Publication: Yerel ekosistemlerden izole edilen ve endüstriyel önemi olan proteinleri üreten bakterilerin izolasyonu, tanımlanması ve ilgili proteinlerin taranması
Abstract
Yerel Ekosistemlerden İzole edilen ve Endüstriyel Önemi olan Proteinleri Üreten Bakterilerin İzolasyonu, Tanımlanması ve İlgili Proteinlerin Taranması Bu çalışmada, Marmara Bölgesinde bulunan sıcak su kaynaklarından ve komposttan izole edilen 146 bakteri kültürü, endüstriyel önemi olan proteaz, endoglukonaz, amilaz ve lipaz gibi enzimleri üretebilirlikleri açısından taranmıştır. Toplam izolatlar içerisinde alkalen proteaz, endoglukonaz, amilaz ve lipaz üretebilen izolatlar sırası ile %80.3, %45.26, %16.06, %16.06 olarak bulunmuştur. Fenotipik özelliklerine göre 146 bakteri kültüründen 19 izolat seçilmiştir. 17 proteaz üreticisi Bacillus cinsi alkalofilik türü bakterilere % 80 benzerlik göstermektedir. Moleküler yöntemle tanılanan Bacillus clausii GMBAE 22 en iyi alkalen proteaz üreticisi olarak bulunmuştur. Bu tez kapsamında, yeni izole edilmiş Bacillus clausii GMBAE 22’ nin ürettiği serin alkalen proteazın saflaştırılması ve karakterizasyonu da çalışılmıştır. Bacillus clausii GMBAE 22’ den elde edilen enzim; protein bakımından zengin ortamda pH 10.5’ da 2 gün 37°C’ de çalkalayıcı inkübatörde üretilmiştir. En yüksek alkalen proteaz enzim aktivitesi hücre kültivasyonunun geç durağan fazında belirlenmiştir. Kültürden filtre edilen enzim; (NH4)2SO4 çöktürmesini takiben, DEAE-Selüloz kromatografisi ile kısmen saflaştırılmıştır. Enzimin saflaştırma katsayısı 2.66, verimi ise %14.69 olarak elde edilmiştir. Enzimin moleküler ağırlığı; SDS-PAGE analizi kullanılarak 25.4 kDa olarak belirlenmiştir. Enzimin optimum sıcaklığı 60°C bulunmuştur; bununla beraber ortama 5 mM konsantrasyonda Ca+2 iyonları ilave edildiğinde optimum sıcaklık 70°C’ ye değişmektedir. Enzimin, pH 10.5’ da 30-40°C arasında 2 saat kararlı olduğu, 50°C’ de ise aktivitesinin %35’ ini kaybettiği belirlenmiştir. Enzimin optimum pH’ sı 12 olarak bulunmuştur. Enzim; 30°C’ de pH 9.0 – 11.0 arasında 24 saat boyunca kararlıdır. Enzim aynı koşullarda pH 12.0’ de %96.70 ve 12.5’ de ise %20.14 oranında aktivitesini koruyabilmiştir. Enzim; PMSF varlığında tamamen inhibe olduğu için bir serin alkalin proteazdır. Km ve kcat değerleri sırasıyla 1.32 mg ml-1, 469.51 dak-1 olarak bulunmuştur. Bu çalışma BSE-073/ 1311 numaralı Bilimsel Araştırma Projeleri Komisyonu (BAPKO) tarafından desteklenmiştir. Fenotipik ve Genotipik Karakterizasyon, Endüstriyel Önemi Olan Enzimler, Bacillus clausii, Serine Alkalen Proteaz, Enzim Saflaştırılması, Enzim Karakterizasyonu. KASIM, 2005 Hülya BAL
Isolated and Identified, Bacterial Cultures were Screened in Order to Estimate Their Ability for Production of Industrially Important Enzymes From Local Ecosystems. In this study, 146 bacterial cultures were isolated from the composts and hot spring waters located in Marmara Region of Turkey. Isolated cultures were screened in order to estimate their ability for production of industrially important enzymes such as alkaline protease, endoglucanase, amylase and lipase. The isolates that are able to produce alkaline protease, amylase, endoglucanase, lipase were found to be 80.3%, 45.26%, 16.06%, 16.06% of the total isolates respectively. 19 of the 146 bacterial cultures were selected for further phenotypic characterisation. 17 alkaline protease producers were shown 80% similarity with the alkaliphilic type species of Bacillus genus. The isolate Bacillus clausii GMBAE 22 was found to be as best alkaline protease producer. It is identified with chemotaxonomic and molecular methods. The purification and characterization of a serine alkaline protease produced by newly isolated Bacillus clausii GMBAE 22 was also studied. The enzyme was produced in protein rich medium by shaking of B. clausii GMBAE 22 cultures for 2 days at pH 10.5 and 37°C. The highest alkaline protease activity was observed at the late stationary phase of cell cultivation. The enzyme in culture filtrate was partially purified by DEAE-cellulose chromatography following the (NH4)2SO4 precipitation step. 2.66-fold purification of enzyme was succeeded with 14.69% yield. The molecular weight of enzyme was found to be 25.4 kDa by SDS-PAGE analysis. Optimum temperature of enzyme was estimated as 60°C, however it is shifted to 70°C after addition of Ca2+ ions in 5 mM concentration. The enzyme was stable between 30-40°C for 2 h at pH 10.5. Only 35% activity loss was observed at 50°C. Optimal pH of the enzyme was found to be 12. Enzyme was also stable in pH 9.0 – 11.0 range for 24 h at 30°C, however, 96.70 and 20.14% activity protections were observed at pH values 12.0 and 12.5 respectively. The strong inhibition of enzyme by PMSF treatment suggested that enzyme is a serine alkaline protease. Km and kcat values were found to be 1.32 mg ml-1, 469.51 dak-1 respectively. This work was supported by The Scientific Research Projects Council (BAPKO) of Marmara Üniversity, with BSE-073/ 1311. Key Words: Phenotypic and Genotypic Characterisation, Industrially Important Enzymes, Bacillus clausii, Serine Alkaline Protease, Enzyme Purification, Enzyme Characterisation. November, 2005 Hülya BAL
Isolated and Identified, Bacterial Cultures were Screened in Order to Estimate Their Ability for Production of Industrially Important Enzymes From Local Ecosystems. In this study, 146 bacterial cultures were isolated from the composts and hot spring waters located in Marmara Region of Turkey. Isolated cultures were screened in order to estimate their ability for production of industrially important enzymes such as alkaline protease, endoglucanase, amylase and lipase. The isolates that are able to produce alkaline protease, amylase, endoglucanase, lipase were found to be 80.3%, 45.26%, 16.06%, 16.06% of the total isolates respectively. 19 of the 146 bacterial cultures were selected for further phenotypic characterisation. 17 alkaline protease producers were shown 80% similarity with the alkaliphilic type species of Bacillus genus. The isolate Bacillus clausii GMBAE 22 was found to be as best alkaline protease producer. It is identified with chemotaxonomic and molecular methods. The purification and characterization of a serine alkaline protease produced by newly isolated Bacillus clausii GMBAE 22 was also studied. The enzyme was produced in protein rich medium by shaking of B. clausii GMBAE 22 cultures for 2 days at pH 10.5 and 37°C. The highest alkaline protease activity was observed at the late stationary phase of cell cultivation. The enzyme in culture filtrate was partially purified by DEAE-cellulose chromatography following the (NH4)2SO4 precipitation step. 2.66-fold purification of enzyme was succeeded with 14.69% yield. The molecular weight of enzyme was found to be 25.4 kDa by SDS-PAGE analysis. Optimum temperature of enzyme was estimated as 60°C, however it is shifted to 70°C after addition of Ca2+ ions in 5 mM concentration. The enzyme was stable between 30-40°C for 2 h at pH 10.5. Only 35% activity loss was observed at 50°C. Optimal pH of the enzyme was found to be 12. Enzyme was also stable in pH 9.0 – 11.0 range for 24 h at 30°C, however, 96.70 and 20.14% activity protections were observed at pH values 12.0 and 12.5 respectively. The strong inhibition of enzyme by PMSF treatment suggested that enzyme is a serine alkaline protease. Km and kcat values were found to be 1.32 mg ml-1, 469.51 dak-1 respectively. This work was supported by The Scientific Research Projects Council (BAPKO) of Marmara Üniversity, with BSE-073/ 1311. Key Words: Phenotypic and Genotypic Characterisation, Industrially Important Enzymes, Bacillus clausii, Serine Alkaline Protease, Enzyme Purification, Enzyme Characterisation. November, 2005 Hülya BAL