Isolasi, Kloning, dan Karakterisasi In Silico ORF Penyandi Isopentenyl Diphosphate Isomerase Tipe 2 (IDI-2) dari Planococcus sp. JS01

Erina Tika Febiyanti, M. Saifur Rohman, S.P., M.Si., M. Eng., Ph.D.; Ahmad Suparmin, S.P., M.Agr., Ph.D.

2026 | Skripsi | MIKROBIOLOGI PERTANIAN

Planococcus sp. JS01 merupakan bakteri halotoleran yang diisolasi dari kawasan Sangiran, Jawa Tengah dan diketahui mampu menghasilkan pigmen karotenoid. Biosintesis karotenoid berlangsung melalui jalur biosintesis isoprenoid yang memerlukan keseimbangan antara isopentenyl diphosphate (IPP) dan dimethylallyl diphosphate (DMAPP), yang diatur oleh enzim isopentenyl diphosphate isomerase tipe 2 (IDI-2). Namun, informasi mengenai gen penyandi IDI-2 pada Planococcus sp. JS01 masih terbatas. Penelitian ini bertujuan mengisolasi dan mengkloning open reading frame (ORF) kandidat penyandi IDI-2 ke dalam vektor ekspresi pET-28a(+), serta mengkarakterisasi kandidat proteinnya secara in silico. ORF kandidat IDI-2 diamplifikasi menggunakan polymerase chain reaction (PCR), kemudian dikloning ke dalam vektor pET-28a(+) melalui metode restriksi–ligasi menggunakan enzim NdeI dan HindIII. Keberadaan DNA insert diverifikasi melalui PCR koloni, PCR verifikasi, dan analisis restriksi, sedangkan karakterisasi in silico meliputi analisis similaritas sekuens, filogenetik, sifat fisikokimia, domain konservatif, dan prediksi struktur tiga dimensi. Hasil penelitian menunjukkan bahwa ORF kandidat IDI-2 berhasil diamplifikasi dan digunakan dalam konstruksi plasmid rekombinan pET-28a(+). Hasil PCR koloni, PCR verifikasi, dan analisis restriksi mengindikasikan keberhasilan penyisipan DNA insert ke dalam plasmid rekombinan, meskipun validasi urutan nukleotida belum dapat dilakukan secara optimal karena kualitas hasil sekuensing yang belum memadai. Analisis in silico menunjukkan bahwa kandidat ORF memiliki kemiripan tinggi dengan protein IDI-2 dari Planococcus plakortidis (WP_269466043.1). Protein putatif hasil translasi diprediksi bersifat stabil, cenderung hidrofilik, mengandung domain konservatif IDI-2_FMN-binding, serta memiliki pola lipatan tiga dimensi yang konsisten dengan karakteristik protein IDI-2. Penelitian ini memberikan dasar molekuler bagi penelitian lanjutan mengenai peran IDI-2 dalam biosintesis karotenoid pada Planococcus sp. JS01.

Planococcus sp. JS01 is a halotolerant bacterium isolated from Sangiran, Central Java, Indonesia, and has been reported to produce carotenoid pigments. Carotenoid biosynthesis proceeds through the isoprenoid biosynthetic pathway, which requires a balanced supply of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), regulated by isopentenyl diphosphate isomerase type 2 (IDI-2). However, information regarding the IDI-2-encoding gene in Planococcus sp. JS01 remains limited. This study aimed to isolate and clone the candidate open reading frame (ORF) encoding IDI-2 into the pET-28a(+) expression vector and to characterize the putative protein through in silico analysis. The candidate IDI-2 ORF was amplified by polymerase chain reaction (PCR) and cloned into pET-28a(+) using restriction–ligation with NdeI and HindIII. The presence of the DNA insert was verified by colony PCR, verification PCR, and restriction analysis, while in silico characterization included sequence similarity, phylogenetic, physicochemical, conserved domain, and three-dimensional structure analyses. The results showed that the candidate IDI-2 ORF was successfully amplified and used for recombinant pET-28a(+) plasmid construction. Colony PCR, verification PCR, and restriction analysis indicated successful insertion of the DNA fragment into the recombinant plasmid, although complete nucleotide sequence validation could not be achieved because of insufficient sequencing quality. In silico analysis revealed that the candidate ORF shared high similarity with the IDI-2 protein of Planococcus plakortidis (WP_269466043.1). The translated putative protein was predicted to be stable, slightly hydrophilic, contain the conserved IDI-2_FMN-binding domain, and exhibit a three-dimensional fold consistent with the characteristic structure of IDI-2 proteins. These findings provide a molecular basis for future studies on the role of IDI-2 in carotenoid biosynthesis in Planococcus sp. JS01.

Kata Kunci : Planococcus sp. JS01, isopentenyl diphosphate isomerase tipe 2 (IDI-2), kloning gen, karakterisasi in silico, karotenoid