercised (Ex), treated with the extract of Diospyros kaki (Dk), and exercised plus treated with the Dk extract (Ex+Dk). Animals of the respective groups were running-exercised for 30 min per day during 8 weeks, and the Dk extract (dose 20 mg/kg) was given by gavage during five days per week within the same period. After the treatment and exercise period, an epilepsy model was produced by penicillin G injection (500 IU) into the left somatomotor cortex, and the electrocorticogram (ECoG) was recorded during 120 min. The mean frequency of spike/wave complexes was significantly smaller in the Ex and Ex+Dk groups from the 65th min of the observation period and, in the Dk group, from the 75th min than the respective value in the C group (P < 0.01, P < 0.05, and P < 0.01, respectively). The differences in the amplitude values and latency to onset of the spike/wave events among all groups did not reach the significance level (P > 0.05). Thus, both the running exercise and Dk extract applications inhibit penicillin-induced epileptiform activity by altering the spike/wave frequency or severity of seizures observed in ECoG recordings. Further studies are needed to determine the effects of physical activity of different intensities and forms and to analyze the active compounds in the Dk extract.Öğe Site-directed mutagenesis of bile salt hydrolase (BSH) from lactobacillus plantarum B14 confirms the importance of the V58 and Y65 amino acids for activity and substrate specificity(Taylor & Francis Inc, 2023) Öztürk, Mehmet; Kılıçsaymaz, Zekiye; Önal, CansuThe bile acids (BAs) de-conjugation is catalyzed by bile salt hydrolase (BSH) enzyme, that is an intestinal bacterial product and a member of the cholylglycine hydrolase (CGH) family. De-conjugated BAs alter BA-mediated signaling pathways such as glucose metabolism, energy homeostasis and lipid absorption and this makes the BSH clinically important. However, BSHs from different sources have a variable substrate preference to eight different bile salts. Although BSH is a well-studied enzyme, its molecular investigations based on BSH substrate recognition are not very well known. In this study, the relationship between substrate specificity of BSH from Lactobacillus plantarum B14 (LpBSH) and its loop II, the aliphatic-hydrophobic V58 and aromatic-hydrophobic Y65 residues in this loop was mutated and analyzed. While PCR-based site-directed mutagenesis was used to substitute V58 and Y65 amino acids for N58, F58, M58, C65, F65 and L65 amino acids, respectively, the BLR (DE3) strain of E. coli was used to express mutant recombinant LpBSHs (mrLpBSHs). Site-directed mutagenesis of LpBSH showed reduced activity of mrLpBSHs against six different BAs. Our results indicated that the V58 and mostly Y65 residues in loop II might be critical for the structural site that is involved in substrate specificity and catalysis. These findings suggested that V58 and Y65 residues of LpBSH might participate in substrate specificity and BSH substrate specificity may be dependent upon the collate group, rather than amino acid moieties. However, more mutagenesis-based investigation on other CGH family members are needed in order to understand the structure and substrate specificity relations of BSHs.
