Çörekotu (Nigella sativa l.) tohumlarından lipaz enziminin ekstraksiyonu

Abstract

Son beş yılda lipaz enzimlerinin, deterjan ve deri sanayiinde yağların biyolojik hidrolizinde; yağ sanayiinde istenilen özelliklerde sıvı yağ ve margarin üretimi için yağların modifikasyonunda ve kimyasal yolla başarılamayan çok çeşitli organik sentezlerin gerçekleştirilmesinde kullanılmış olması, endüstriyel lipaz enzimlerinin karakterizasyonu ve üretimi üzerine çalışmaların hızlanmasına neden olmuştur. Literatürde, mikrobiyal ve hayvansal orijinli lipazlar üzerinde çok sayıda çalışma bulunmasına karşılık, bitkisel lipazlarla ilişkin sınırlı sayıda çalışmaya rastlanmıştır. Bu çalışmada, çörekotu tohumlarından tampon çözeltiler ile lipaz enziminin ekstraksiyonu amaçlanmıştır. Bu amaçla, orijinal ve çimlenmiş çörekotu tohumları Fosfat ve Tris-HCL tampon çözeltileri ile ekstrakte edilmiştir. Çimlenmenin, tampon çözelti pH'mın ve cinsinin ekstraksiyon verimine ve elde edilen lipaz ekstraktlannın aktivitelerine olan etkilerinin belirlenmesiyle, optimum ekstraksiyon koşulları saptanmıştır. Çimlenmenin, lipaz enziminin ekstraksiyon verimini 1,4 kat ve aktivitesini ise 2 kat arttırdığı belirlenmiştir. Çimlenmiş tohumlarla yürütülmüş ekstraksiyon deneylerinde, tampon çözelti pH'mın ekstraksiyon verimi ve lipaz aktivitesine büyük etkisi olduğu saptanmıştır. Fosfat tampon çözeltisi için pH 6, Tris-HCl için pH 7 optimum ekstraksiyon pH'ları olarak belirlenmiştir. pH 6 fosfat tampon çözeltisi ile %21 verimle spesifik akti vitesi 0.64 U/mg protein olan lipaz enzimi elde edilirken, Tris-HCl (pH 7) tampon çözeltisi ile çalışıldığında elde edilen lipaz enziminin verimi %18 ve spesifik aktivitesi 0.74 U/mg protein olmaktadır. Çörekotu lipaz enziminin optimum sıcaklığının 40°C olduğu; pH 6 ve pH 9'da iki optimum pH değeri gösterdiği de belirlenmiştir. VIII

Lipases (or acylglycerol ester hydrolases, EC 3.1.1.3) are unique enzymes and they are extensively used as additives in detergents (to hydrolize fats under alkaline conditions); in the production of enhanced flavor additives (to release short chain fatty acids from butterfat feedstocks); in the synthesis of several valuable chemical compounds which could not be produced by chemical reactions and in the manufacture of engineered fats with desired nutropharmaceutical and/or functional properties (to change the qualitative and quantitative profile of fatty acid residues). Selectivity of certain lipases for or against particular fatty acids/acyl moieties has been utilized for the enrichment of such fatty acids or their derivatives from naturally occurring mixtures via selective hydrolysis, esterification and interesterification. For example, the polyunsaturated fatty acids, especially Eicosapentaenoic acid (20:5- EPA) and Docosahexaenoic acid (22: 6-DHA) have been reported to have beneficial effects in cardiovascular diseases, autoimmune disorders and other inflammations, such as arthritis, asthma. The main source of these fatty acids is marine oils and due to complex nature of fish oils, EPA and DHA could not be separated from fish oils by conventional separation methods. The ability of the some lipases to discriminate against EPA and DHA has been utilized in the production of concentrates enriched in EPA and DHA. The different physiological and pharmaceutical activities for both enantiomers of one molecule are well known in medical chemistry. As examples, S-phenylalanin is bitter but the R-isomer is sweet; R-talidomide is a good sedative drug but S-isomer is teratogenic; S-verapamil is a strong controller of certain kinds of cardiac arrythmia and the R-verapamil is a potent antitumoral agent. This different or opposite activity of both enantiomers is a real problem because there are more than 500 drugs currently marketed as racemic mixture with negligible information available on the properties of the individual stereoisomers. Due to the enantioselectivity of certain lipases to the one of the isomer, lipases have succesfully used in the synthesis of chiral drugs over the last ten years. Lipases can be obtained from mammals, yeasts, bacterias and higher plants. To date, most of the industrial lipase enzymes have been produced from fungal resources and they are well characterized. However, lipases from higher plants have not yet been investigated in this regard and they may display properties different to those from fungal resources and they could be exploited for industrial or technical purposes. IX Cereal grains and oilseeds are cheap and alternative sources of lipases. Seeds generally contain proteins and depending on the plant species, mainly starch or triacylglycerols as food reserve for germination. In the mobilization of these three major reserves during germination, they are hydrolyzed initially by specific proteases, amylases and lipases, respectively. Most investigations on plant lipases have been carried out on oleoginous seeds in which lipase activity is generally found to became prominent upon germination. Oilseed including, sunflower, cotton, corn, rape, mustard contained only alkaline lipase activity, which increased drastically during germination. Castor bean contains acid lipase in dormant seeds. In addition to the acid lipase, alkali lipase was found in germinating castor bean endosperm. The pattern of acid and alkaline lipases in castor bean did not seem to be common in other oil seeds. Lipase from germinating seedlings of oilseed rape has been used as biocatalyst for esterification and interesterification of lipids. In recent studies conducted at chemical Technologies in chemical engineering department, Nigella Sativa (Black cumin) seeds have been shown as a good source of lipase enzyme which can be an effective biocatalyst in hydrolysis and esterification reactions of triglycerides. Nigella sativa L., also known as black cumin or fennel flower, is a member of the Ranunculaceae family and native to some parts of the mediterranean region. It is cultivated in India and Turkey, the major plantation areas being Afyon, Burdur and İsparta. Presently, the seeds are sold at local and export markets for use as condiment or native medicine; furthermore, about 5 tonnes per year of seed oil is being exported in recent years for similar purposes. In this study, the extraction and the characterization of lipase enzyme from Nigella Sativa seeds were investigated. The extraction was performed by phosphate and Tris-HCl buffer solutions of different pH values. The effects of pH and the types of the buffer solutions on the yield and the activity of the lipase extracts were examined. Samples of Denizli origin Nigella sativa seeds were purchased from a locak market in Istanbul. Moisture content was determined by oven-drying at 105°C. Total nitrogen content was determined using the standard Kjeldahl method. Crude protein was expressed as 6.25xN. Crude fiber was determined according to the standard ADCS method, by calculating the loss in weight of dried residue remaining after digestion of a fat-free sample with 0.25 M H2SO4 and 0.6 M NaOH under specified conditions. Ash content was determined by incineration of the sample in a muffle furnace at 600°C for 16 h. Total fat content was obtained by the soxhlet extraction X method using hexane as described by ADCS method. Carbohydrate content was obtained by subtracting the sum of protein, fat, ash and moisture from 100. The proximate composition of Nigella sativa seeds indicated that seeds were composed of 23.3% protein, 35.4% oil, 9.4% moisture, 4.0% ash, 6.7% crude fiber and the rest being composed of other carbohydrates. The crude fiber content of 6.7% in black cumin seeds makes it a source of dietary fiber which would be helpful in reducing gastrointestinal disorders. The lipase extracts were obtained as follows: 60 g seeds were homogenized for 20 min using a blender with 300 ml buffer solution at 4°C. The homogenate was filtered through nylon cloth. The homogenate was then centrifuged for 30 min at 10,000 g, yielding supernatant liquid and crude particulate fraction. The supernatant liquid is called as "lipase extract" throughout this text. The protein content of lipase extract was determined spectrophotometrically according to the biuret method using a Randox Total Protein Reagent. Bovine serum albumin was used for establishing the standard curve. Lipase activity of extract was determined by the titrimetric method using olive oil emulsion. Fir preparation of the substrate, 5 g olive oil, 5 g gum arabic and 95 mL 0.89% (W/V) NaCI solution were emulsified with a blender at room temperature for two times 5 minutes. Emulsions were always prepared immediately before use. The assay mixture for lipase activity contained 5 mL substrate emulsion, 0.5 mL 0.1 M CaCL; solution, 1 mL lipase extract (containing 5- 10 mg of protein) and 0.89% NaCI solution, made up to a final volume of 10 mL. The mixture was incubated at 37°C in a shaker for a period of 10 min. The reaction was stopped by adding 20 mL of etanol/acetone (1:1,V/V) mixture. The fatty acids liberated were measured by titration with 0.05 N KOH using a thymolphthalein as an indicator. Corrections were made for endogenous fatty acid production (assay mixture without substrate emulsion) and nonenzymatic fatty acid production(assay mixture without enzyme preparation). The lipase activity was expressed as jumol fatty acids released per mg protein in minute. To investigate the effect of germination on the protein content and specific activity of the lipase extract, set of experiments was conducted with germinating seeds. Nigella sativa seeds were soaked in water for 24 hr at 26 C. The end of this imbibition period was designated day zero of germination. Germination was carried out in moist papers at 26°C in darkness for seven days. The seed samples, taken during the germination, were extracted with water and analyzed for protein and lipase activity assays. The results indicated that the maximum lipase activity in lipase extract was reached at day 4 of seedling growth. Therefore, the subsequent experiments were conducted with 4 days germinated seeds. To see the effects of pH and the types of buffer on the protein content and activity of the lipase extracts, two sets of extraction studies were conducted with phosphate and Tris-HCl buffer solutions. The pH of the solutions was changed from 5 to 10 and from 6 to 9 for phosphate and Tris-HCl buffer solutions, respectively. It was observed that the solubility of the seed proteins was minimum at pH 7 for both buffer solutions. With decreasing or increasing the pH, the protein content of the XI lipase extract increased. The lipase extract obtained by pH 6 phosphate buffer displayed maximum lipase activity whereas the highest activity was observed in the lipase extract obtained by pH 7 Tris-HCl. Tris-HCl solutions dissolved relatively less amount of protein from seeds than those of phosphate solutions, but the lipase extracts obtained by Tris-HCl buffer solutions exhibited higher specific activity than those of corresponding solutions obtained by phosphate solutions. This fact may be explained by the selective dissolving of lipase containing proteins in Tris-HCl buffer solutions. The optimal extraction pH was established as 6 and 7 for phosphate and Tris-HCl buffers, respectively. In conclusion, we can say that phosphate and Tris-HCl buffers are suitable buffers for extraction of lipase enzyme from Nigella sativa seeds. Studying by pH 6 phosphate and pH 7 Tris-HCl buffer solutions, enzyme proteins could be extracted from seeds in a yield of 21 and 18 % based on protein content of seeds, respectively. The corresponding extracts had lipase activities of 0.64 and 0.74 U/mg protein, respectively. The lipase extract in pH 6 phosphate buffer was further purified separately by acetone and ammonium suphate precipitation methods. Pure acetone was added dropwise to 100 ml lipase extract, containing 1177 mg proteins with a specific activity of 0.64 U/mg protein, until the crystals were seen. Then the mixture was held -15°C for 30 min. The precipitate thus obtained was separated by centrifugation at 10.000 rpm for 15 min and redissolved in phosphate buffer (pH 6) solution. The resulting lipase solution contained 727 mg proteins, and its specific activity was found to be 1.85 U/mg protein. This treatment increased the lipase activity about 3 -fold. The another 200 ml sample of the same lipase extract was also concentrated by adding ammonium sulphate to a definite concentration (0-30 %, 30-60 %, and 60-80 %) and incubated at 4°C for 30 min. The precipitates thus obtained were separated by centrifugation at 10.000 rpm for 15 min. These precipitates were then divided into two equal parts. One part was dissolved in Tris-HCl (pH 6) and the other was dissolved phosphate (pH 6) buffer solutions. The results indicated that the ammonium sulphate fractionation method gave the best results. The protein fraction precipitated at 60-80 % ammonium sulphate saturation, exhibited 10.71 U/mg protein of activity, in Tris-HCl solution. Finally, the optimum pH and temperature of the Nigella sativa lipase were determined. The titrimetric method was also used for he determination of the pH optimum of lipase activity. The text mixture contained in a final volume of 10 mL: XII 5 mL olive oil emulsion, 0.5 mL 0.1 M CaCİ2 solution, 0.8 mL enzyme ekstract and 3.7 mL buffer, adjusted to different pH values varying from 4 to 10. For the determination of optimum temperature, samples of lipase extract were incubated at 30, 35, 40, 45, 50 and 55°C separately for 30 min in previously determinated optimum pH. Then, the lipase assays of these samples were determinated by the same method exhlained above. Two lipases were found in extracts from Nigella sativa seeds, like castor beans. Acid lipase had optimal activity at pH 6. Alkaline lipase displayed high activity ot pH 9. The results also showed that the activity of the acid lipase was about three times that of alkaline lipase. The optimum temperature of Nigella sativa lipase enzyme was found to be 40°C. As conclusion, phosphate and Tris-HCl buffer solutions can be used for extraction of lipase enzyme from Nigella sativa seeds.