Enzim izalasyonu ve katalitik etkisinin incelenmesi
Isolation and characterization of notive lipase
- Tez No: 46605
- Danışmanlar: PROF.DR. H. AYŞE AKSOY
- Tez Türü: Yüksek Lisans
- Konular: Kimya Mühendisliği, Chemical Engineering
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 1995
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 82
Özet
ÖZET Endüstriyel ölçüde yüksek sıcaklık ve basınç altında gerçekleştirilen bir çak kimyasal dönüşme enzimlerin kata litik etkisi altında çak daha ılımlı koşullarda gerçekleşe bilmektedir. Ayrıca bu proseslerden bazılarının zehirli ve çok pahalı çözücüler varlığında gerçekleşmesi de enzim lerin kimyasal proseslerde kullanım imkânlarının araştırıl masının önemini arttırmaktadır. Enzimler bitkisel maddeler den, hayvansal organlardan ve mikroorganizmalardan izale edilip saflaştırılarak kullanılırlar. Yağlı tohumlar önem li bir enzim kaynağıdır. Bu çalışmada çörekatu tohumu ve tohum pres artığı li- paz kaynağı alarak hidroliz deneylerinde kullanılmış ayrı ca yağlı tohumlara uygulanan asetonla çöktürme yöntemi kul lanılarak enzim izole edilmiş ve aseton tozu olarak isimlen dirilen bu ham enzimin hidroliz reaksiyanundaki etkileri de incelenmiştir. Elde edilen sonuçlara göre 1 :1 yağ-su oranında, 50 C ve pHrfi'da tohum ve pres artığı aynı aktivitayi göstermiş ve hidroliz reaksiyonları yaklaşık 8 saatte dengeye ulaş mıştır. Asetonla çöktürme yönteminde ise pH'ı 6 olan tam pon çözelti ile parçalanan tohumlardan kazanılan aseton tozunun çak daha aktif olduğu ve hidroliz reaksiyonunun 3 saat sonunda dengeye ulaştığı belirlenmiştir. Ticari ve farklı pozisyon spesif ikliğine sahip enzimlerle yapılan reaksiyonların sonunda çörekatu tohumu lipazının pozisyon spesifikliği göstermediği belirlenmiştir. xı
Özet (Çeviri)
ISOLATION AND CHARACTERIZATION OF NATIVE LIPASE SUMMARY All farms of life live by enzymes and produce enzymes. Cells of animal and plant tissues and cells of mikroorganisms have been used as sources of active materials. All enzymes are proteins with enourmous catalytic activity produced by living cells, the enzyme activity varying considerably to the source of the enzyme. Over 2000 different kinds are known, each catalyzing a different kind of chemical reaction. They have very complex and specific three-dimensional confor mation in which the chains are folded. This conformation is necessary far the activity of the enzyme. Enzymes, like other proteins, can undergo denaturation. lilhen this occurs their activity is lost. Some enzymes depend far activity only on their polypeptide structure; others also require one or more nonprotein components, called cafactors. The cofactor maybe a metal ion (Fe, Zn, etc.) or an organic molecule called a coenzyme (NAD, FAD, etc. ). Part of the coenzyme structure is a vitamin molecule. If the coenzyme is bound tightly to the enzyme, it is called a prosthetic group. If the nonprotein portion, the prostatic group, is separated from the protein portion by dialysis, the enzyme becomes inactive. The activity is reestablished', however, if the two portions are united. In many enzymes, the coenzyme portion is the reactive part. The reaction catalysed by enzyme proceeds as follows : k1 E + S v N ES C N E + P k2 The free enzyme then combines in rapid succession with new substrate molecules. Depending on the number of its active group, one enzyme molecule may react XIIsimultaneously with several molecules of the subtrate. The substance, or substances, undergoing accelerated chemical change under the auspices of the enzyme are called its substrates. An enzyme can catalyze an inde finite amount of chemical change without aldered by the reaction. However, because most isolated enzymes are relatively unstable, they often gradually lose activity under the conditions employed far their study. They are such efficient catalysts that they accelerate chemical reactions measurably. Like other chemical reactions, enzyme-catalyzed reactions proceed only when accompanied by a decrease in free energy. Most enzymes are highly specific; they catalyze only one specific reaction or act upon only one isomer of a particular compound. Each exhibits different substrate specificity and requires different reaction conditions. Enzymes are usually classified and named according to the reaction they catalyze. A classification system has been developed by the International Enzyme Commission. Each enzyme is identified by four numbers. The first number indicates the type of reaction the enzyme catalyzes and are divided into six main classes: 1 - Oxidoreductases (oxidation-reduction) 2- Transferases (transfer of chemical groups) 3- Hydrolases (hydrolysis) k- Lyases (addition to double bands) 5- İsamerases ( isamerizatian ) 6- Ligases (formation of new bands) The second and the third number specify the reaction in mare detail and the fourth number designates the origin of the enzyme. Furthermore, each enzyme has been given a recommended name and a systematic name. Factors governing catalytic activity: 1- Temperature: The temperature dependence of enzyme- catalyzed reactions exhibits an optimum. The optimum is generally between kU and SO G. 2- Value of pH: All enzymes have an optimum pH range for activity. The optimum depends not only on pH but also an lianic strength and type of buffer. For almost xmenzymes, the pH optimum lies in the range from 5 ta 7. 3- Activation: Many chemical effectors activate or inhibit the catalytic activity of enzymes. Enzyme activa tion by many inorganic ions has been adequately described. The activating ion may be involved directly in the reaction by complexing the coenzyme or cosubstrate. k- Inhibition: In vivo and in vitro inhibition studies of enzymatic reatians contributed important knowledge to various fields of biochemistry. Depending on the type of inhibitory effect, the following mechanisms of enzyme inhibition may be distinguished: Irreversibl inhibition, rsversibl inhibition, competitive inhibition, non competitive inhibition, uncompetitive inhibition, substrate inhibition. Enzymes are localized on the cell membrane, in the nucleus, and in other subcellular particles. Enzymes can be isolated from their original locations and are active outside of the living cell. Enzymes are obtained from three major sources : plant, animal, microbial. Enzymes are very complex proteins, and their high degree of specificity as catalysts is manifest only their native state. The native conformation is attained under specific conditions of pH, temperature, and ionic strength. Hence only mild and specific methods can be used for enzyme isolation. Figure 1 shows the sequence of steps involved in the recovery of enzymes. The enzyme must therefore first be extracted from, the cells or tissue in which it is found and separated from many proteins and other substances present in the crude extract. Because of the instability of most enzymes, however their isolation requires special techniques. For intracellular enzymes, which are being isolated today in increasing amounts, the first step involves grinding to rupture the cells. If the enzyme is present in cells, the cells must be broken. After cell disruption, the next step is separation of extracellular or intracellular enzymes from cells, respectively. This operation is rather difficult because of the small size : of bacterial cells and the slight difference between the density of the cells and that of the fermentation medium-? Filtration, Cantrif ugation, extraction, flocculation and Flotation is used for separation of solid matter in industry. xivFermentation Animal Organs Plant material Micr oorgan isms Grinding Intra cellular snzyme Extraction Extra cellular enzyme Disruption Filtration Concentration Purification Drying Final Product FIGURE 1. Sequence of Steps in The Isolation of Enzymes. The enzyme concentration in starting material is often very loiu. The volume of material to be processed is generally very large, and substantial amounts of waste material must be removed. Thus, if economic purifi cation is to be achieved, the volume of starting material must be decreased by concentration. Only mild concentra tion procedures that do not inactivate enzymes can be employed. These include thermal methods, precipitation, and to an increasing extant, membran filtration. For many industrial applications, partially purified enzyme preparations will suffice; however, enzymes for xvanalytical. purposes and far medical use must be highly purified. Special procedures employed for enzyme puri fication are crystallization, electrophoresis, and chromatography. The purpose of the present study is to investigate the izolation and characterisation of native lipase of Nigella sativa seed. Far the izolation of enzyme(lipase), the method based on change is solubility was used and the hydrolysis of triglyceride catalysed by acetone powder was investigated for the characterisation of enzymes. Nigella sativa seeds of Turkish origin were purc hased from herbal shop in İstanbul and used as a lipase source for the acetone powder preparation. Lipolytic hydrolysis reactions were carried out in a three-necked flask (250 ml) equipped with a stirrer, a temperature controller, and a thermometer. Optimum reaction conditions were determined at previous studies as pH:S, T:50 C, oil/ buffer solution: 1/1 and SO % seed content based on oil. In all experiments, 80 g used frying oil and 80 ml phosphate buffer solution (pH:6) were placed into the reaction flask and heated to 50QC by stirring. At the reaction temperature lipase was added to the mixture as catalyst. Ground Nigella sativa seed (60 %), press residue of seed (46.6 %) and acetone powder (7.3 % ) at equivalent quantities based on oil weight used as lipalysis catalyst. Samples were withdrawn at predetermined time inter vals and placed in a 90°C water bath for 15 min to inactivate the enzyme. Then, they were centrifuged to saperate the lipase, and the oil phase was dried using anhydrous Na“S0,. Acid values (AV) of the oil samples were determined by titration. The degree of hydrolysis was calculated by the following equation: Hydrolysis % = 100. ( AU2~AU1 )/(SU-AU1 ) Where AIL and l\\l”are acid value of samples at the initial time and time t, respectively, and SV is the saponification value of used frying oil. At the same time, the composition of hydrolyzed oil samples was investigated by TLC-FID. Complete separation of the lipid mixture was achieved into triglyceride (TG) xvienzîm İzolasyonu ve katalîtîk etkîsînîn İncelenmesi Nursel ÇERÇÎO?LU Anahtar Kelimeler : Enzim, Lipaz, Enzim İzolasyonu, Aseton Tozu, Hidroliz. Özet : Endüstriyel ölçüde yüksek sıcaklık ve basınç altında ger çekleştirilen bir çok kimyasal dönüşme, enzimlerin katalitik etkisi altında çok daha ılımlı koşullarda gerçekleşebilmektedir. Enzimler, bitkisel maddelerden, hayvansal organlardan ve mikroorganizmalardan izole edilip saf laştırılarak kullanılırlar. Yağlı tohumlar önemli bir enzim kaynacıdır. Bu çalışmada çöreotu tohumu, tohum pres artığı ve tohumdan asetonla çöktürme yöntemi ile elde edilen aseton tozu kullanılarak trigliseridlere enzimatik hidroliz reaksiyonları uygulanmıştır. Elde edilen sonuçlara 1:1 yağ-su oranında, 50°C ve pH:6'da tohum ve pres artığı aynı aktiviteyi göstermiş ve hidroliz reaksiyonları yaklaşık 8 saatte dengeye ulaşmıştır. Asetonla çök türme yönteminde ise pH'ı 6 olan tampon çözelti ile parçalanan tohumlardan kazanılan aseton tozunun çok daha aktif olduğu ve hidroliz reaksiyonunun 3 saat sonunda dengeye ulaştığı belirlen miştir. ISOLATION AND CHARACTERIZATION OF NATIVE LIPASE Nursel ÇERÇİO?LU Keywords : Enzyme, Lipase, Hydrolysis, Enzyme Isolation, Acetone powder. Abstract : Enzyme catalyzed reactions are used more and more in the technology instead of those performed in a classical chemical reactions. Advantages of enzyme catalyzed reactions include mild reaction conditions and less thermal damage of reactants and products. Our goal was the isolation and characterization of plant lipases. For the isolation of enzyme was used the method based on change in solubility and the hydrolysis of trigliceride catalyzed by acetone powder, was investigated for the characterization of enzymes. The results suggest that ground and pressed seed catalyzed the reaction with the similar effect. In order to increase the activity of acetone powder, Nigella Sativa seed was ground with buffer solution at different pH values (5,6 and 8) and then, acetone powder was precipitated from ground seed with similar method. In hydrolysis reactions catalysed by these powders under the same counditions (pH:6, T:50°C), it was observed that the acetone powder prepared from pre-treated seeds by buffer solutions (pH:6) had higher activity than the other.fatty acid(FA), 1, 3-diglycerids (1,3-DG), 1, 2-diglyceride (1,2-DG), 2-monaglyceride (2-MG) and 1 monoglyceride (1-MG). In the reactions catalysed by ground Nigella sativa seed and pressed seed the hydrolysis degrees were calculated as 83 % and 81. B % at the optimum conditions in B-h reaction time. As can be seen, in the enzymatic hydrolysis of used frying oil by ground Nigella sativa seed and pressed seed the similar results were obtained by giving an important catalytic activity. Far the investigation of the potential application of Nigella sativa seed“acetone powder”in the enzyme- catalyzed hydrolysis lipase“acetone powder”was prepared by charging blender, equipped with a 10.00 ml glass container, with 100 g of Nigella sativa seed, followed by 200 ml of acetone, then ground for 3.5 min. The resulting mixture was transferred into a 1000 ml beaker, which was cooled by salt-ice mixture and the glass container was rinsed with 150 ml of acetone. The acetone was added to the original mixture, then stirred for 1 min. The suspension containing low density material was carefully decanted into a Buchner funnel equipped for vacuum filtration. The seed residue was washed with two 250 ml portions of acetone. Acetone wash was added to the filter cake. The grayish filter cake was transferred into a 1000 ml beaker, washed with two 150 ml portions of acetone, followed by another 100 ml portion. The combined acetone wash was vacuum filtered to afford a liqht grayish powder. The powder was transferred and spread on to an appropriately sized filter paper, then air-dried under a hood to give 12.8 g of grayish powder (12.B %). The powder was stared at k C until required. Acetone powder, prepared according to this method developed by Ayorinde and co-workers, was used in hydrolysis reactions (7.8 % based on oil weight as catalyst. After 8 h af reaction time, the hydrolysis degree was calculated as 36.6 %. It was shown that rate of the hydrolysis reaction catalysed by acetone powder decreased, because of the negative effect af acetone on enzymes. It was observed that raw lipase (acetone powder), prepared under cooling with ice-salt mixture was more active than other that prepared cooling with ice and therefore ice-salt mixture was used for the coaling in the preparation of acetone powder. These results suggest that ground and pressed seed catalysed the reaction with the similar effect and hydrolysis degree of xviithe reaction increased by increasing acetone powder content. The hydrolysis degrees at 7.8 %, 15 %, 25%, and 35 % acetone powder content based on the oil weight were calculated as 36.6 %, 58. 6 %, 75.2 % and 79.7 % after B h of reaction time. In order to increase the activity of acetone powder, Nigella sativa seed was ground with buffer solution at different pH values (5,6 and 8) and then, acetone powder was precipitated from ground seed with similar method. In the hydrolysis reactions catalysed by these powders under the same conditions (pH:6, T:50 C) an equilibrium was achieved in 2 hours and, it was observed that the acetone powder prepared from pre-treated seeds by buffer solutions had higher activity than the other. In order to investigate the specificity of Nigella sativa seed lipase commercial lipases, lipase D“Amana 100”and Cylindraceae Type 111(1,3 specific and non specific) were used in the hydrolysis reactions (pH:6, T:50DC and T:37DC). Results showed that the ' Nigella sativa seed lipase (acetone powder) is non specific. xvixi
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