Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Crude Pectinolytic Enzymes Production in Fed-Batch Shake Flask Cultivation(01. Izmir Institute of Technology, 2023) Esen, Büşra Nur; Uzuner, Sibel; Uzuner, Sibel; Taşkent Sezgin, HümeyraThe use of waste in the production of enzymes, which is one of the products with high added value, is one of the right strategies to reduce the production cost of the product and sustainability movement. In this study, the production of polygalacturonase (PGase) and pectin lyase (PLase) enzymes from Bacillus subtilis ATCC 6633 in fed batch submerged fermentation, the conditions and composition of the fermentation medium and the effects of pretreatment methods (thermal, thermo-chemical, microwave assisted dilute acid (MW- DA)) on the conversion of fermentable sugar from black carrot pulp were investigated. The MW-DA was chosen the best with higher fermentable sugar content (FSC). The three different powers (300, 600, 850 W) and 3 different treatment time (30, 60, 90 s) were examined by Taguchi design. The highest FSC was found at 300 Watt for 30 seconds. MW-DA followed by ES produced the most fermentable sugar (0.493 g/g, 87.3% conversion). The amount of fermentable sugar was enhanced from 15.8% to 87.3% when MW-DA treatment is combined with enzymatic saccharification (ES). Yeast extract, whey and pea protein were examined as nitrogen sources. According to the enzyme activity results obtained, the fermentation medium was modified with pea protein. Certain concentrations (2.5%, 5%, 10%, 15%) were fed to the fermentation medium. The highest PGase activity was determined at the 15% feed concentration and 72th hours (164.34±2.26 U/L) whereas the highest PLase activity was obtained at 72th hours (188.22±1.72 U/L) at 5% feed concentration.Master Thesis The Effect of Enzyme Use on the Formation of Carbonyls and Structural Properties of Cakes(01. Izmir Institute of Technology, 2021) Er, Ayşe Ege; Ceylan, Çağatay; Şanlı Mohamed, GülşahEnzymes are used as additives to improve the quality parameters of cakes. However, high temperature conditions produce carbonyl-containing compounds as precursors of toxic maillard reaction products. In this study three food grade enzymes were used as agents to decrease the formation of carbonyl-containig compounds while preserving the cake quality factors. For this purpose transglutaminase, lipase and amylase enzymes were used. All of the three enzymes lowered the amounts of carbonyls with the largest decrease by lipase of 31.83% (p<0.05) with respect to the control cake. Transglutaminase and lipase addition changed the carbonyl profile of cakes. Both transglutaminase and lipase caused important changes in protein secondary structures with large increases in alpha helix, turns and anti-parallel beta structures, however, amylase did not cause such large changes. The three enzymes used caused the lipid/protein ratio to decrease. The level of lipid unsaturation did not change for transglutaminase and lipase, however, the level unsaturation decreased in the case of amylase indicating the formation of dicarbonyls was via Maillard reaction not due to lipid peroxidation. However, the GC-MS analysis results indicated that there was no change in the formation of neither the Maillard reaction products nor the lipid oxidation products in the head space analysis. The amorphous structure of the starch in cake samples increased depending on the enzyme concentration used.Master Thesis Chemical Characterization of Caldanaerobacter Subterraneus Subsp. Tengcongensis Heme-Nitric Oxide/Oxygen Binding Protein(01. Izmir Institute of Technology, 2020) Erdal, Merve; Sürmeli, Nur Başak; Sürmeli, Nur BaşakHemoproteins, which contain the heme prosthetic group , take part in different biological processes in many stages of life. Their ability to catalyze important biosynthesis reactions makes them good candidates for understanding and elucidating complex mechanisms for biocatalysis. In this study, the catalytic properties of thermophilic Thermoanaerobacter tencogensis nitric oxide/oxygen binding protein, a heme protein reshaped by rational design, were investigated and chemical characterization was carried out. The peroxidase activity of the enzyme was investigated by the oxidation reactions of guaiacol, amplex red and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS). Kinetic parameters of the reactions were determined. These obtained results demonstrated that, in the presence of H2O2, wild type and Y140H TtH-NOX proteins are able to catalyze oxidation reactions of guaiacol, Amplex red and ABTS. Comparison of the kinetic parameters showed that Y140H mutant catalyzed the guaiacol and ABTS oxidation 3-fold and 15 -fold faster than wild type enzyme, respectively. The stability of TtH-NOX proteins were investigated in the presence of organic solvents. Results were demonstrated that WT TtH-NOX was more stable than Y140H mutant in the presence of organic solvents In addition to these, for the first time, thermophilic TtH-NOX proteins were immobilized with a novel enzyme immobilization method and organic-inorganic hybrid nanostrucrures were obtained. Copper ion incorporated TtH-NOX-based hybrid nanoflowers were synthesized at different pH values. SEM and EDX analysis of TtH-NOX-based hybrid nanoflowers proved that free TtH-NOXs were immobilized successfully.Master Thesis Directed Evolution of a Cytochrome P450 Enzyme To Increase Peroxidation Activity(Izmir Institute of Technology, 2019) Başlar, Muhammet Semih; Sürmeli Eraltuğ, Nur BaşakDirected evolution, mimicking the natural selection, is a powerful tool to create novel enzymes. Evolution of natural enzymes to achieve desired properties are performed in iterative rounds of random mutagenesis followed by a screening/selection method. Enzyme activity can be enhanced with substituting the active site amino acids in the enzyme. CYP119, a member of cytochrome P450 protein family, is a thermophilic enzyme extracted from Sulfolobus acidocaldarius that exhibits monooxygenase, peroxidase and oxidoreductase activity. These properties give CYP119 a potential to be used in production of fine chemicals and pharmaceuticals. Herein, two different mutant libraries of CYP119, containing substituted amino acids at Thr213-Thr214 and Val151- Phe153 positions, constructed via combinatorial active site saturation test (CAST), and screened for improved peroxidation activity. Additionally, fluorescence based Amplex Red peroxidation activity assay using hydrogen peroxide as cofactor of CYP119 was optimized. Screening of mutant libraries resulted four improved CYP119 mutant enzymes from Thr213-Thr214 mutant library. Val151-Phe153 mutant library did not yield any improved peroxidation activity mutants which indicated amino acid substitutions at 151- 153 positions do not have any effect on peroxidation activity of CYP119. Furthermore, effect of substituted amino acids at predetermined positions were analyzed. Substrate, Amplex Red, makes single or double hydrogen bond when molecular docking was performed on improved mutant enzymes also distance of nitrogen atom in Amplex Red to heme iron is closer than wild type CYP119 in improved mutant enzymes. Thus, increasing the peroxidation activity of mutant CYP119 enzymes.Master Thesis Optimization of Expression and Isolation of a Thermophilic P450 Enzyme(Izmir Institute of Technology, 2018) Aslantaş, Yaprak; Sürmeli, Nur Başak; Şanlı Mohamed, GülşahCytochrome P450 enzymes (CYP or P450) are monooxygenases that catalyze the oxidation of hydrocarbons with high efficiency and selectivity, and many other reactions like hydroxylation, epoxidation, reduction, demethylation. CYP119, is a thermophilic P450 from Sulfolobus acidocaldarius. Thanks to thermophilic properties, CYP119 has potential to be widely used as a biocatalyst in production of fine chemicals and pharmaceuticals. However, production and purification of CYP119s is quite difficult and time consuming. Here, through recombinant protein production techniques, the optimum production and purification of heat-tolerant CYP119 has been successfully carried out. N-terminal and C-terminal histidine tags were cloned to CYP119. Protein expression was induced in Escherichia coli BL21 (DE3) cells with isopropyl β-D-1-thiogalactopyranoside (IPTG). δ-aminolevulinic acid (ALA) was also used to increase the heme biosynthesis. Different IPTG and ALA concentrations, expression temperature and duration were used to optimize production. CYP119 was isolated and purified with Ni-NTA affinity column. The thermostability of purified N (N-His-CYP119) and C (C-His-CYP119) terminal His-tagged were compared with wild type CYP119 (Wt-CYP119). Oxidation reaction of CYP119 and variants carried out and compared at 25 °C and 65 °C. Also, epoxidation of styrene was performed with N-His-CYP119 in different temperatures. The effects of histidine tags on stability and activity of the CYP119s were observed. Here, conditions for the production of CYP119 were optimized and the histidine tags were found to cause changes in stability and function of proteins. This project will lead to increase in the production of the important enzyme CYP119, which will increase its utilization in the industry.Master Thesis Generation of Mutant Libraries for Directed Evolution of a Thermophilic P450 Enzyme(Izmir Institute of Technology, 2018) Haklı, Emre; Sürmeli, Nur Başak; Bedir, ErdalDirected evolution, inspires from natural selection, is a frequently utilized approach in protein engineering for designing enzymes. It allows iterative evolution of existing proteins towards the ones with desired characteristics by the application of random mutagenesis in the laboratory. However, library construction constitutes the most fundamental part of directed evolution. Application of different construction methods affects both the number and diversity of variants created and the screening/selection techniques used. Early procedures including error-prone PCR, mutator strains, chemical mutagens and gene shuffling have been successful in whole gene mutagenesis yet have been required more screening/selection effort by leading larger libraries. On the other hand, recent approaches such as use of degenerate primers and site saturation mutagenesis have decreased the screening/selection effort by allowing random mutagenesis of amino acids located at specific positions in the polypeptide chain. Especially, active site residues of biocatalysts were chosen as targets and the catalytic efficiencies were enhanced. CYP119, a member of cytochrome P450 protein family, from Sulfolobus Acidocaldarius is a thermostable enzyme capable of catalyzing peroxidation, monooxygenation and oxidoreduction reactions. Here, a library of mutants consist of CYP119 variants was created via application of combinatorial active site saturation test (CAST) in amino acid positions 213 – 214 and an effective fluorescence-based method was developed to screen the library for increased peroxidase activity while utilizing hydrogen peroxide as oxidant. After screening of mutant library, a variant with Thr213Arg – Thr214Ile substitutions showed 1.32-fold increased peroxidase activity in the catalysis of Amplex Red compared to wild type CYP119.Master Thesis Purification and Biochemical Characterization of Xylanase Expressed in Thermophilic Geobacillus Sp.(Izmir Institute of Technology, 2015) Algan, Müge; Köksal, Mustafa; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; Köksal, MustafaXylanase is an enzyme that catalyzes the degradation of the linear polysaccharide β-1,4-xylan into xylose and breaks down the hemicellulose structure of plant cell wall. The xylanolytic property of the enzyme makes it preferable for many biotechnological applications in industry. This enzyme is possibly produced by some bacterial and fungal microorganisms. In this study, briefly, xylanase enzyme was expressed in thermophillic Geobacillus sp. and purified by cold acetone precipitation and gel filtration chromatography. Molecular weight of our xylanase was found as 40.1 kDa by SDS-PAGE and this protein band was verified by Native-PAGE activity staining. Finally, it was characterized using biochemical methods. For characterization studies, Km and Vmax values were calculated from Lineweaver-Burk plot as 10.2 mg/ml and 31.7 U/ml, respectively. The optima temperature and pH for enzyme activity were investigated using beechwood xylan as substrate and found as 55°C and 8.0, respectively. Furthermore, effects of some metal ions, various chemical reagents and organic solvents on enzyme activity were also determined and we observed that Ca2+, Mn2+ and Co2+ affected the activity positively while Zn2+, Cd2+, Fe3+, EDTA, SDS, CHAPS and DTT shielded the activity. And only β-mercaptoethanol caused a significant change amoung organic solvents. Lastly, that the enzyme has a long shelf-life was confirmed assaying the samples taken from enzyme stocks stored at +4°C and room temperature for six weeks.Master Thesis Immobilization of Lipase From Candida Rugosa on Hydrophobic and Hydrophilic Supports(Izmir Institute of Technology, 2001) Öztürk, Banu; Harsa, Hayriye ŞebnemThe aim of this study is to find the optimum conditions for the immobilization of Candida rugosa lipase and also to select the most suitable support maximizing the immobilized enzyme yield under these optimum conditions.Prior to the immobilization studies, optimum working conditions of soluble Candida rugosa lipase were investigated. Candida rugosa lipase expressed maximum activity in pH 6.5 phosphate buffer with 1.0 M NaCl solution at 30 0C. Indeed, lipase was fully active between 4 and 37 0C. Under these conditions, activity of free lipase was 12.2 U/mg enzyme.Immobilization studies were carried out under constant temperature and stirring rate. Immobilization of lipase on hydrophobic support, octyl-sepharose, was very rapid and the equilibrium was reached in 10 minutes. Immobilized enzyme ratio was maximized by the use of pH 7.0 phosphate buffer with 1.0 M NaCl at 37 0C. Also, optimum solid-liquid ratio was found to be 0.1 when 1 ml support was added to 9 ml of enzyme solution. Effect of enzyme loading on lipase immobilization on octyl-sepharose was investigated at two different temperatures. At 20 0C and in the specified range of enzyme concentration (0.25-75 mg/ml), the experimental data fitted well to the linear isotherms with a C value of 138.2. Also, at 4 0C, results were similar and the experimental data could be expressed with the linear isotherm with a C value of 209.1. Under these conditions, activity of immobilized enzyme was 410 U/ml hydrated support.In the case of immobilization on hydrophilic support, chitosan, the reaction was much slower and the amount of the immobilized enzyme was maximized at the end of 24th hour. Optimum conditions of immobilization were determined as pH 6.5 and 20 0C. In this case, addition of salt decreased the immobilization and, therefore, low ionic strengths favored immobilization. Again, optimum solid-liquid ratio was found to be around 0.1 when the 1 ml support was added to 9 ml. of enzyme solution. The effect of enzyme loading was investigated at 20 0C and in the specified range of concentration, data fitted well to the Langmuir isotherm where the constants were calculated to be qm.200 U adsorbed enzyme/ml hydrated support and K. 72.5 U/ml solution.The storage stability of chitosan immobilized lipase was investigated at 4 0C under two different conditions. When immobilized lipase was kept dry for 25 days, 50 % of the initial activity remained. However, if the same lipase was kept in phosphate buffer of pH 6.5, activity loss was only 10 % after a period of 2 months. Complementary work was also carried out to find the differences in the behavior of free and chitosan immobilized lipase against different substrates. The results showed that the presence of 33 % olive oil and 45.5 % corn oil in substrate yielded the maximum activity for free and chitosan immobilized lipases, respectively.Master Thesis Molecular Cloning, Overexpression and Biochemical Characterization of Bacterial Amylase for Biotechnological Processes(Izmir Institute of Technology, 2012) Burhanoğlu, Tülin; Karakaya, Hüseyin Çağlar; Şanlı Mohamed, GülşahAmylases are the enzymes that act on glycosidic bond of starch and related polysaccarides. They comprise 25% of enzyme utilised in a variety of industry. It is used to obtain maltose, glucose and maltodextrins in various lenghts during industrial processes. Amylases are widely distributed enzymes in bacteria, fungi, higher plants and animals. Thermophilic enzymes are widely demanded in order to be stable at harsh process conditions. Isolating these enzymes from thermophilic microorganism is increasing trend because of ease of enzyme production. In this study α-amylase gene region from a thermophilic Bacillus sp. isolated from Balçova Geotermal region in İzmir was cloned to compotent E. coli BL 21 cells. Additionally protein expression was reinforced with pKJE7 chaperone plasmid. Cloned gene was sequenced and found as 1542 bp in length. Thermophilic amylase that has a 59.9 kD molecular weight was expressed and purified from this recombinant strain. Mass spectrometric analysis were performed and the enzyme was matched with α-amylase family protein of Geobacillus thermodenitrificans NG80-2 using NCBInr database. The aminoacid sequence of this enzyme was seen to be similar 92% with our obtained enzyme. According to the results of characterization studies, the amylase enzyme was seen to have highest activity at pH 8.0 and 60°C. The enzyme was also showed to have resonable activity between pH5 and 9. 85% of the enzyme activity was retained at 70°C. Furthermore, amylase activities at 65 and 85°C were observed to remain stable for 5 and 2 hours, respectively. It was also showed that the activity was stable and pH7 and 9 for 6 hours. The effects of some metal ions, chemical agents and organic solvents on enzyme activity were examined so, Co+2, Mg+2,Ca+2 was determined to be as inducer for the enzyme activity. Conversely the activity was inhibited by Cu+2. Furthermore methanol, DDT and Triton X-100 was found to have no effect on the enzyme activity.