Partial purification and biochemical characterization of B-Lactamase from penicillin G resistant strains of streeptococcus thermophilus
Penisillin G'ye dayanıklı streptococcus thermophilus susundan elde edilen B-Laktamazin kısmi saflaştırılması ve biyokimyasal karakterizasyonu
- Tez No: 38638
- Danışmanlar: PROF.DR. FARUK BOZOĞLU
- Tez Türü: Yüksek Lisans
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Penicillin G resistance, P-lactamase, S. thermophilus, partial purification IV, Penicillin G resistance, P-lactamase, S. thermophilus, partial purification IV
- Yıl: 1995
- Dil: İngilizce
- Üniversite: Orta Doğu Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 71
Özet
oz PENISILLIN G' YE DAYANIKLI STREPTOCOCCUS THERMOPHILUS SUSUNDAN ELDE EDİLEN p-LAKTAMAZIN KISMI SAFLAŞTIRILMASI VE BİYOKİMYASAL KARAKTERKASYONU CHIRICA, LAURA-CRISTINA Yüksek Lisans Tezi, Biyokimya Tez Yöneticisi: Prof. Dr. Faruk Bozoglu Yardimci Tez Yöneticisi: Doç. Dr. Tülin Güray Mayis, 1995, 71 Sayfa Bilindiği gibi, bakteriler J3-laktam antibiyotiklerinin öldürücü etkisinden kurtulabilmek için bir yada birkaç P-laktamaz semizlemektedirler. Bu, bakteriler tarafından çok geniş ve genellikle en etkin şekilde uygulanan bir mekanizmadir. Gerçekte, bu mekanizma antibiyotiklerin tip ve gida endüstrisinde kuUanimi açisindan çok önemlidir. Bu, çalismada, Penisilin G dayanikli Streptococcus ihermophilus suslarindan elde edilen iki değişik p-laktamaz (E.C 3.5.2.6) ilk defa olarak kismi saflastirilmis, biyokimyasal özellikleri incelenmiştir. Kismi saflastirma işlemi SephadexG150 SF kolon kromatografisinde yapilmis ve enzim I ve enzim ffnin bagil aktiviteleri sirasiyla 3.06 ve 2.97 ünite/mg protein2.97 units/mg protein. The purification fold was 2.45 for fraction I and 2.32 for fraction II. Both fractions of p-lactamase showed specificity to penicillins but not for cephaloridines. The kinetic constants for both fractions were determined by using benzylpenicillin (penicillin G) as a substrate. Fraction I exhibited higher substrate affinity, p-lactamase having low KM value of 3.44 uM, whereas KM for fraction II was 4.76 uM. The Vmax values were 8.33 umole/min/mg protein and 3.125 umole/min/mg protein for fraction I and II, respectively. The two fractions of P-lactamase were inhibited by iodine, copper sulphate, iron sulphate but not by EDTA. Typical pH-activity curves were obtained and the shape of the curves and the optimal pH range, between 6 and 7, were similar for both fractions of p-lactamase. The effect of temperature was tested and an optimal temperature of 40 to 45° C was found for both fractions of P-lactamase. The freeze-dried form of the enzymes was found to be much more stable than the P-lactamase in dilute solutions.
Özet (Çeviri)
ABSTRACT PARTIAL PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF p-LACTAMASE FROM PENICILLIN G RESISTANT STRAINS OF STREPTOCOCCUS THERMOPHILUS CHIRICA, LAURA-CRISTINA M.S., in Biochemistry Supervisor: Prof. Dr. Faruk Bozoglu Co-supervisor: Assoc. Prof. Dr. Tülin Güray May, 1995, 71 pages It has. been known that, the synthesis of one or several P-lactamases represents the most wide spread and often, the most efficient mechanism devised by bacteria to escape the lethal action of P-lactam antibiotics. In fact, this mechanism is very important for the use of antibiotics in medicine and also in food industry. In this study, two different fractions of P-lactamase (E.C 3.5.2.6) from penicillin G resistant strains of Streptococcus thermophilus were partially purified for the first time, and some biochemical properties were examined. Partial purification was carried out on Sephadex G150 SF column chromatography and the specific activities were found as 3.06 units/mg protekand m2.97 units/mg protein. The purification fold was 2.45 for fraction I and 2.32 for fraction II. Both fractions of p-lactamase showed specificity to penicillins but not for cephaloridines. The kinetic constants for both fractions were determined by using benzylpenicillin (penicillin G) as a substrate. Fraction I exhibited higher substrate affinity, p-lactamase having low KM value of 3.44 uM, whereas KM for fraction II was 4.76 uM. The Vmax values were 8.33 umole/min/mg protein and 3.125 umole/min/mg protein for fraction I and II, respectively. The two fractions of P-lactamase were inhibited by iodine, copper sulphate, iron sulphate but not by EDTA. Typical pH-activity curves were obtained and the shape of the curves and the optimal pH range, between 6 and 7, were similar for both fractions of p-lactamase. The effect of temperature was tested and an optimal temperature of 40 to 45° C was found for both fractions of P-lactamase. The freeze-dried form of the enzymes was found to be much more stable than the P-lactamase in dilute solutions.
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