Analisis perbandingan aktivitas fibrinolitik Bacillus subtilis strain G8 dan IFP1.1 menggunakan metode presipitasi aseton dan amonium sulfat = Comparison of the fibrinolytic activity of Bacillus subtilis strain G8 and IFP1.1 with ammonium sulfate and acetone precipitation method

Sihombing, Caitlyn Christvania (2023) Analisis perbandingan aktivitas fibrinolitik Bacillus subtilis strain G8 dan IFP1.1 menggunakan metode presipitasi aseton dan amonium sulfat = Comparison of the fibrinolytic activity of Bacillus subtilis strain G8 and IFP1.1 with ammonium sulfate and acetone precipitation method. Bachelor thesis, Universitas Pelita Harapan.

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Abstract

Terdapat berbagai sumber enzim fibrinolitik yang dapat digunakan sebagai penanganan terhadap sumbatan pembuluh darah (trombosis), salah satunya adalah nattokinase yang berasal dari Bacillus subtilis natto. Untuk dapat mengisolasi enzim fibrinolitik, diperlukan proses presipitasi protein terlebih dahulu. Dua jenis metode presipitasi protein diuji terhadap B. subtilis G8 dan B. subtilis IFP1.1 yang telah diisolasi sebelumnya. Penelitian ini bertujuan untuk menganalisis perbedaan aktivitas fibrinolitik dari ekstrak protein yang dipresipitasi menggunakan metode presipitasi aseton dan ammonium sulfat, untuk menentukan metode yang lebih unggul dalam mengisolasi protein fibrinolitik. Prosedur dari penelitian ini antara lain preparasi kultur cair B. subtilis; presipitasi protein dengan metode ammonium sulfat dan aseton; kuantifikasi dan penyetaraan konsentrasi ekstrak protein; uji degradasi gumpalan darah; uji degradasi gumpalan euglobulin; uji degradasi fibrin; zimografi fibrin; dan pembuatan euglobulin agar plate. Hasil yang didapat adalah ekstrak protein dari metode aseton yang memiliki konsentrasi lebih tinggi dibandingkan dengan metode ammonium sulfat. Selain itu, ekstrak protein B. subtilis IFP1.1 yang dipresipitasi aseton memiliki aktivitas degradasi gumpalan darah paling tinggi, dibandingkan dengan B. subtilis G8 maupun metode presipitasi ammonium sulfat. Zimografi fibrin terhadap ekstrak protein B. subtilis IFP1.1 menunjukan adanya aktivitas protein fibrinolitik yang lebih tinggi pada ekstrak protein metode aseton, sedangkan tidak terdapat perbedaan pada ekstrak protein B. subtilis G8 pada kedua metode presipitasi. Penelitian ini menunjukkan adanya perbedaan pada masing-masing metode presipitasi protein serta tingkat aktivitas protein fibrinolitik yang lebih tinggi didapat dari ekstrak protein B. subtilis IFP1.1 yang dipresipitasi dengan metode aseton dibandingkan dengan metode ammonium sulfat. / There are many fibrinolytic enzyme sources that can be used to treat blockages in the blood vessels (thrombosis), and one of the examples is nattokinase from Bacillus subtilis natto. To isolate the fibinolytic enzymes from its source, a protein precipitation method is needed. Two types of protein precipitation method were tested in this study to isolate proteins from B. subtilis G8 and IFP1.1, two strains of bacteria that have been isolated previously. This research aimed to analyze the fibrinolytic activity of the protein extracted from acetone and ammonium sulfate precipitation method, to determine the preferable method to isolate fibrinolytic proteins. The procedures of this research include preparations of B. subtilis liquid culture; protein precipitation with ammonium sulfate and acetone method; protein concentration quantification and equalization; whole blood clot degradation assay; euglobulin clot degradation assay; fibrin degradation assay; fibrin zymography; and the experiment to create euglobulin agar plate. The results obtained from this research were higher protein concentration from the acetone precipitation method compared to ammonium sulfate method. Besides that, acetone extract from B. subtilis IFP1.1 had the highest whole blood clot degradation percentage compared to B. subtilis G8 or the protein precipitated with ammonium sulfate. Furthermore, fibrin zymography with protein extract from B. subtilis IFP1.1 showed higher fibrinolytic activity from the acetone protein extract, and no difference in activity for G8 with both precipitation method. This study indicated there are differences between both precipitation methods, and that higher fibrinolytic activity for B. subtilis IFP1.1 can be achieved with the acetone precipitation method compared to ammonium sulfate method.

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