Kloning Gen Penyandi Protease Netral (NPR) Termostabil dari Geobacillus Sp. DS3 dengan Sel Inang Escherichia Coli DH5?

Ilmi Octaviani Geopany, Dr. rer. nat. Lucia Dhiantika Witasari, S.Farm, Apt., M.Biotech.; Prof. Widya Asmara

2024 | Tesis | S2 Bioteknologi

Enzim protease netral termostabil dihasilkan oleh bakteri termofilik yang dapat tumbuh optimal pada kondisi lingkungan yang bersuhu tinggi. Enzim protease yang dihasilkan oleh bakteri ini sangat bermanfaat dalam bidang industri seperti: deterjen, kulit, tekstil, makanan maupun obat-obatan. Geobacillus sp. DS3 merupakan bakteri termofilik yang tumbuh optimal pada suhu 55oC – 65oC dan bersifat neutrofilik yang dapat tumbuh optimal pada lingkungan dengan pH 6,2 – 7,5. Tujuan dari penelitian ini yakni kloning gen penyandi protease netral (NPr) termostabil yang diproduksi oleh Geobacillus sp. DS3 termofilik. Tahapan penelitian yang dilakukan adalah kloning protease netral (NPr) termostabil dari Geobacillus sp. DS3 ke plasmid pETSUMO menggunakan metode kloning TA. Kemudian, plasmid rekombinan ditransformasi menggunakan metode heat shock ke sel kompeten Escherichia coli DH5? Selanjutnya, dilakukan analisis transforman dengan metode koloni PCR dan sequencing untuk mengetahui sekuen dari fragmen DNA dan orientasinya. Selanjutnya, dibuat pohon filogentik untuk mengetahuai kekerabatan antara gen penyandi protease netral, protease asam dan protease basa. Hasil dari penelitian ini adalah bahwa plasmid rekombinan pETSUMO NPr termostabil dapat dikloning dengan Escherichia coli DH5? dan gen penyandi NPr dari Geobacillus sp. DS3 memiliki kekerabatan paling dekat dengan M4 family metallopeptidase Geobacillus vulcani.

Thermostable neutral protease enzymes are produced by thermophilic bacteria that can grow optimally in high-temperature environmental conditions. Protease enzymes produced by these bacteria are very useful in industrial fields such as detergents, leather, textiles, food, and medicine. Geobacillus sp. DS3 is a thermophilic bacteria that grows optimally at a temperature of 55oC - 65oC and is neutrophilic which can grow optimally in an environment with a pH of 6.2 - 7.5. The purpose of this study was to clone the gene encoding thermostable neutral protease (NPr) produced by Geobacillus sp. DS3. The stages of the research carried out were cloning thermostable neutral protease (NPr) from Geobacillus sp. DS3 to the pETSUMO plasmid using the TA cloning method. Then, the recombinant plasmid was transformed using the heat shock method into competent cells of Escherichia coli DH5?. Furthermore, transformant analysis was carried out using the colony PCR method and sequencing to determine the sequence of DNA fragments and their orientation. Furthermore, a phylogenetic tree was made to determine the kinship between the genes encoding neutral protease, acid protease, and alkaline protease. The results of this study are that the thermostable pETSUMO NPr recombinant plasmid can be cloned with Escherichia coli DH5? and the NPr-encoding gene from Geobacillus sp. DS3 has the closest kinship to the NPr-encoding gene from Bacillus subtilis protease. The results of this research are that the thermostable pETSUMO NPr recombinant plasmid can be cloned with Escherichia coli DH5? and the gene encoding NPr from Geobacillus sp. DS3 is the closest relative to M4 family metallopeptidase Geobacillus vulcani.

Kata Kunci : enzim protease netral termostabil, plasmid rekombinan pETSUMO NPr, Geobacillus sp. DS3, Escherichia coli DH5?