Bazı Bacillus izolatlarının poligalakturonaz üretimlerinin araştırılması
Investigation of polygalacturonase enzyme production by Bacillus strains isolated from various sources
Yazar: KUDRET BULUT
Danışman: DOÇ. DR. AYŞE AVCI
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Bu çalışmada, Van Gölü'nden izole edilen alkali özellikteki Bacillus izolatlarının poligataktruronaz enzim üretimleri araştırılarak en iyi enzim üreticisi izolat seçilmiş ve bu izolatin enzim üretim koşulları optimize edilmiştir.  Çalışmada 25 izolat kullanılmış ve enzim üretimleri pektinaz içeren besiyeri ortamında geliştirilen bakterilerin üzerine iyot çözeltisi damlatılarak kolonilerin etrafında oluşan şeffaf zon belirlenmiştir. En büyük zon çapının görüldüğü VGA7 izolatı en iyi üretici bakteri olarak seçilmiş ve bununla enzim üretim çalışmaları yapılmıştır.  İzolatın muhafazası %25 gliserol içeren nutrient broth içerisinde yapılmış olup aktifleştirme işlemi önce nutrient agar ardından nutrient broth içerisinde 33 ˚C'de 24 saatte yapılmıştır. Aktif kültürden üretim ortamına %5 oranında aşılama yapılmıştır. Enzim üretiminde pektin içeren bazal bir besiyeri (10 g/L maya özütü, 10 g/L pektin, 1,5 g/L NaCl, 2 g/L K2HPO4, 0,1 g/L MgSO4.7H2O) hazırlanmıştır. Enzim üretimine sıcaklık, pH, pektin konsantrasyonu, karbon ve azot kaynaklarının etkisi belirlenmiştir. Sıcaklığın belirlenmesi için 25, 30, 33, 37 ve 40 ˚C'de inkübasyonlar yapılmıştır. pH etkisinin belirlenmesi için bazal besiyerinin başlangıç pH'sı, 7, 8, 9, 10 ve 11'e ayarlanarak 33 ˚C'de 24 saat inkübasyon gerçekleştirilmiştir. Pektin konsantrasyonunun etkisinin belirlenmesi için besiyerine 2, 5, 10, 15 ve 20 g/L oranlarında pektin eklenmiştir. Besiyerinde kullanılan pektin yerine gam arabik, sakkaroz, glukoz, früktoz, nisaşta ve laktoz eklenerek üretimler gerçekleştirilerek karbon kaynaklarının etkisi belirlenmiştir. Farklı azot kaynaklarının etkisinin belirlenmesi için yapılan çalışmada ise maya özütü yerine aynı oranda yağsız süt tozu, sarı mercimek unu, soya unu, pepton, kazein ve amonyum sülfat eklenmiştir. İnkübasyonlar sonrasında alınan örnekler 10000 rpm'de santrifüjlenerek hücreler çöktürülmüş ve süpernatantta enzim aktivitesi tayini yapılmıştır. Enzim aktivitesi tayini için pektin içeren tampon çözeltisinde enzim eklenerek 50 ˚C'de 30 dakika inkübe edilmiş süre sonunda oluşan indirgen şeker miktarı DNS yöntemi ile belirlenmiştir. Elde edilen sonuçlara göre, enzim üretiminin en yüksek olduğu sıcaklık 33 ˚C, pH 9, 20 g/L pektin varlığında olmuştur. Karbon kaynaklarında en iyi olanın pektin olduğu belirlenmiş olup bunu laktoz takip etmiştir. Azot kaynaklarından ise en etkili olanın maya özütü olduğu belirlenmiştir. Ayrıca enzimin optimum sıcaklığı ve pH'ı da belirlenmiştir. Enzimin optimum pH'ı 12, sıcaklığı ise 70 ˚C olarak belirlenmiştir.  Sonuç olarak bu çalışma ile Van Gölü'nden izole edilen ve pH 12 gibi ekstrem koşullarda aktivite gösteren poligalakturonaz enzimi üretimi gerçekleştirilmiştir. Elde edilen bilgiler ışığında enzimin uygulama alanları ile ilgili çalışmalar ileride gerçekleştirilebilir.

Abstract:
Pectic substances, together with other structural components, are found in the cell wall of the plant and are among the basic compounds that give rigidity to tissues. The function of pectic substances is to provide structural integrity to the cell and make them cohesive. They are are mainly composed of D-galacturonic acid units bound by α(1-4) glycosidic bonds in which some hydroxyl groups are methylated. These molecules are classified based on their esterification levels or carboxylic groups. If 75% of the carboxylic acids are methylated, it is called pectin, if less than 75% is methylated, it is called pectic acid, and if there is no methyl esterification, it is called polygalacturonic acid. Pectin is the general name of these groups.
Pectinases are generally the name given to a group of enzymes that have the ability to catalyze pectic compounds through depolymerization (hydrolases and lyases) and deesterification (esterases) reactions. Pectinases are classified according to the substrate they act on, the way they break down the substrate they act on, and whether the enzyme breaks down sequentially or randomly. Polygalacturonases are enzymes belonging to the pectinase family and catalyze the breakdown of polygalacturonic acid into monomers or dimers. Pectinases, which have a 25% share in the global enzyme market, have an important place in the food industry. Areas of use include clarification of fruit juices, tea and coffee fermentation, textile and paper industry. Although many plants are sources of polygalacturonase, microbial-derived enzymes are preferred in industry today due to their ease of production, economical and sustainable nature. Molds, yeast and bacteria can be used in the production of polygalacturonase, and mold-based enzymes are mostly produced. In recent years, there has been a trend towards bacterial enzymes due to their superior properties such as high temperature application and resistance to alkaline and acid conditions.
In this study, the polygatactruronase enzyme production from alkaline Bacillus isolates isolated from Lake Van was investigated and the best enzyme producer isolate was selected for further studies at which the enzyme production conditions of this isolate were optimized. A total of 25 isolates were tested in the study. They were grown on agar medium containing pectin at 33 oC for 24.  At the end of the incubation period, Petri dishes were covered with Iodine solution and they were observed for the formation of transparent zones around the colonies. The isolate having the highest zone that was coded as VGA7, was selected for further studies. The isolate was maintained in nutrient broth containing 25% glycerol, and the activation of the bacterium was performed on nutrient agar plates followed by nutrient broth at 33 ˚C for 24 hours. Volume of inocultaion was 5% made from the active culture into the production medium. In the enzyme production studies, a basal medium containing 10 g/L yeast extract, 10 g/L pectin, 1.5 g/L NaCl, 2 g/L K2HPO4, 0.1 g/L MgSO4.7H2O was used. The subsequent tests were performed by making slight modification on that medium. The effects of temperature, pH, pectin concentration, carbon and nitrogen sources on the enzyme production were determined. In order to determine the effect of temperature on the enzyme production, incubations were carried out at 25, 30, 33, 37 and 40 ˚C for 24 hours. The effect of pH on the enzyme production was performed by adjusting the initial pH of the basal medium to 7, 8, 9, 10 and 11 by using 2 N HCl or 2 N NaCl and incubation was carried out for 24 hours at 33 ˚C. To determine the effect of pectin concentration, pectin was added to the basal medium at rates of 2, 5, 10, 15 and 20 g/L and inbutaions were performed at 33 ˚C for 24 hours. The effect of carbon sources was determined by adding pectin with gum arabic, sucrose, glucose, fructose, starch and lactose instead of pectin used in the basal medium. In the study conducted to determine the effect of different nitrogen sources, the same amount of skim milk powder, yellow lentil flour, soy flour, peptone, casein and ammonium sulfate was added instead of yeast extract. The samples taken after the incubations were centrifuged at 10000 rpm to precipitate the cells and enzyme activity was determined in the supernatant. For enzyme activity determination, enzyme was added to the glisin-NaOH buffer solution (pH 10) containing pectin and incubations were performed at 50 ˚C for 30 minutes. At the end of the incubation 2 mL DNS solution was added to stop the reaction and also to apply DNS reducing sugar determination method to detect the amount of reducing sugars generated by the enzyme. The reducing sugar amount was determined as galacturonic acid and for this a standard curve was constructed using galacturonic acid.
According to the results, enzymatic activity was observed at the the temperatures studied, however the highest activity (1.95 U/mL) was detected when the temperature was 33 ˚C, and the lowest (1.61 U/mL) was at 25 ˚C.   At the higher tempertures the the activity was hight enough which was 1.89 U/mL at 40 ˚C. The pH studies showed that the enzyme was highly alkaline which had lower activity at pH 7 and it increased significantly with increasing pH. The highest enzyme activity was obtained when the initial pH of the medium was 9 (1.93 U/mL), while the minimum was at pH 7 (1.33 U/mL).  Effect of pectin concentration studies showed that enzyme activity increased with the increasing pectin concentration and the highest activity was observed at 20 g/l pectin concentration, however, the increase in the activity with concentration was not statisticalyy significant. Regarding the effects of various carbon sources on the enzyme production, all the tested carbon sources led to enzyme production in a range between 1.52 and 1.95 U/mL. The highest enzyme activity was detected with pectin. In addition, enzymatic activity with lactose as substrate was also high and those with glucose, sucrose, fructose and starch were statisticaly similar. Among the nitrogen sources tested yeast extract was found to be the best one for the production of enzyme and the lowest enzyme activity were obtained with pepton. Natural nitrogen sources including skim milk powder, soy bean flour and lentil flour had also promising enzymatic activities that had 75.3 to 81% relative activity compared to yeast extract. These results indicated that natural nitrogen sources which cheaper and easily available can be evaluated in future studies. Ammonium sulfate was tested as inorganic nitrohen source and it had 67.1% relative activity compared to yeast extract.
Additionally, optimum temperature and pH of the enzyme were also determined. To determine the optimum temperature enzymatic activities were determined at 30, 40, 50, 60, 70, and 80 ˚C. The lowest activity was found at 30 ˚C and it increased as the temperature was increased up to 70 ˚C which was determined as the optimum. On the other hand at 80 ˚C, the activity started to decrease. The optimum pH of the enzyme was determined by preparing the substrate (pectin) in various buffer solutions [Citrate buffer (pH 4, 5, 6), Tris-HCl buffer (pH 7, 8, 9), and glycin-NaOH buffer (pH 10, 11, 12)]. Enzmatic activity analysis were performed using the mentioned buffer at 50 ˚C for 30 minutes and reducing sugars genetrated by the enzyme was determined with DNS method. The activity was lowest at pH 4 and it increased with increasing ph values. The highest enzymatic activity was obtained at pH 12. This study showed that the enzyme is extremely alkaline. As a result, it was shown that a novel Bacillus sp. VGA7 isolated from Lave Van was a good source of extremely alkaline polygalaturonase enzyme. In the light of the information obtained, studies on the application areas of the enzyme can be carried out in the future.