Isolasi dan karakterisasi enzim protease pada isolat bakteri termofilik dari sumber air panas Gonoharjo Boja

Abstract

Telah diisolasi dan di karakterisasi enzim protease dari isolat bakteri termofilik somber air panas Gonoharjo Boja. Is°Iasi bakteri dilakukan dengan metode goresan menggunakan medium nutrien agar. Is°lat bakteri ditumbuhkan pada medium nutrien agar, dan enzim diproduksi pada medium skim milk broth. lsolasi enzim dilakukan dengan metode sentrifuge, dan difraksinasi bertingkat menggunakan amonium sulfat, yaitu tingkat kejenuhan 0 — 20 % ( Fl ), tingkat kejenuhan 20 — 40 % (F2 ), tingkat kejenuhan 40 — 60 % ( F3 ), dan tingkat kejenuhan 60 — 80 % F4 ), dilanjutkan dialisis dalam buffer fosfat. Parameter yang diamati adalah aktivitas enzim, aktivitas spesifik, dan kadar protein. :Fraksi yang diperoleh diuji dengan substrat kasein, menggunakan spektrofotometer UV¬Vis pada panjang gelombang optimum kasein dan hasilnya diekstrapolasikan terhadap kurva standar kasein, untuk mendapatkan nilai aktivitas enzim protease. Karakterisasi yang dilakukan meliputi penentuan temperatur, pFl, dan waktu inkubasi optimum. Hasil penelitian menunjukkan bahwa tingkat kejenuhan 20 — 40 % F2 ) rnemiliki tingkat kemurnian yang tertinggi dengan aktivitas spesitik 3,011 unit/mg. Temperatur optimal 44 'V; pH — 7,5; dan waktu inkubasi 15 menit. The isolation and characterization of protease from thermophilic bacteria isolate of Gorioharjo Boja hot spring have been done. The bacteria isolation has been carried out by streak method using nutrien agar medium. Bacteria isolate was grown on nutrien agar medium, and the enzyme was produced on the skim milk broth Medium. The enzyme isolation has been carried out by centrifuge, and fractinating the phase has been done using the ammonium sulfate, i.e 0 — 20 % saturated degree ( Fl ), 20 — 40 % saturated degree ( F2 ), 40 — 60 % saturated degree ( F3 ), and 60 — 80 %. saturated degree ( F4 ), followed by dialysis in p.hospate buffer. The parameters observed were enzyme activity, specific activity, and the proteine content. The fraction obtained were then analyzed using caseine substrate by UV-Vis spectrophotometer in caseine optimum wavelength. The results were extrapolized on the caseine standard curve to obtain protease enzyme activity value. The enzyme characterization was done 'by determined the optimum temperature, pH, and incubation time. The results showed that F2 (20 — 40% saturated degree) give the highest purity which has specific activity of 3.011 unit/mg. The optimal temperature was 44°C; = 7.5; and incubation. time was 15 minutes