Karakterisasi enzim kitinase intraseluler dari bakteri providencia stuartii = Characterization of intracellular chitinase from providencia stuartii bacteria

Giovanni, Gian (2018) Karakterisasi enzim kitinase intraseluler dari bakteri providencia stuartii = Characterization of intracellular chitinase from providencia stuartii bacteria. Bachelor thesis, Universitas Pelita Harapan.

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Abstract

Kitinase adalah enzim yang dapat menghidrolisis senyawa polimer kitin dengan acak pada ikatan glikosidik dan mampu menghasilkan turunan kitin menjadi kitin oligosakarida dan monomer N-asetil glukosamin yang dihasilkan oleh bakteri kitinolitik. Tujuan dari penelitian ini adalah menentukan pH optimum dan suhu optimum enzim kitinase yang diisolasi dari bakteri Providencia stuartii berdasarkan besarnya aktivitas enzim kitinase, serta bertujuan untuk menentukan berat molekul kitinase. Enzim kitinase intraseluler diperoleh dari isolasi sel bakteri Providencia stuartii melalui sonikasi dan sentrifugasi dengan menggunakan substrat koloidal kiti pada media. Purifikasi enzim kitinase intraseluler dilakukan dengan pengendapan protein menggunakan amonium sulfat 70% dan diteruskan dengan dialisis menggunakan kantung selofan 14 kDa untuk mendapatkan enzim kitinase intraseluler yang lebih murni. Analisis pH optimum dan suhu optimum kitinase intraseluler menggunakan metode spektrofotometri UV-VIS pada panjang gelombang 540 nm, serta menentukan berat molekul dengan SDS-PAGE. Hasil analisis aktivitias enzim kitinase berdasarkan pH menunjukkan bahwa pH optimum kitinase ada pada pH 5 dengan besar aktivitas enzim 7,17 ± 0,11 U/ml , sedangkan suhu optimum kitinase ada pada suhu 40oC dengan besar aktivitas enzim 5,14 ± 0,21 U/ml. Besar berat molekul yang dimiliki kitinase dari Providencia stuartii sebesar 38,76 kDa. / Chitinase is an enzyme that has ability to hydrolize the polymer of chtin randomly at glycosidic bond and produce chitin derivations such as chitin ologisaccharides and N-asetilglucosamine monomer, produced by chitinolytic bacteria. The aims of this research were to determine the optimum pH and temperature of chitinase that is isolated from Providencia stuartii bacteria based on its enzymatic activity and to determine the molecular weight of chitinase using electrophoresis method. Intracellular chitinase enzyme is obtained from the isolation of Providencia stuartii bacteria cell with sonication and centrifugation using colloidal chitin in media. Intracellular chitinase enzyme purification was done with protein precipitation using 70% amonium sulphate and continued with dialysis using 14 kDa cellophane tube to obtain purer intracellular chitinase enzyme. The optimum pH and temperature of intracellular chitinase were determined using UVVIS spectrophotometer at 540 nm wave-length and the molecular weight was determined using SDS-PAGE. The result showed that the optimum pH was 5 with 7.17 ± 0.11 U/ml enzyme activity, while the optimum temperature was 40oC with 5.14 ± 0.21 U/ml enzyme activity. The molecular weight of chitinase from Providencia stuartii was 38.76 kDa.

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