Organik çöplerden elde edilen besiyerinde üreyen Apergillus niger' den proteaz enzimi üretimi ve karakterizasyonu
Protease enzyme production and characterization from Aspergillus niger, reproduced in media from organic waste
Yazar: BETÜL TEKYILDIZ
Danışman: PROF. DR. ARZU ÇAĞRI MEHMETOĞULU
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Endüstriyel enzimler ile ilgili çalışmalar teknolojinin gelişmesi, kullanılan ürünlerin kullanım alanlarının artması ve ekonomik değerinin yüksek olması gibi sebeplerden kaynaklı önemlidir. Günümüz dünyasında, nüfus her geçen gün artmaktadır ve bu nedenle artan atık oluşumu gerçeğini düşünmek elzemdir çünkü oluşan atık gereksinimleri karşılanabilirse, üretim maliyetinde önemli ölçüde azalma sağlanabilmektedir. Bu nedenle çalışmamızda proteazın kolay, hızlı ve düşük maliyetli şekilde üretimi için elzem parametreler optimize edilmiştir. Bu tez çalışması kapsamında; optimum proteaz enzimi üretmek için meyve ve sebze atığı, karbonhidrat bazlı ve protein (hayvansal veya bitkisel) oranı yüksek organik atıklarından besiyerleri hazırlanarak enzimin bu besiyerlerinde üreme potansiyelleri farklı pH (5,0, 5,5, 6,0) ve farklı inkübasyon sürelerinde (24, 48, 72 saat) Taguchi yöntemi ile araştırıldı.  Taguchi yöntemiyle hesaplanan en yüksek sinyal/gürültü  (S/N) oranına sahip olan seviyeler dikkate alındığında, proteaz enzim üretimi için 30 °C'de 24 saat inkübasyon süresi,  pH 5,5 değeri, ve %50: 50 bitkisel ve hayvansal protein oranına sahip besiyeri şartlarının optimum şartlar olduğu belirlenmiştir. Aktiviteyi en fazla etkileyen faktörün protein oranı olduğu ve daha sonra sırasıyla inkübasyon süresi ve pH değerinin aktiviteyi etkilediği belirlendi. İnkübasyon süresinin uzaması, pH değeri ve hayvansal protein oranının artması, aktivite değerinde azalmaya sebep olduğu görüldü. 
A. niger' den üretilen proteaz enzimi TPP (üçlü faz ayırma) yöntemi ile saflaştırılmış ve biyokimyasal karakterizasyonları incelenmiştir. Bu yöntem geniş bir uygulama alanına sahip, çok yönlü bir biyoayırma aracı haline gelen, yüksek geri kazanım ve saflık seviyelerine sahip bir dizi enzim ve proteini saflaştırmak için kullanılır. TPP sistemi ile proteaz enzimi 1,0:1,5 (ham enzim çözeltisi: t-bütanol oranı) ve %80 doygun amonyum sülfat konsantrasyonu ile %235,7 verimle 13,15 kat saflaştırıldı. Enzimin alt fazda toplandığı tespit edildi ve karakterizasyon çalışmaları için alt faz kulanıldı. A. niger' den saflaştırılan proteaz enziminin optimum ve stabil pH'ları sırasıyla 10,0 ve 9,0; optimum ve stabil sıcaklığı ise 60°C olarak tespit edildi. Proteaz enziminin okside edici ajanlar ve yüzey aktif maddeleri varlığında gösterdiği aktivite değerleri incelendiğinde SDS' in ciddi bir aktivite kaybına sebep olduğu, diğer maddelere nazaran %1'lik H_2 O_2 varlığında aktivitesini koruduğu tespit edildi. Tween 20, Tween 80 ve TrionX-100 varlığında yakın sonuçlar göstererek belli oranlarda azalmaya sebep oldu.  Serin proteaz inhibitörü fenilmetilsülfonilflorid (PMSF) varlığı aktiviteyi sıfıra yakın bir değerde inhibe ederken, β-merkaptoetanol aktivitede artışına sebep oldu. Metaloenzim inhibitörü olan Etilen Diamin Tetra Asetik Asitin (EDTA) özellikle %5 konsantrasyonunda aktiviteyi azaltması enzimin metal bağlama bölgesine sahip olduğunu göstermiştir. Enzimin substratlarla olan ilişkisi incelendiğinde kazein ve azokazeinin aktivitede değişiminde olumlu etkileri olurken, hemoglobin, jelatin ve BSA' nın aktivitede azalmaya sebep olduğu görülmüştür.

Abstract:
Within the scope of this thesis; In order to produce the optimum protease enzyme, media are prepared from fruit and vegetable waste, carbohydrate-based and high protein (animal or vegetable) organic wastes, and the growth potential of the enzyme in these media is at different pH (5.0, 5.5, 6.0) and different incubation times ( 24, 48, 72 hours) were investigated by the Taguchi method. Considering the levels with the highest signal-to-noise (S/N) ratio calculated by the Taguchi method, an incubation time of 24 hours at 30 °C, a pH value of 5.5, and a 50:50 vegetable and animal protein ratio for optimum enzyme production medium was found to be used. It was determined that the factor affecting the analysis the most was the protein ratio, which had the greatest value in the removal efficiency, and then the incubation time and pH value affected the activity, respectively. Proteins are polymers formed as a result of the polymerization of amino acids, and the amount and type of each amino acid differs according to the protein source. For example, the amino acids contained in animal and vegetable proteins are different, and the differences also affect the type of protease. Proteases are classified as serine protease, cysteine protease, acidic protease and metalloproteases depending on the functional amino acid radical in their active sites. Among the vegetable proteins in the composition of the medium prepared in our study, pulses contain amino acids such as acidic acid, lysine, glutamic acid and arginine. Animal proteins contain all 20 amino acids, including cysteine and serine, enzymes that protease uses to break down protein [33]. Looking at the results, the medium with equal amounts of vegetable and animal protein was preferred for protease production, but it is still known that animal proteins are richer in amino acids compared to vegetable proteins for enzyme production. When the amount of animal protein is increased to 70%, an increase in activity occurred, but the yield obtained at an equal rate could not be achieved at this value. 60%-40% rate had a significant decrease in protease enzyme activity. For this reason, it would be a more logical approach to provide a balance rather than giving weight to one of the vegetable or animal proteins. It was observed that the prolongation of the incubation period, the increase in the pH value and the ratio of animal protein caused a decrease in the activity value. As the incubation period increases, the decrease in enzyme production is due to the other compounds in the medium and the deterioration of the solid substrate structure used over time. Enzyme production by the microorganism in a short time provides an important advantage. Enzyme production increases linearly as the optimum incubation time approaches, but gradually decreases after the peak growth time. The reason for this can be shown that the decrease and deterioration of the nutrients in the fermentation medium and the accumulation of  waste materials in the environment accelerate the transition of bacteria to the death   phase and the production in the death 
phase is lower than the stationary phase. After calculating the most productive medium, the composition of the medium was examined and according to the analysis results; The humidity in the broth was found to be 97.96%. Ash determination results were calculated as 0.41%. The protein ratio of the medium is 0.6 g/100g; total sugar 3.61 g/100; oil 0.26g/100g; polysaccharide was found to be 0.81 g/100 g.
Protease enzyme produced from A. niger is partially purified and biochemically purified by triple phase separation (TPP) system, which is used to purify a range of enzymes and proteins with high recovery and purity levels, which has become a versatile bioseparation tool with a wide range of applications. characterizations were studied. To find the ammonium sulfate/t-butanol ratio at which purification is best, use different ammonium sulfate saturations (30%, 40%, 50%, 60%, 70% and 90%) and different homogentate/t-butanol ratios (1.0:0). ,5, 1.0:1.0, 1.0:1.5 and 1.0:2.0 ) enzyme activities were calculated. After these calculations, the protease enzyme was purified 13.15 times with a yield of 235.7% with 1.0:1,5 (raw enzyme solution: t-butanol ratio) and 80% saturated ammonium sulfate concentration by TPP system. The molecular weight of the purified protease enzyme was calculated using the Maldi Tof device and its molecular weight was found to be 15.56 kda.
This phase was used in characterization studies since it was determined that the enzyme was collected in the lower phase. In order to see the pH value showing the optimum effect on the purified protease enzyme, enzyme activity values were measured by using buffers at different pH values containing 0.65% casein. 0.1 M sodium phosphate was used for pH 6.0-8.0, Tris-HCl for pH 7.0-9.0 and glycine-NaOH for pH 9.0-11.0. It was determined that the protease enzyme had the best activity in pH 10 glycine-NaOH (pH 9.0-11.0) buffer. Some buffers were used for stable pH determination on the purified protease enzyme. 0.1 M sodium phosphate was used for pH 6.0-8.0, Tris-HCl for pH 7.0-9.0 and glycine-NaOH for pH 9.0-11.0 and activity values were checked. It was determined that the protease enzyme had the best activity in pH 9 100 mM Tris-HCl buffer. In order to determine the optimum temperature on the protease enzyme, the activity values were checked at temperatures between 20°C and 90°C. The obtained data were shown in graphs and charts. In the light of these results, it was determined that the optimum temperature was 60°C. In order to determine the stable temperature value of the protease enzyme, the activity values were measured between 20°C and 90°C and incubation times of 15, 30, 60 and 90 minutes. Graphs and charts were created with the results obtained from the analyzes. It was observed that protease provided the best activity at 60°C temperature and at the end of 60 minutes with a value of 98.91%. Compared to other temperature values, it was observed that it was the temperature that best preserved its activity for varying periods of time. At the end of the 120 min incubation period at 20 °C, 30 °C and 90 °C, a visible loss of activity was detected in the activity. In order to examine the effect of oxidizing agent H2O2 and surfactants on the purified protease enzyme, surfactants such as SDS, Tween-20, Tween-80, Trion X-100 at 1% and 5% concentrations and H2O2 (oxidizing agent) ) were prepared and the activity was  determined  by interacting these solutions with the enzyme for 30 minutes. When the results were examined, it was observed that 1% SDS caused a significant loss of activity in the enzyme activity, while 5% SDS completely inhibited the activity of the enzyme. It was determined that the enzyme activity was preserved up to 87% in the presence of 1% H2O2. As a result, it was determined that as the concentration of the surfactants increased, the effect on the activity of the enzyme increased. In order to examine  the effect of protease enzyme on inhibitors, 1 mM and 5 mM solutions from 
DTNB, EDTA, PMSF and β-mercaptoethanol, and 1 mM and 8 mM solutions from urea were prepared and kept for 30 minutes by interacting with the enzyme, and activity was measured at the end of the time. Enzyme untreated with inhibition solution was used as control and accepted as 100. When the results are examined, it is seen that the enzyme inhibits approximately 95% of its activity in the presence of 1% PMSF, and inhibits almost all of it at 5%. The presence of β-mercaptoethanol at two concentrations caused an increase in activity. A significant decrease in enzyme activity, also in the presence of EDTA, led to the conclusion that the enzyme has a metal binding site. The inhibitory effect of PMSF, a serine-specific inhibitor, on the enzyme indicates that there is an alkaline serine protease in its active site. The fact that the alkaline serine protease enzyme, which was examined in our study, is very popular in the world and has a wide area of use in the industry, shows the importance of the study. In order to determine the activity values of the protease enzyme on various substrates, activity measurements were made using 0.65% concentration casein, BSA, hemoglobin, gelatin and azokazein substrate solution prepared in 100 mM pH 8.5 Tris-HCl buffer. The obtained values are shown in Table 3.11. The protease enzyme showed the best activity in the presence of casein. Casein was followed by azokazein hemoglobin, gelatin, and finally BSA.
The results obtained in the study are important because the researches of biotechnology on industrial enzymes are also gaining importance. The reason for this can be shown as the development of enzyme technology day by day, expanding product usage areas and very high economic value. Some of these studies are on the production of enzymes from waste because in today's world, the population is increasing day by day and therefore it is inevitable to consider the fact of increasing waste generation and take the necessary action. Especially due to the inherent biodegradable properties of fruits and vegetables, the amount of waste obtained from them amounts to about 50 tons per year, of which only 0.5% is allocated as input for various processes, the rest is thrown away. For this reason, nowadays, the focus is on regeneration of resources from wastes, which are abundant as a by-product of any process.
The results of the present study clearly show that organic wastes, which are inexpensive substrates, can be used as a potential source for protease production. Proteases account for more than 60% of global industrial production, and therefore this study gains importance in terms of the enzyme market, because isolation of microorganisms that produce new alkaline proteases that accelerate reactions and have high resistance to adverse conditions is also important. In addition, rapidly developing industrialization provides the world with a perennial raw material need for products. In this regard, if the generated waste requirements can be met, a significant reduction   in the production cost can be achieved. Therefore, in our study, essential parameters were optimized for the easy, fast and cost-effective production of protease.