Optimasi Metode Isolasi Gen amyS Penyandi a-Amilase dari Bacillus paralicheniformis ATCC 12759

Abstract

Bakteri pembentuk endospora dikenal sebagai penghasil enzim termostabil, termasuk a-amilase termostabil yang banyak digunakan dalam industri berbasis pati. Sebanyak 60% produksi a-amilase dihasilkan oleh genus Bacillus. Namun, penggunaan bakteri native dalam industri membutuhkan energi besar, sehingga diperlukan teknologi DNA rekombinan. Penelitian ini bertujuan mengoptimasi proses isolasi gen meliputi isolasi DNA genom hingga konfirmasi urutan nukleotida gen amyS dari B. paralicheniformis ATCC 12759 melalui sekuensing Sanger. Gen a-amilase diisolasi menggunakan metode PCR dengan sepasang primer spesifik, yaitu amyS_F1 (5`-ATGAAACAACACAAACGGC-3`) dan amyS_R1 (5`-CTATCTTTGAACATAGATCGAAACC-3`). DNA genom menunjukkan hasil optimum setelah penambahan volume proteinase K, tanpa inkubasi setelah isopropanol, dan buffer TE bersuhu 25 ? untuk resuspensi DNA. Sementara itu, isolasi gen amyS tervisualisasikan dengan pita tunggal ukuran sekitar 1,5 kb pada konsentrasi cetakan DNA 25 ng dan suhu annealing 54,7 ?. Konfirmasi hasil amplifikasi gen menunjukkan urutan nukleotida memiliki tingkat kesamaan 100% dengan gen amyS referensi.

Endospore-forming bacteria are recognized as producers of thermostable enzymes, including thermostable a-amylase which is widely used in starch-based industries. Approximately 60% of a-amylase production is derived from the genus Bacillus. However, the use of native bacteria in industrial applications requires high energy consumption; therefore, recombinant DNA technology is necessary. This study aimed to optimize the gene isolation process, from genomic DNA extraction to nucleotide sequence confirmation of the amyS gene from B. paralicheniformis ATCC 12759 through Sanger sequencing. The a-amylase gene was isolated using the PCR method with a pair of specific primers, namely amyS_F1 (5`-ATGAAACAACACAAACGGC-3`) and amyS_R1 (5`-CTATCTTGAACATAGATCGAAACC-3`). Genomic DNA yielded optimum results following the addition of proteinase K, precipitation with cold isopropanol without an incubation, and dissolution using TE buffer at 25 ?. Meanwhile, isolation of the amyS gene was visualized as a single band of approximately 1,5 kb at a DNA template concentration 25 ng and an annealing temperature of 54,7 ?. Confirmation of the gene amplification product revealed that the nucleotide sequence exhibited 100% identity to the reference amyS gene sequence.