Publication: Ülkemizden izole edilen bacillus licheniformis ba17’den elde edilen alkalen proteaz enziminin saflaştırılması ve karakterizasyonu
Abstract
Ülkemizden İzole Edilen Bacillus licheniformis BA17’den Elde Edilen Alkalen Proteaz Enziminin Saflaştırılması ve Karakterizasyonu Bu çalışmada Van Gölü’nden izole edilen B. licheniformis BA17 alkalen proteaz enziminin saflaştırılması ve karakterizasyonu araştırılmıştır. B. licheniformis BA17’den proteaz üretimi için (w/ v) %1 maltoz, %0.8 yeast, %0.02 MgSO4.7H2O, %0.1 K2HPO4, %1 NaNO3 içeren besi yeri kullanılmıştır. Alkalen proteaz üretimi BA17’nin 37°C’de ve 180rpm çalkalama hızında 24 saat büyütülmesi ile gerçekleştirilmiştir. Ultrafiltrasyon ve iyon değiştirme (DEAE selüloz) kromatografisi adımları ile enzim %58 verimde ve 5.4 kat saflaştırılmıştır. Enzimin moleküler ağırlığı 19.7 kDa, Ca+2 iyonu varlığında ve yokluğunda optimum sıcaklığı 60°C olarak bulunmuştur. Saf enzimin 40°C, 50°C, 60°C ve 70°C sıcaklıklarda yarılanma ömrü sırası ile 38 saat, 93 dakika, 14 dakika ve 6 dakika olarak bulunmuştur. Enzimin optimum pH’sı 10 olarak belirlenmiş ve pH 8.0-10 aralığında 30oC sabit sıcaklıkta 2.5 saat inkübe edildiğinde aktivitesini kaybetmediği, pH 10.5, 11, 11.5, 12, 12.5 ve 13 değerlerinde, aynı koşullarda inkübe edildiğinde ise sırasıyla %15, %15, %36.6, %50.7, %54.3 ve %90 oranında aktivite kaybının olduğu gözlenmiştir. 30°C sıcaklıkta 5mM konsantrasyonunda Cu+2, Mg+2, Mn+2 ve K+1 iyonu varlığında enzim aktivitesinin sırasıyla %23, %4, %6 ve %12 oranında arttığı, 5mM Ca+2 ve NH4+1 iyonu varlığında enzim aktivitesinin etkilenmediği, 5mM Co+2, Fe+2 ve Na+1 iyonu varlığında ise enzim aktivitesinin sırasıyla %6, %27 ve %5 oranında azaldığı belirlenmiştir. 5mM (NH4)2SO4’ın enzim aktivitesinde %17 oranında kayba neden olduğu gözlenmiştir. %5 SDS(w/ v) ve %1 Triton X-100(v/ v) varlığında enzimin aktivitesini kaybettiği, %1 Tween 20(v/ v) varlığında ise aktivitesinin %23.5 oranında arttığı gözlenmiştir. 5mM PMSF varlığında aktivitesinin tamamen kaybolması sebebi ile enzimin serin proteaz olduğu, 5mM EDTA varlığında aktivitede görülen %81 oranındaki kayıp sebebi ile enzimin Ca+2 bağlama bölgesine sahip olduğu belirlenmiştir. 10mM NBS, 10mM TLCK ve TPCK, 10mM etilasetimidat ve iodoasetik asit varlığında aktivitede görülen kayıp sebebi ile triptofan, histidin ve lizin rezidülerinin aktif bölgede bulunduğu belirlenmiştir. Enzimin sentetik substratlardan kemotripsin substratı olan N-Suc-Ala-Ala-Pro-Phe-PNA’yı hidrolizlediği belirlenmiştir. Enzimin kazein hidrolizi için, Km ve Vm değerleri sırasıyla 0.209mg/ ml ve 5.5μmol tirosin/ ml/ dak olarak hesaplanmıştır. Alkaline proteaz, Bacillus licheniformis, saflaştırma, karakterizasyon
Purification and Characterization of Alkaline Protease Produced By Bacillus licheniformis BA17 Isolated from Turkey In this work, purification and characterization of alkaline protease produced by B. licheniformis BA17 isolated from Van Lake in Turkey was investigated. Medium containing (w/ v) 1% maltose, 0.8% yeast extract, 0.02% MgSO4.7H2O, 0.1% K2HPO4, and 1% NaNO3 was used for alkaline protease production. B. licheniformis BA17 was grown overnight at 37°C and 180rpm. Purification fold and yield obtained by using the ultrafiltration and ion exchange chromatography in series were 5.4 and 58 %, respectively. The molecular weight of enzyme was found to be 19.7 kDa by using SDS-PAGE electrophoresis, and the optimum temperature was 60°C in the presence and absence of Ca+2 ion. The half-life of the pure enzyme was 38 hours, 93 minutes, 14 minutes and 6 minutes at 40°C, 50°C, 60°C and 70°C, respectively. The optimum pH of enzyme was determined as 10, and activity was not affected when the enzyme was incubated for 2.5 hours at 30°C and at pH between 8.0 and 10. Under the same incubation conditions, 15%, 15%, 36.6%, 50.7,% 54.3% and 90% of the activity was lost at pH 10.5, 11, 11.5, 12, 12.5 and 13, respectively. The catalytic activity of enzyme increased 23%, 4%, 6% and 12% in the presence of 5mM Cu+2, Mg+2, Mn+2 and K+1 ions at 30°C, respectively but activity did not change in the presence of 5mM Ca+2 and NH41+ ions. However activity decreased 6%, 27% and 5% in the presence of 5mM Co+2, Fe+2 and Na+1 ions, respectively. 17% of the enzyme activity was lost in the presence of 5mM (NH4)2SO4. In the presence of 5% SDS (w/ v) and 1% Triton X-100(v/ v), the enzyme retained its original activity, but in the presence of 1% Tween 20 (v/ v) enzyme activity increased by 23.5 %. Enzyme activity was inhibited by PMSF, suggesting that the enzyme is a serine protease. The enzyme has a Ca+2 binding site since 81% of the activity was retained in the presence of 5 mM EDTA. The inhibition of enzyme activity in the presence of 10mM NBS, 10mM TLCK and TPCK, 10mM ethylacetimidate and iodoacetic acid, suggested the presence of tryptophan, histidine and lysine residues at the active site It was also found that enzyme hydrolyzed synthetic kemotrypsin substrate “N-Suc-Ala-Ala-Pro-Phe-pNA”. Km and Vm values of the enzyme for casein hydrolysis were found approximately 0.209 mg/ ml and 5.5 μmol tyrosine/ ml/ min respectively. Key words: Alkaline protease, Bacillus licheniformis, purification, characterization
Purification and Characterization of Alkaline Protease Produced By Bacillus licheniformis BA17 Isolated from Turkey In this work, purification and characterization of alkaline protease produced by B. licheniformis BA17 isolated from Van Lake in Turkey was investigated. Medium containing (w/ v) 1% maltose, 0.8% yeast extract, 0.02% MgSO4.7H2O, 0.1% K2HPO4, and 1% NaNO3 was used for alkaline protease production. B. licheniformis BA17 was grown overnight at 37°C and 180rpm. Purification fold and yield obtained by using the ultrafiltration and ion exchange chromatography in series were 5.4 and 58 %, respectively. The molecular weight of enzyme was found to be 19.7 kDa by using SDS-PAGE electrophoresis, and the optimum temperature was 60°C in the presence and absence of Ca+2 ion. The half-life of the pure enzyme was 38 hours, 93 minutes, 14 minutes and 6 minutes at 40°C, 50°C, 60°C and 70°C, respectively. The optimum pH of enzyme was determined as 10, and activity was not affected when the enzyme was incubated for 2.5 hours at 30°C and at pH between 8.0 and 10. Under the same incubation conditions, 15%, 15%, 36.6%, 50.7,% 54.3% and 90% of the activity was lost at pH 10.5, 11, 11.5, 12, 12.5 and 13, respectively. The catalytic activity of enzyme increased 23%, 4%, 6% and 12% in the presence of 5mM Cu+2, Mg+2, Mn+2 and K+1 ions at 30°C, respectively but activity did not change in the presence of 5mM Ca+2 and NH41+ ions. However activity decreased 6%, 27% and 5% in the presence of 5mM Co+2, Fe+2 and Na+1 ions, respectively. 17% of the enzyme activity was lost in the presence of 5mM (NH4)2SO4. In the presence of 5% SDS (w/ v) and 1% Triton X-100(v/ v), the enzyme retained its original activity, but in the presence of 1% Tween 20 (v/ v) enzyme activity increased by 23.5 %. Enzyme activity was inhibited by PMSF, suggesting that the enzyme is a serine protease. The enzyme has a Ca+2 binding site since 81% of the activity was retained in the presence of 5 mM EDTA. The inhibition of enzyme activity in the presence of 10mM NBS, 10mM TLCK and TPCK, 10mM ethylacetimidate and iodoacetic acid, suggested the presence of tryptophan, histidine and lysine residues at the active site It was also found that enzyme hydrolyzed synthetic kemotrypsin substrate “N-Suc-Ala-Ala-Pro-Phe-pNA”. Km and Vm values of the enzyme for casein hydrolysis were found approximately 0.209 mg/ ml and 5.5 μmol tyrosine/ ml/ min respectively. Key words: Alkaline protease, Bacillus licheniformis, purification, characterization