KLONING DAN ANALISIS IN SILICO ORF THERMOLYSIN-LIKE PROTEASE DARI Bacillus sp. T3

NUZULYA RAMADHANI, M. Saifur Rohman, S.P., M.Si. M.Eng, Ph.D; Dr. Ir. Jaka Widada M.P

2018 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Thermolysin merupakan enzim protease yang bersifat termostabil. Perlunya biaya yang besar dalam menemukan dan menumbuhkan bakteri termofilik menyebabkan munculnya upaya untuk mendapatkan protease termostabil dari bakteri mesofilik. Pada penelitian ini dilakukan kloning, dan analisis in silico ORF thermolysin-like protease dari Bacillus sp. T3. untuk mengetahui struktur dan karakter dari enzim tersebut. ORF gen penyandi thermolysin-like dari Bacillus sp. T3 telah berhasil dikloning dengan menggunakan metode Polymerase Chain Reaction (PCR). ORF thermolysin-like hasil kloning yang berukuran sekitar 1500 bp berhasil ditransformasikan ke dalam E. coli BL21 (DE3) dengan menggunakan plasmid pET28a. Hasil uji keberhasilan transformasi dilakukan dengan metode PCR koloni dan menghasilkan ukuran yang sama dengan hasil kloning sebelumnya. Analisis struktur dan karakter ORF thermolysin-like dari Bacillus sp. T3 menggunakan BLAST, ExPASy ProtParam, MEGA6, dan PSIPRED. Dari hasil analisis sekuen diketahui bahwa ORF thermolysin-like protease dari Bacillus sp. T3 memiliki panjang sekuen 506 asam amino, pI pada kisaran 7, mengandung jumlah asam amino muatan positif yang lebih banyak dibandingkan negatif dengan perbandingan rasio 1,019, dan rasio asam amino arginine dan lysine sebesar 0,368. Nilai-nilai tersebut mendekati nilai pada hasil analisis kelompok bakteri mesofil. Pada analisis secara struktural, ORF thermolysin-like protease Bacillus sp. T3 memiliki zinc binding site pada sisi aktif dengan residu katalitik berupa His-142, His-146, dan Glu-166. Dari keseluruhan analisis in silico yang telah dilakukan, dapat disimpulkan bahwa ORF thermolysin-like protease dari Bacillus sp. T3 memiliki karakter yang dekat dengan kelompok bakteri mesofil.

Thermolysin is a protease enzyme which is termostable. The need for a high cost in discovering and growing the thermophilic bacteria has led the efforts to produce thermostable protease of mesophilic ones. Therefore, this research was conducted in cloning and in silico analysis of ORF thermolysin-like proteases from Bacillus sp. T3 to find out the structure and character of these enzymes. ORF of thermolysin-like encoder gene from Bacillus sp. T3 was successfully cloned by using Polymerase Chain Reaction (PCR) method. ORF of thermolysin-like cloning results, measuring around 1500 bp was successfully transformed into E. coli BL21 (DE3) using plasmids pET28a. Analysis of transformed cells was done by using colony PCR method that had the same size as previous cloning result. Analysis of the structure and the character of the ORF thermolysin-like from Bacillus sp. T3 was done by using BLAST, ExPASy ProtParam, MEGA6, and PSIPRED. Sequence analysis results shows that ORF of thermolysin-like protease from Bacillus sp. T3 has a sequence of amino acids 506 long, pI in the range 7, contains more positive charges of amino acids than negative ones by ratio 1.019, and 0.368 for the ratio of arginine and lysine amino acids. These numbers are quite close to the value of the analysis results of mesophile bacteria groups. In structural analysis, ORF of thermolysin-like protease from Bacillus sp. T3 has a zinc binding site on the active side of the catalytic residues in His-142, HIS-146, and Glu-166. From overall in silico analysis that have been done, it can be inferred that the ORF thermolysin-like proteases from Bacillus sp. T3 has close characters with group of mesophile bacteria.

Kata Kunci : ORF, thermolysin, thermolysin-like protease, Bacillus sp. T3, kloning, analisis in silico.