Matrik Alumina-Agarosa untuk pemurnian enzim lipase dari kecambah niji wijen (Sesamum indicum)

MINARNI, Wiwik Sri, Prof.Dr.Ir. Tranggono, M.Sc

2005 | Tesis | S2 Teknologi Hasil Perkebunan

Immobilized metal affinity chromatography metode pada penelitian ini, untuk pemurnian lipase. Matrik berdensitas tinggi dibuat dengan melapisi alumina menggunakan agarose secara teknik emulsi. Imminodiacetic acid (IDA) dan butanediol diglycidyl ether (BDGE) diimobilisasikan ke matrik. BDGE berikatan dengan matrix alumina-agarose. Variasi konsentrasi BDGE adalah 20, 30, 40, 50, dan 60 % yang akan berinteraksi dengan ion logam (Cu2+, Zn2+, Ni2+, Fe2+). Hasil penelitian menunjukkan bahwa ion logam Cu2+ dan konsentrasi BDGE 40% dihasilkan adsorbsi protein paling tinggi dengan elusi konsentrasi imidazole yang rendah. Konsentrasi NaCl pada buffer mempengaruhi adsorbsi protein pada immobilized metal affinity. Dilaporkan bahwa peningkatan konsentrasi NaCl akan meningkatkan jumlah adsorbsi protein. Aktivitas spesifik lipase tertinggi adalah pada 0,5 mM NaCl pH 7. Strategi elusi mempengaruhi faktor purifikasi. Tahap pertama elusi menggunakan 2, 20 dan 100 mM imidazole menghasilkan faktor purifikasi 3,6. Tahap kedua elusi menggunakan 10 dan 50 mM imidazole menghasilkan faktor purifikasi tinggi yaitu 7,7. Tahap ketiga elusi menggunakan 20 dan 60 mM imidazole menghasilkan faktor purifikasi 6,3.

immobilized metal affinity chromatography method research on purification of lipases was carried out employing. A high-density matrix was prepared by coating alumina particles with agarose using an emulsion technique. In addition, iminodiacetic Acid (IDA) and butanediol diglycidyl ether (BDGE) were immobilized, and bound on the alumina-agarose matrix. Various concentration of BDGE i.e 20, 30, 40, 50 and 60 percents, were used which were expected to interact with some metal ions (Cu2+, Zn2+, Ni2+, Fe2+). Result showed that the metal ion Cu2+ and concentration 40 % BDGE was found to adsorb the highest concentration of protein with low concentration imidasol in the elution. Concentration of NaCl in buffer, affect protein adsorption in the immobilized metal affinity system. Was noted to As the NaCl concentration increased, the amount of adsorbed protein also increased. Highest specific activity of lipase was reached at 0,5 mM NaCl and pH 7. The elution strategi affected purification factor. Although the first-step elution using 2, 20 and 100 mM Imidazole concentration resulted in a low value of purification factor i.e. 3,6, how ever second-step elution using 10 and 50 mM Imidazole resulted in high-performance purification with a value of 7,7. Finally, in the third-step elution using 20 and 60 mM imidazole, a low purification factor of 6,3 was obtained.

Kata Kunci : Teknologi Enzim,Pemurnian Enzim Lipase,Kromatografi, Immobilized metal affinity chromatography, Proteins, Iminodiaccetic acid, metal ion