LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans
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Yazar "Karagüler Gül, Nevin" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
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ÖgeBiochemical characterisation of truncated amylopullulanase from Thermoanaerobacter brockii brockii(Graduate School, 2022-06-24) Kayrav, Aycan ; Karagüler Gül, Nevin ; 521201105 ; Molecular Biology-Genetics and BiotechnologyThe last point reached as a result of the chemical transformation carried out by nature is life. Enzymes are responsible biomolecules for the chemical transformation that makes life possible. People had benefited from the reactions carried out by enzymes to obtain various products in many fields throughout history, from the times when they did not know the existence of these catalysts. Over time, people had understood how great potential these biocatalysts have with the structure and working principles. Since then the characterisation of novel enzymes and the usage area of these enzymes have been expanded day by day. Today, more than 3.000 enzymes are used in biotechnological and industrial fields and it is reported that the size of this enzyme market has reached 6 billion USD in 2021. The enzymes that dominate this market with a share of 30 % are those used in the food and beverage industry and the ones that have the upper hand in this group are the enzymes used in starch processing applications. Starch, the main component of many agricultural products and the primary source of carbohydrates for most people, is the main source of carbon in the world. In addition to starch, oligosaccharides, disaccharides and monosaccharides obtained as a result of hydrolysis are also used in various fields such as food, pharmaceuticals and biofuels. To obtain these hydrolysis products, starch is subjected to a three-step process: gelatinisation, liquefaction and saccharification. The viscous solution which is obtained by hydrolysis of α-1,4- and α-1,6- glycosidic bonds in the starch structure and gelatinisation is achieved by using α-amylase, β-amylase, glucoamylase and pullulanase enzymes during the liquefaction. During all these processes to remain the enzymes stable for a long time and maintain their activity, pH adjustment and the addition of Ca2+ ionsare required . Remove both the formed salt as a result of pH adjustments and excess Ca2+ ions causes additional steps and all of these lead to an increase in the cost of production. To eliminate these steps and perform a one-step liquefaction-saccharification process can be possible with an alternative enzyme. Amylopullulanases (E.C. 3.2.1.1/41) are enzymes capable of hydrolysing both α-1,4- and α-1,6- glycosidic bonds, while those obtained from extremophilic organisms can maintain their activities under harsh industrial conditions. In this study, the amylopullulanase (TbbApu) enzyme belonging to the Thermoanaerobium brockii brockii organism, is one of the strong candidates for starch hydrolysis processes, is examined. Previously, for the recombinant production of TbbApu, the whole apu gene had been obtained by using the primary walking method by our group and the optimisation of expression studies was in progress. As a result of the studies, pET-28 a (+) vector and E. coli BL21 (DE3) were chosen as the appropriate expression vector and and E. coli BL21 (DE3) the appropriate host, respectively. Additionally, the variants TbbApuΔSH3 without SH3 domain and TbbApuΔCBM20 without CBM20 domain were constructed to investigate the effect of SH3 and CBM20 domains. In the scope of this thesis, apart from TbbApuΔSH3 and TbbApuΔCBM20 variants, four additional truncated constructs namely TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1 and TbbApuΔX25-2-CBM20 were also obtained to disclose the effects of X25 domain. The biochemical characterisation, raw starch binding ability and kinetic studies of TbbApu and its six variants have been accomplished. The function of the X25 domain on substrate binding, activity and stability of the enzyme has been revealed for the first time with this study. The genes belonging to each construct were amplified by PCR with specific forward and reverse primers that have SacI and NotI restriction sites at their 5' ends and using the whole apu gene as a template. As a result of the amplification, tbbApuΔX25-1-SH3, tbbApuΔX25-2-SH3, tbbApuΔX25-1-CBM20 and tbbApuΔX25-2-CBM20 genes with a length of 4065, 3750, 3375 and 3060 base pairs, respectively, were obtained. Then, the obtained genes and the pET-28 a (+) expression vector were cut with SacI and NotI enzymes to form sticky ends and the ligation reactions were set up for the insertion of the genes into the pET-28 a (+) expression vector. Then, transformation was performed using competent E. coli BL21 (DE3) host cells. For the determination of positive colonies from the colonies obtained as a result of the transformation, half of the colonies were inoculated on agar plates containing red pullulan. 1 µM IPTG and 40 µg/mL kanamycin were also included in the agar plate for induction and selection respectively. For the control of detected positive colonies from the PRR plate, the other half of the colonies were inoculated into Luria-Bertani (LB) medium and plasmid isolation was performed from these cells. Then, the isolated plasmids were cut with FastDigest SacI enzyme and linearised to determine the length of the plasmids. By linearisation, tbbApuΔX25-1-SH3 - pET-28 a (+) vector with a length of 9434 base pairs; 9119 base pairs long tbbApuΔX25-2-SH3 -pET-28 a (+) vector, 8744 base pairs tbbApuΔX25-1-CBM20- pET-28 a (+) vector and 8429 base pairs tbbApuΔX25-2-CBM20- pET-28 a (+) vector were obtained from all selected colonies. After the cloning, TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20 were over-expressed using magic media. To purify these recombinant enzymes, purification was achieved in three steps by using metal affinity chromatography, ion exchange chromatography and heat purification respectively. Then, the pullulanase and α-amylase activities of TbbApu and its six variants were checked by the red pullulan and starch-containing polyacrylamide gel. The biochemical characterisation of the enzymes was completed. As a result of the studies, the optimum reaction temperature for pullulanase activities was determined as 70 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, 75 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3 and TbbApuΔX25-1-CBM20 and also 80 °C for TbbApuΔX25-2-CBM20 domain. The optimum reaction temperature for α-amylase activities was specified as 75 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, and 80 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20. The pure variants were incubated for 24 hours at variable temperatures to examine the effect of temperature on the stability of enzymes. It was observed that the most stable temperature of 60 °C for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3 and TbbApuΔX25-2-SH3, 50 °C for TbbApuΔX25-1-CBM20 and 40 °C for TbbApuΔX25-2-CBM20. Optimum pH values for both activities were found to be 6.5 for TbbApu, TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20, 6 for TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-SH3, and 7 for TbbApuΔX25-2-CBM20. It has been determined that the enzymes reached 80 % of their α-amylase activities with pH 5 and protected it up to pH 8. When the effects of different pH values on the stability of the enzymes were examined, the most stable pH according to pullulanase activities was found as pH 4 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-CBM20, pH 6 for TbbApuΔX25-2-SH3, pH 6.5 for TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20 after 24 hours of incubation. According to their α-amylase activities, the most stable pH was found as pH 3 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, pH 6 for TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20, pH 7 for TbbApuΔX25-1-SH3. In addition, it is represented that TbbApu and its variants can maintain their stability between pH 3 and 8 and TbbApu can be used in starch hydrolysis processes without pH adjustment. When the effect of metal ions was examined, while Mn2+ and Co2+ ions increased both pullulanase and α-amylase activities of TbbApu and its variants whereas, Mg2+, Zn2+ and Al3+ ions decreased both activities of all enzymes except pullulanase activity of TbbApuΔCBM20 variant. Although α-amylase enzymes used in starch processing are Ca2+ ion-dependent, TbbApu and its variants are not dependent on Ca2+ ion for activity and stability, making the enzyme and its variants a strong candidate for starch hydrolsis process. Also, in the presence of 20 % and 50 % hexane and acetone, with some exceptions, both activities of TbbApu and its variants were increased and 20 % and 50 % butanol, DMF and DMSO presence strongly inhibited both activities of the enzymes. When the effects of organic solvents on the stability of enzymes were examined, it was observed that, with some exceptions, the stability of the enzymes increased in the presence of 20 % and 50 % acetone and hexane, compared to both pullulanase and α-amylase activities, after 24 hours of incubation. In the case of inhibitors and detergents, it was observed that inhibitors moderately inhibited pullulanase and α-amylase activities of TbbApu and its variants with some exceptions, whereas all inhibitors increased the pullulanase activity of TbbApuΔCBM20 and nonionic and anionic enzymes inhibited both activities moderately with. However, the activities of all enzymes were strongly inhibited in the presence of CTAB, which is a cationic detergent. Then, the kinetic parameters of the enzymes were elucidated. The results imply that, truncation SH3 domain improves both α-amylase and pullanase activities of the enzyme. However, truncation of CBM20 and X25 domains from TbbApu caused to loss of affinity and specificity of the enzyme to soluble starch and to shift in the specificity of the enzyme to pullulan. The removal of the SH3 domain and also CBM20 domain, the carbohydrate-binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. The removal of the SH3 domain and also CBM20 domain, the carbohydrate binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. However, sequential removal of X25 and SH3 domains resulted in 27.3 % and 58.4 % reduction in raw starch binding ability, while removal of CBM20 and X25 domains resulted in a 90 % decrease in raw starch binding ability. Thus, it was revealed that the X25 domain is involved in binding raw starch. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose as a result of pullulan hydrolysis and maltotriose starch hydrolysis, respectively. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose in pullulan hydrolysis and maltotriose in starch hydrolysis, respectively.
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ÖgeRecombinant production and characterization of chitinase enzyme from Pseudomonas mandelii KGI_MA19(Graduate School, 2022-06-24) Saraç Cebeci, Emine Tuğçe ; Karagüler Gül, Nevin ; 521201111 ; Molecular Biology-Genetics and BiotechnologyBiological catalysts, generally found in protein structure, that catalyze the biochemical reactions necessary for life are called enzymes. They increase the reaction rate by lowering the activation energy of the reaction catalysed. In recent years, enzymes have gained a large share in different industrial areas, like food, agriculture, pharmacy, cosmetics, waste removal, leather processing, detergent, and medical applications. Enzymes can minimize the formation of unwanted by-products, they are environmentally friendly and cheap, and have biodegradable properties. They are also considered safe for the cleaning, health, and food industries. In addition to their preferred properties, enzymes that remain active at low/high temperatures, in the presence of organic solvents, at different salt concentrations and pH values, and with an affinity for different substrates are attracting the attention of the industry. Meeting the increasing demand for the use of biomolecules with different properties in the industry is possible with the development of the physical and biochemical properties of the biocatalysts defined today with the help of protein engineering, metagenomics, advanced DNA technologies, nanotechnology, and finally, the discovery of new biocatalysts. Scientific research has been increasing in this direction, especially because enzymes from living things that live in extreme conditions can be used in a wide range of industries. Chitin (C8H13O5N)n is an inelastic, hard, white, non-elastic biopolymer formed by the bonding of N-Acetyl-D-glucosamine monomers (Glc-NAc) with β-1,4 glycosidic bonds, which ranks second after cellulose among the most abundant biopolymers in nature. It is a nitrogen-containing polysaccharide. Chitin is found in the cell wall of fungi, the outer shells of insects, the shells of sea creatures such as lobster, crab, shrimp, and the mouth areas of cephalopods such as cuttlefish and octopus which have a high annual production in the world. According to the Food and Agriculture Organization (FAO, 2019) data, 10.5 million chitin-rich shellfish (12.3%) are grown in aquaculture. The need for converting chitin wastes, which are rising in quantity by the day, into biologically useful products develops. In recent years, the employment of biocatalysts in the removal of chitin wastes instead of chemical methods, which are poisonous to the environment and expensive, has allowed for a safe conversion for the environment. Chitinase enzymes (EC 3.2.1.14), in the hydrolase class, catalyse the destruction of β-1-4 glycosidic bonds in chitin (C8H13O5N)n and separate N-acetyl-D-glucosamine molecules from the polysaccharide chain. The chitinase enzyme, which is one of the most common hydrolase enzymes in nature, is found in insects, plants, fungi and viruses. It is also commonly found in different bacterial genera such as Aeromonas, Arthrobacter, Bacillus, Chromobacterium, Flavobacterium, Pseudomonas, Sanguibacter, Serratia, and Streptomyces. With the increase in green and environmentally-friendly technology in recent years, the interest in chitinases is increasing day by day. Chitinase enzymes, which have agricultural importance, replace chemicals and pesticides used in agriculture with their ability to be used as biocontrol agents. It creates a positive effect on the marine ecosystem by recycling marine waste. Due to their pharmaceutically antifungal and antibacterial properties, they are used in the production of anti-inflammatory and anti-fungal drugs, anti-cancer and immune-enhancing agents, wound dressings, contact lenses, and surgical sutures. It is a bioprotective additive used to increase shelf life in the food industry. Antarctica with the highest elevation and lowest temperature has recently become a popular research area. 70% of the freshwater resources on the Antarctic continent, which has not been touched by human hands until lately, are in the form of ice. It also features powerful winds, extremely cold temperatures, and is exposed to low temperatures in the winter and high UV rays in the summer. It has a natural and distinct habitat as a result of these factors. Because of the harsh circumstances, it is unavoidable to uncover new species and enzymes and genes. Extremophiles are living systems that can thrive in harsh environments. Biocatalysts derived from extremophilic organisms and demonstrating catalytic activity even under adverse circumstances have been dubbed extremozymes. Psychrophiles and psychrotolerants are extremophilic microorganisms that can live at 0-20 °C and 0-30 °C, respectively. Because of their low energy needs, flexibility, and high catalytic activity, cold-compatible enzymes derived from psychrophilic and psychrotolerant organisms are crucial for commercial applications. Biotechnological approaches help the identification of novel and diverse extremophiles and extremozymes from Antarctica that has not been described in the literature yet. In the study, which was carried out using Antarctic sediment samples collected within the scope of the 2nd National Antarctic Science Expedition (TAE-2), 12 sediment samples taken from 8 different regions were cultured and enriched. Subsequently, freeze-thaw stress was applied to each cultured sample. After the applied freeze-thaw stress, it was observed that five cultures were resistant to stress. Two morphologically different colonies were selected from each stress-resistant sample, DNA isolations were made and 16S rRNA analyzes were carried out. The sample with the lowest similarity rate according to 16S rRNA analysis was sent for whole-genome analysis and according to the results of the analysis, a new strain of Pseudomonas mandelii, Pseudomonas mandelii KGI_MA19, was identified. Phenotypic and biochemical characterizations of the identified KGI_MA19 strain were performed. The next step was the recombinant production and the characterization of the chitinase enzyme of the psychrotolerant Pseudomonas mandelii KGI_MA19. The gene region of the chitinase enzyme of Pseudomonas mandelii KGI_MA19 was amplified by polymerase chain reaction (PCR) by designing gene-specific primers containing SacI and NotI restriction sites. The amplified gene region of interest and the pET-28a (+) vector were cut with SacI and NotI restriction enzymes. T4 DNA ligase enzyme was used for ligation of the cut PCR product and expression vector. Plasmid pET-28a (+) containing the PCR product was transformed into Escherichia coli BL21 (DE3) competent cells. The colony PCR method was applied to determine the colonies containing the relevant gene as a result of the transformation, and plasmid isolation of two colonies selected from among the colonies thought to be positive was performed. Then, to understand whether the colonies contain the gene product or not, the plasmid was cut with the EcoRV endonuclease enzyme, which has a fast-cutting feature. The DNA sequencing results showed that the chitinase gene-specific PCR product was correctly inserted into the pET-28a (+) plasmid. The Magic MediaTM (Invitrogen), which is commercially available and used in the expression of E.coli cells, was used to monitor the expression level of the chitinase gene in E.coli BL21 cells. At the end of Magic Media incubation, cells were treated with lysis (0.1M Tris-HCl, pH 8.0, 0.3M NaCl) buffer, and homogenization was performed by ultrasonication using the M73 probe. A high amount of produced chitinase enzyme was purified by the His-Tag affinity chromatography method using the His-Trap column. The activity of the chitinase enzyme obtained in high purity was determined by the 3,5-Dinitrosalicylic acid (DNS) method which allows one to determine the amount of reducing sugars expected to be released as a result of the reaction performed by the usage of colloidal chitin as a substrate. Biochemical characterization including optimum pH, pH stability, and optimum temperature, thermal stability has been completed. A new, psychrotolerant strain, Pseudomonas mandelii KGI_MA19, was identified from sediment samples from the Antarctic King George Island, and its molecular, phenotypic, morphological, and biochemical characterization was completed. In addition, the chitinase enzyme which has an increasing impact in the industrial field has been successfully produced and biochemically characterized by using recombinant DNA methods. The cold-adaptive chitinase enzyme of the Pseudomonas mandelii KGI_MA19 strain, which was obtained for the first time within the scope of this thesis, is thought to be a promising potential biocatalyst for industrial applications.