Eksplorasi Protein Toksin Bacillus thuringiensis dari Tanah di Kabupaten Tangerang

Abstract

AbstrakBacillus thuringiensis (Bt) merupakan bakteri gram positif berspora penghasil protein toksin yang bersifat sebagai insektisidal dan sitosidal. Tujuan dari penelitian ini adalah mengeksplorasi protein toksin yang dihasilkan dari isolat lokal B.thuringiensis hasil sampling di beberapa tempat di wilayah Kabupaten Tangerang dan memprediksi karakteristik protein tersebut berdasarkan bobot molekulnya.Beberapa wilayah yang dijadikan titik sampling antara lain : Ciherang, Ciputat, Pasar Kemis, Kosambi, Parung Secab, Puspiptek, Pamulang, dan Cileduk. Isolasi B.thuringiensis dari sampel tanah dilakukan dengan metode Travera et al. (1987) menggunakan medium T3.Isolat yang diperoleh dikultivasi selama 72 jam pada suhu 28 o C hingga sporulasi dan dihasilkan protein kristal yang kemudian ditentukan kandungan proteinnya dengan metode Lowry. Analisis profil protein dilakukan dengan SDS-PAGE pada konsentrasi resolving gel 12 % dan stacking gel 4 %.Dari hasil penelitian diperoleh 6 sampel tanah yang positif mengandung B.thuringiensis, yaitu sampel Ciherang, Pasar Kemis, Kosambi, Parung Secab, Pamulang dan Cileduk. Analisis SDS-PAGE pada masing-masing sampel menunjukkan 5 jenis protein toksin yang diduga bersifat insektisidal, yaitu Cry 15, Cry 23, Cry 25, Cry 30, dan Cry 35, dan 3 jenis protein yang diprediksi bersifat sitosidal yaitu PS-2 (Cry 46), PS-3 (Cry41), dan PS-4 (Cry 45). AbstractBacillus thuringiensis (Bt) is a gram-positive sporulated bacterium produces protein toxin as an insecticidal and sitosidal. The purpose of this study was to explore these protein toxin produced by local isolates isolated from several places in the district of Tangerang and predict the characteristics of these proteins based on molecular weight. Some areas are used as sampling points are: Ciherang, Chester, Pasar Kemis, Kosambi, Parung Secab, Puspiptek, Pamulang, and Cileduk. B.thuringiensis were isolated by T3 medium. Cultivated isolates obtained during 72 hours at 28 o C until sporulation and crystal protein is produced which is protein content then quantification by Lowry method. Protein profile analysis performed by SDS-PAGE which use 12% resolving gel concentration and 4% stacking gel. From the results obtained 6 positive samples of soil containing B. thuringiensis, i.e Ciherang, Pasar Kemis, Kosambi, Parung Secab, Pamulang and Cileduk. SDS-PAGE analysis result showed five types of proteins are predicted to be insecticidal toxin, which is Cry 15, Cry 23, Cry 25, Cry 30 and Cry 35, and 3 types of proteins are predicted to be sitosidal the PS-2 (Cry 46), PS-3 (Cry41), and PS-4 (Cry 45).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis of toxin proteins of Bacillus thuringiensis isolated from soil at the Liwa Botanical Garden

“…The description of the phase and mechanism of B. thuringiensis Cry protein is provided in Figure 3. The results of the assumption of molecular weight with SDS PAGE are in accordance with what was done by [9]. The mechanism of action of the Cry protein is through direct ingestion by insects.…”

“…The SDS PAGE results shown Table 3 confirms previous research findings done by [9]. The mechanism of action of the Cry protein is through direct ingestion by insects.…”

“…Isolates that have been re-cultured in Luria Bertani media were then transferred to B. thuringiensis sporulation selective media, namely T-3 media, until they sporulated for seven days, resulting in the colonies displayed in Figure 1. The isolate colonies formed shown in Figure 1 confirm the findings of previous researchers that B. thuringiensis incubated in the T-3 selective medium were grew uniformly with yellowish-white color, rough and slightly shiny [9]. On medium T-3, the colonies that grew are colonies that have produced spores.…”

“…The isolate samples of B. thuringiensis used in this study were taken from collections of Microbiology Laboratory at the Department of Biology, University of Lampung. Those bacterial isolates were isolated by Janah et al, 2022 from soil at the Liwa Botanical Garden, West Lampung Regency the Lampung Province of Indonesia [7] Two isolates coded Bt3 and Bt5 were selected and rejuvenated with Luria Bertani media [8], containing 10 g tripton, g yeast extract, 10 g NaCl in 15 g agar [9]. Isolates were taken aseptically using ose needle, inoculated into Luria bertani agar slant medium by streak method and incubated at room temperature for 24 hours.…”

“…The results of protein quantification that have been separated from isolate spores using a spectrophotometer at a wavelength of 595 nm are presented in Table 2. The separation of spores and crystalline proteins is done by adding a mixture of 1% SDS 0.01% beta mercaptoethanol [9]. This mechanism is also described by [10] that the crystalline proteins mixed with spores will be separated due to the presence of beta mercaptoethanol which is able to break disulfide bonds in proteins without affecting their primary structure through the reduction of S-S bonds by producing two cysteine residues [13].…”