PEMURNIAN ENZIM SEFALOSPORIN-C ASILASE DAN OPTIMASI PROSES KROMATOGRAFI PENUKAR ION
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Abstract
Sefalosporin-C asilase (CCA) merupakan enzim yang berperan penting dalam konversi satu tahap sefalosporin-C menjadi 7-ACA. Proses purifikasi merupakan salah satu cara untuk meningkatkan aktivitas spesifik enzim CCA. Proses purifikasi dimulai dari memecah sel, diikuti dengan tahap presipitasi menggunakan amonium sulfat, dialisis, kromatografi penukar ion (IEC) dan kromatografi eksklusi. Optimasi proses IEC pada tahap elusi juga dilakukan untuk membandingkan elusi enzim CCA secara gradien dan isokratik. Proses purifikasi pada penelitian ini mampu meningkatkan kemurnian enzim hingga 33,66 kali, dengan aktivitas spesifik sebesar 3,00 U/mg dan perolehan enzim sebesar 41,41%. Hasil optimasi IEC pada proses elusi secara isokratik lebih efisien dari segi waktu (hanya membutuhkan 3 kolom volume (CV) dibandingkan dengan secara gradien (sampai 9 CV). Hasil SDS-PAGE menunjukkan bahwa CCA rekombinan merupakan enzim dengan 2 macam bentuk yaitu enzim aktif, yang terdiri dari subunit α (25 kDa) dan β (58 kDa), dan enzim tidak aktif berupa prekursor (83 kDa). Proses presipitasi menggunakan amonium sulfat 30% tersaturasi dapat mengendapkan prekursor CCA.
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