Isolasi, seleksi aktinomisetes Khitinolitik dan karakterisasi enzim khitinase

NUGROHO, Agustinus Joko, Prof.Dr.Ir. Sebastian Margino

2006 | Tesis | S2 Bioteknologi

Khitinase (EC.3.2.1.14) merupakan enzim hidrolitik yang mampu merombak khitin secara acak, baik dalam bahan maupun organisme tertentu. Enzim ini sebagian besar dihasilkan oleh kelompok aktinomisetes yang masuk dalam genus Streptomyces, Nocardia, Microsomonas, Actinoplanes dan Streptosporangium. Tujuan penelitian ini adalah: (1) memperoleh aktinomisetes indigenus dengan kemampuan khitinolitik tinggi melalui isolasi dan seleksi, dan (2) isolasi, purifikasi, dan karakterisasi enzim khitinase isolat unggul. Penelitian dilaksanakan dalam 5 tahapan, yaitu: (1) isolasi dan seleksi aktinomisetes khitinolitik, (2) karakterisasi isolat unggul, (3) optimalisasi kondisi pertumbuhan, (4) purifikasi enzim (fraksionasi ammonium sulfat dan DEAE Cellulose), dan (5) karakterisasi enzim. Sebanyak 84 isolat berhasil diisolasi dari tanah, limbah udang, dan kompos. Isolat IK yang diklasifikasikan sementara dalam Streptomyces sp. IK, memiliki aktivitas enzim tertinggi. Kondisi optimal pertumbuhan Streptomyces sp. IK pada medium pH 7, konsentrasi khitin 0,2% (b/v), jumlah inokulum 10% (v/v), dan waktu inkubasi 4 hari. Aktivitas spesifik khitinase meningkat setelah dilakukan pemurnian dengan fraksionasi ammonium sulfat dan kromatografi penukar ion (DEAE Cellulose). Hasil karakterisasi enzim khitinase memiliki berat molekul 71 kDa, Vmaks 4,26 μg/jam, nilai Km 2,92 mg/ml, aktivitas khitinase optimal pada pH 6,7 dan suhu 370C.

Chitinase ( EC.3.2.1.14) is a hydrolytic enzyme which cleave randomly inside the chains of chitin. Organism has the ability of producing chitinases including those actinomycetes such as Streptomyces, Nocardia, Microsomonas, Actinoplanes and Streptosporangium. The purpose of this research was to characterize and purify the chitinase from indigenous actinomycete. This research was conducted in five step activities, those were: (1) isolation and selection of chitinolytic actinomycetes, (2) characterization of excellent strain, (3) optimization of growth condition, (4) purification of enzyme (precipitation using ammonium sulfate and DEAE Cellulose), and (5) characterization of enzyme. There are 84 strains of chitinolytic actinomycetes have been isolated from soil, shrimp waste, and compost. Strain IK classified in Streptomyces sp. IK, have the highest enzyme activity. The optimal condition of Streptomyces sp. IK, of pH medium was 7, concentration of chitin 0.2% (w/v), with inoculum concentration of 10% (v/v), and incubation time at 4 day. Specific activity chitinase mount after purified by ammonium sulfate precipitation and DEAE-Cellulose ion-exchange chromatography. The apparent molecular weight of the purified protein was 71 kDa, Km value and Vmax of the protein for colloidal chitin were 2.92 mg/ml and 4.26 μg/h, respectively. Chitinase was optimally active at pH of 6.7 and at 370C

Kata Kunci : Aktinomisetes,Kitinase,chitinase, characterization, purification, actinomycetes