Pengendalian hayati nematoda sista kuning (NSK) globodera rostochiensis secara terpadu: Isolasi, seleksi bakteri proteolitik dan karakterisasi protease

JUMI'ATI, Prof. Dr. Ir. Sebastian Margino

2010 | Tesis | S2 Bioteknologi

Nematoda Sista Kuning (Globodera rostochiensis) adalah hama kentang baru (ditemukan di Indonesia pada tahun 2003) dan menimbulkan kerusakan hingga 70%. Pengendalian Nematoda Sista Kuning (NSK) secara kimiawi cukup efektif, tetapi menimbulkan resistensi, terbunuhnya organisme bukan sasaran dan pencemaran lingkungan. Oleh karena itu pengendalian hayati menggunakan mikroorganisme merupakan pengendalian alternatif yang lebih ramah lingkungan.Kelebihan agen pengendali hayati produk bakteri proteolitik karena menghasilkan protease yang dapat menghidrolisis protein, sehingga dapat merusak telur NSK yang memiliki komponen vitelin (protein). Tujuan penelitian ini adalah: (1) isolasi dan seleksi bakteri proteolitik indigenus yang berpotensi tinggi dalam merusak telur NSK; (2) isolasi, purifikasi, dan karakterisasi enzim protease ekstraseluler isolat terpilih.Penelitian ini dilakukan dalam 5 tahapan yaitu: (1) isolasi dan seleksi bakteri proteolitik; (2) karakterisasi isolat terpilih; (3) optimalisasi kondisi pertumbuhan untuk produksi protease; (4) purifikasi enzim (fraksionasi ammonium sulfat, DEAE Cellulose dan Sephacryl S-300); (5) karakterisasi enzim. Sebanyak 131 isolat berhasil diisolasi dari berbagai sumber (tanah pembuangan limbah udang, ayam, sapi, kompos, dan rhizosfer tanaman tomat dan kentang. Isolat TBRSN-1 yang teridentifikasi sementara sebagai Bacillus sp. mempunyai kemampuan merusak telur NSK sebesar 84,62%. Protease diproduksi Bacillus sp. TBRSN-1 pada kondisi optimal pertumbuhan yakni pada medium pH 7, konsentrasi substrat skim milk 1% (b/v), jumlah inokulum 5% (v/v), agitasi 150 rpm, suhu 30°C dan waktu inkubasi 60 jam. Aktivitas protease meningkat setelah dilakukan pemurnian dengan fraksionasi ammonium sulfat, kromatografi penukar ion (DEAE Cellulose) dan kromatografi gel filtrasi (Sephacryl S-300). Hasil karakterisasi menunjukkan bahwa protease ekstraseluler Bacillus sp. TBRSN-1 memiliki berat molekul 48,1 kDa, Vmaks 4,03 μg/jam, Km 7,83 mg/ml, aktivitas protease optimal pada pH 7 dan suhu 30°C.

Potato Cyst Nematode (PCN) is one of the potatoes pest which first found in Indonesia in 2003 and caused the economic lost impact to the farmer up to 70%. Due to the problem caused by chemical control (resistency, killed non target organisms and environmental pollution), development of an alternative control which has friendly environmental is great importance i.g. biological control. Bacteria producing protease is a good choice for controlling GCN since GCN eggshell cangisting of protein. The purpose of this research was to isolate proteolytic bacteria has high ability damage GCN eggshell, purify and characterize the protease of selected bacteria. This research was conducted in five step activities, those were; (1) isolation and selection of proteolytic bacteria; (2) characterization of excellent isolate; (3) optimization of its growth condition; (4) purification of proteolytic enzyme (precipitation using ammonium sulfat, DEAE Cellulose and Sephacryl S- 300), and (5) characterization of the enzyme.There are 131 strains of proteolytic bacteria have been isolated from several sources (shrimp, chicken, cow and compost). Isolate TBRSN-1 was determinate as Bacillus sp. TBRSN-1, has the highest enzyme activity and damage ability to GCN eggshell (84.62%). Optimal condition of Bacillus sp.TBRSN-1 for producing protease was pH 7.0, skim milk 1% (w/v), inoculum concentration of 5% (v/v), gitation 150 rpm, temperature 30°C, and incubation 60 hours. Specific activity of protease was increased after purified by ammonium sulfate precipitation, DEAE ellulose ion-exchange chromatography and Sephacryl S-300 gel-filtration chromatography. The apparent molecular weight of purified protein was 48.1 kDa, Km value and Vmax of the enzyme for casein substrate were 7.83 mg/ml and .03 μg/h, respectively. The protease was optimally active at pH 7 and 30°C.

Kata Kunci : Bakteri proteolitik,Protease,Purifikasi, proteolityc bacteria, protease, purification.