PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection
Permanent URI for this collectionhttps://hdl.handle.net/11147/7645
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Article Citation - Scopus: 6Functional Characterization of a Novel Cyp119 Variant To Explore Its Biocatalytic Potential(John Wiley and Sons Inc, 2022) Sakalli, T.; Surmeli, N.B.Biocatalysts are increasingly applied in the pharmaceutical and chemical industry. Cytochrome P450 enzymes (P450s) are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. P450s catalyze reactions using nicotinamide adenine dinucleotide phosphate (NAD(P)H) cofactor and electron transfer proteins. Alternatively, P450s can utilize hydrogen peroxide (H2O2) as an oxidant, but this pathway is inefficient. P450s that show higher efficiency with peroxides are sought after in industrial applications. P450s from thermophilic organisms have more potential applications as they are stable toward high temperature, high and low pH, and organic solvents. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius. In our previous study, a novel T213R/T214I (double mutant [DM]) variant of CYP119 was obtained by screening a mutant library for higher peroxidation activity utilizing H2O2. Here, we characterized the substrate scope; stability toward peroxides; and temperature and organic solvent tolerance of DM CYP119 to identify its potential as an industrial biocatalyst. DM CYP119 displayed higher stability than wild-type (WT) CYP119 toward organic peroxides. It shows higher peroxidation activity for non-natural substrates and higher affinity for progesterone and other bioactive potential substrates compared to WT CYP119. DM CYP119 emerges as a new biocatalyst with a wide range of potential applications in the pharmaceutical and chemical industry. © 2021 International Union of Biochemistry and Molecular Biology, Inc.Article Citation - WoS: 6Citation - Scopus: 6Functional Characterization of a Novel Cyp119 Variant To Explore Its Biocatalytic Potential(Wiley, 2021) Sakallı, Tuğçe; Sürmeli, Nur BaşakBiocatalysts are increasingly applied in the pharmaceutical and chemical industry. Cytochrome P450 enzymes (P450s) are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. P450s catalyze reactions using nicotinamide adenine dinucleotide phosphate (NAD(P)H) cofactor and electron transfer proteins. Alternatively, P450s can utilize hydrogen peroxide (H2O2) as an oxidant, but this pathway is inefficient. P450s that show higher efficiency with peroxides are sought after in industrial applications. P450s from thermophilic organisms have more potential applications as they are stable toward high temperature, high and low pH, and organic solvents. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius. In our previous study, a novel T213R/T214I (double mutant [DM]) variant of CYP119 was obtained by screening a mutant library for higher peroxidation activity utilizing H2O2. Here, we characterized the substrate scope; stability toward peroxides; and temperature and organic solvent tolerance of DM CYP119 to identify its potential as an industrial biocatalyst. DM CYP119 displayed higher stability than wild-type (WT) CYP119 toward organic peroxides. It shows higher peroxidation activity for non-natural substrates and higher affinity for progesterone and other bioactive potential substrates compared to WT CYP119. DM CYP119 emerges as a new biocatalyst with a wide range of potential applications in the pharmaceutical and chemical industry.Article Citation - WoS: 40Citation - Scopus: 50Hyaluronidase 1 and Ss-Hexosaminidase Have Redundant Functions in Hyaluronan and Chondroitin Sulfate Degradation(American Society for Biochemistry and Molecular Biology, 2012) Gushulak, Lara; Hemming, Richard; Martin, Dianna; Seyrantepe, Volkan; Pshezhetsky, Alexey; Triggs-Raine, BarbaraHyaluronan (HA), a member of the glycosaminoglycan (GAG) family, is a critical component of the extracellular matrix. A model for HA degradation that invokes the activity of both hyaluronidases and exoglycosidases has been advanced. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to HA breakdown. Herein, we used mouse models to investigate the contributions of the endoglycosidase HYAL1 and the exoglycosidase β-hexosaminidase to the lysosomal degradation of HA. We employed histochemistry and fluorophore-assisted carbohydrate electrophoresis to determine the degree of HA accumulation in mice deficient in one or both enzyme activities. Global HA accumulation was present in mice deficient in both enzymes, with the highest levels found in the lymph node and liver. Chondroitin, a GAG similar in structure to HA, also broadly accumulated in mice deficient in both enzymes. Accumulation of chondroitin sulfate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulfate breakdown. Extensive accumulation of HA and chondroitin when both enzymes are lacking was not observed in mice deficient in only one of these enzymes, suggesting that HYAL1 and β-hexosaminidase are functionally redundant in HA and chondroitin breakdown. Furthermore, accumulation of sulfated chondroitin in tissues provides in vivo evidence that both HYAL1 and β-hexosaminidase cleave chondroitin sulfate, but it is a preferred substrate for β-hexosaminidase. These studies provide in vivo evidence to support and extend existing knowledge of GAG breakdown.Article Citation - WoS: 62Citation - Scopus: 67Thermal Stability of Carbonic Anhydrase Immobilized Within Polyurethane Foam(John Wiley and Sons Inc., 2010) Kanbar, Bora; Özdemir, EkremThermal stability of carbonic anhydrase (CA) immobilized within polyurethane (PU) foam was investigated. The catalytic activity of the enzyme was estimated by using p-nitrophenyl acetate (p-NPA) as the substrate in tris buffer containing 10% acetonitrile. The immobilized CA was stable during the repeatable washings and stability tests over 45 days stored in tris buffer at ambient conditions indicating that the CA was covalently attached to the polyurethane (PU) foam by crosslinking. The immobilized CA was found to be 98% stable below 50°C, whereas a drastic decrease was seen at temperatures between 50 and 60°C. The optimum temperature for the immobilized CA was found to be 45°C and it lost its activity completely at 60°C. Thermal deactivation energies for the free and immobilized CA were estimated to be 29 and 86 kcal/mol, respectively. The association of unfolded CA with the polymeric backbone chains of the PU foam was also addressed. It was concluded that the immobilized CA was highly stable at temperatures less than 50°C and could be used in biomimetic CO sequestration processes. © 2010 American Institute of Chemical EngineersArticle Citation - WoS: 56Citation - Scopus: 63Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance During S Phase(American Society for Biochemistry and Molecular Biology, 2006) Koç, Ahmet; Mathews, Christopher K.; Wheeler, Linda J.; Gross, Michael K.; Merrill, Gary FredericThioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Δtrx1 Δtrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Δtrx1 Δtrx2 yeast. In contrast to wild-type cells, Δtrx1 Δtrx2 cells were unable to accumulate or maintain high levels of dNTPs when α-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Δtrx1 Δtrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.Article Citation - WoS: 30Citation - Scopus: 42Effect of Various Process Parameters on Morphology, Rheology, and Polygalacturonase Production by Aspergillus Sojae in a Batch Bioreactor(American Institute of Chemical Engineers, 2007) Öncü, Şelale; Tarı, Canan; Ünlütürk, SevcanThe effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 ± 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 ± 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 ± 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.Article Citation - WoS: 4Citation - Scopus: 4Pentobarbital-Mediated Regulation of Alternative Polyadenylation in Drosophila Glutathione S-Transferase D21 Mrnas(American Society for Biochemistry and Molecular Biolog, 2004) Akgül, Bünyamin; Tu, Chen-Pei D.Two nearly identical, gstD21(L) and gstD21(S) mRNAs whose polyadenylation sites differ by 19 nucleotides, are transcribed from the intronless glutathione S-transferase D21 gene in Drosophila. Both mRNAs are intrinsically very labile, but exposure to pentobarbital renders them stabilized beyond what can be attributed to transcriptional activation. We have reconstituted this PB-mediated mRNA stabilization in a transgene (D21L) that contains the full-length gstD21(L) sequence. We have also constructed a similar transgene (D21L-UTR), which matches D21L but excluded the native 3′-UTR. D21L-UTR produces a relatively stable RNA, whose stability is unaffected by pentobarbital. Following pentobarbital treatment of wild-type flies, the levels of gstD21(L) and gstD21(S) mRNAs hold at a relatively constant ratio (L/S) of 1.4 ± 0.2. In transgenic flies, heat shock induction of D21L mRNA changed the L/S ratio to 0.6 ± 0.1, and it was further reduced to 0.3 ± 0.1 as D21L mRNA accumulated in the presence of PB. The ratio returned nearly normal (1.1 ± 0.1) as the D21L mRNA decayed over 12 h after terminating induction. In constrast, when D21L-UTR was present, the ratio remained constant (1.7 ± 0.2) even under various induction conditions and during recovery. Thus, the 3′-UTR, which was the critical difference between these two transgenes, must have some role in determining the L/S ratio. Induced D21L mRNA alone is not sufficient to cause reversible changes in the ratio. Such changes require the presence of pentobarbital. Therefore, pentobarbital may regulate this L/S ratio by affecting the choice of polyadenylation sites for the gstD21 mRNAs through sensing the concentrations of the native 3′-UTR sequences.Article Citation - WoS: 5Citation - Scopus: 5Evidence for a Stabilizer Element in the Untranslated Regions of Drosophila Glutathione S-Transferase D1 Mrna(American Society for Biochemistry and Molecular Biology Inc., 2002) Akgül, Bünyamin; Tu, Chen-Pei D.The neighboring genes gstD1 and gstD21 share 70% sequence identity, gstD1 encodes a 1,1,1-trichloro-2,2-bis-(P-chlorophenyl)ethane dehydrochlorinase; gstD21, a ligandin. Both of their mRNAs are inducible by pentobarbital but otherwise behave very differently. Intact gstD21 mRNA is intrinsically labile, but becomes stabilized when separated from its native untranslated region (UTR). In contrast, whereas gstD1 mRNA is very stable in its entirety, without its native UTRs it becomes even more labile than that of gstD21. Decay patterns from four chimeric D1-D21 mRNAs, designed to reveal the individual importance of each molecular region to stability, strongly indicate the presence of destabilizing elements in the coding region ofgstD1 mRNA. Thus, the UTRs of this molecule must contain a dominant stabilizer element that overrides the destabilizing influence of the coding region and confers overall stability to the entire molecule. The suspected presence of such a stabilizer element in gstD1 mRNA extends a concept from mRNA metabolism in yeast and cultured mammalian cells to include a multicellular organism, Drosophila melanogaster. The complementary presence of destabilizing and stabilizer elements on the same mRNA reveals a regulatory mechanism by which an abundant mRNA can be further induced by a chemical stimulus, or otherwise be returned to normal levels during recovery.