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تاثير بعض مكونات القهوة على فعالية انزيم ? - Galactosidases المستخلص من بكتريا الفم المرتبطة بالبخر الفموي == Effect of Some Coffee Components on The Activity of ? - Galactosidase Isolated From Oral Bacteria Associated with Halitosis
Author name:
اميرة مريبي زرزور عبد الفضلي
Supervisor name:
عودة مزعل الزاملي | قاسم نجم ثويني
General topic:
Chemistry
Specific topic:
Chemistry
Degree:
Doctorate
University:
University of Babylon - College Of Science
Language:
English
University location:
Babylon
Abstract:
This study includes extraction and purification of α-galactosidase enzyme from Erysipelothrix rhusiopathiae associated with halitosis infections & estimating the inhibition behaviors of some coffee components (caffeine and chlorogenic acid) on the activity of this enzyme. These isolates were collected from outpatients attending Dental Clinical in Al-Zahra Hospital , Al-Kut/ Wasit province, Iraq during (March to October 2014) with age of (21-60 years old ), clinically diagnosed as having halitosis; from 100 patients (30 : 30%) had positive bacterial culture. Thirteen (43.3%) of these 30 patients were females; (6 : 46.1%) of them with gram negative bacteria whereas (7 : 53.8 %) were having gram positive bacteria, while (17 : 56.6%) were collected from males, (8 : 47.0%) having gram negative bacteria, and ( 9 : 52.9%) with gram positive bacteria. Among the gram negative bacteria isolated, four(20.0%) isolates belonged to Veillonella spp., 3 to Porphyromonas endontalis (15.0%), and 2 to Prevotella intermedia (10.0%). Whereas gram-positive isolates were 4 belonged to Erysipelothrix rhusiopathiae (20.0%), 4 to Streptococcus salivariu ( 20.0%), and 3 to Streptococcus oralis (15.0%). Further identification of these isolates was performed depending on commercial systems (Api20E ), in addition to Vitek2 system which also has the ability to identify the ability of bacteria to produce galactosidase and determine its type (either α-galactosidase or β-galactosidase). So that Vitek2 system helped us to select an isolate with ability to produce α-galactosidase. | Identification of Erysipelothrix rhusiopathiais was based on gram staining, colonial morphology, hemolytic and biochemical properties. Identification of the isolates was confirmed by Api20E system and automatic Vitek2 system. These 4 isolates were designated by the | II | researcher as E. rhusiopathiae IrqA1, E. rhusiopathiae IrqA2, E. rhusiopathiae IrqA3, and E. rhusiopathiae IrqA4. | Erysipelothrix rhusiopathiae IrqA2 was used for extraction and purification of α -galactosidase. This isolate was selected because it had a higher enzyme activity (730 U/ml). α-galactosidase enzyme has been extracted from a selected Erysipelothrix rhusiopathiae IrqA2 isolates and its concentration of protein was estimated to be 19.56 mg/ml as a crude protein. The activity of the enzyme was measured using ( p-nitrophenyl- α -D-galactopyranoside) as substrate , then purification steps included precipitation by 40% ammonium sulfate ( yielded 4.74mg/ml protein) with activity 515.2U/ml ,dialysis , DEAE- cellulose chromatography of partially purified α-galactosidase after ammonium sulfate precipitation showed one peak with maximum activity 331.15U/ml ( 0.537mg/ml of protein) .After ion-exchange chromatography, SDS-PAGE showed protein band at 55KDa) represented subunit of the partially purified enzyme . Kinetics parameter were calculated , Km (5.5 mM )& Vmax (8.3 μmol /min). The enzyme showed maximum activity at 45°C at pH 6.5 . | The effect of caffeine on partially purified α -galactosidase activity revealed non-competitive inhibition (decrease Vmax 6.2μmol/min, unchanged Km value 5.5 mM ). However, the effect of chlorogenic acid showed an uncompetitive inhibition on enzyme activity .Which reflected that the enzyme has a single catalytic site since the change in Km (3.1 mM ) & Vmax (4.1 μmol/min) was observed . Worldwide and based on literature review, this is the first study for extraction of α-galactosidase enzyme from this bacterium.
