ISOLASI, KLONING DAN EKSPRESI OPEN READING FRAME LUCIFERASE-LIKE MONOOXYGENASE ENZYME TIPE 2 DARI Priestia megaterium PSA14

ABIDAH TAUCHID, M. Saifur Rohman, M. Si., M. Eng., Ph. D.

2021 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Luciferase-like monooxygenase (LLM) memiliki kemampuan untuk mengkatalisis reaksi oksidasi Baeyer-Villiger khususnya dalam proses biosintesis senyawa organik. Analisis in silico, menunjukkan bahwa Priestia megaterium PSA 14 memiliki empat (4) open reading frame LLM. Penelitian ini bertujuan untuk mendapatkan open reading frame LLM tipe 2 Priestia megaterium PSA 14 dan klon tersebut pada vektor pET28a(+) serta mendapatkan protein rekombinan LLM tipe 2 Priestia megaterium PSA 14 pada bakteri Escherichia coli BL21 (DE3). Open reading frame LLM tipe 2 diamplifikasi dengan metode PCR kemudian dikloning dengan menyisipkan open reading frame kedalam vektor pET28a(+), kemudian vektor pembawa open reading frame ditrasformasikan kedalam Escherichia coli BL21(DE3) serta diekspresikan menggunakan 12% SDS-PAGE. Hasil penelitian menunjukkan bahwa hasil amplifikasi open reading frame memiliki persentase similaritas sebesar 96.88% terhadap LLM class flavin dependent oxidoreductase Priestia megaterium. Open reading frame berhasil dikloning kedalam vektor pET28a(+) dan menunjukkan persentase similaritas sebesar sebesar 99.70% dengan MsnO8 family LLM class flavin-dependent oxidoreductase Priestia megaterium. Rekombinan LLM tipe 2 berhasil diekspresikan dalam system Escherichia coli BL21(DE3). Berat molekul protein rekombinan LLM tipe 2 sebesar ����¯�¿�½������±43,95 kDa bila dianalisis dengan 12% SDS-PAGE.

Luciferase-like monooxygenase (LLM) is an enzyme that has capability to catalyze the Baeyer-Villiger oxidation reaction, in organic compounds biosynthesis. In silico studies showed that the Priestia megaterium genome contains four open reading frames of LLM. Therefore, the objectives of this work were to obtain an open reading frame type 2 LLM from Priestia megaterium PSA 14 genome and that clone into the pET28a(+) vector and to obtain protein recombinant type 2 LLM from Priestia megaterium PSA 14 genome in Escherichia coli BL21 (DE3). The open reading frame of type 2 LLM was amplified by using PCR and then cloned it into the pET28a(+) vector. The resultant plasmid was then used to transform Escherichia coli BL21(DE3). The expression of recombinant protein of type 2 LLM was examined by using 12% SDS-PAGE. The results showed that the open reading frame of type 2 LLM from Priestia megaterium PSA 14 was successfully amplified and showed percent similarity of 96.88% to LLM class flavin dependent oxidoreductase Priestia megaterium. Open reading frame was also successfully cloned into pET28a(+) vector and showed percent similarity of 99.70% to MsnO8 family LLM class flavin-dependent oxidoreductase Priestia megaterium and expressed in Escherichia coli BL21(DE3) system. The molecular weight of the recombinant type 2 LLM exhibited molecular weight of ����¯�¿�½������±43.95 kDa, when it was analyzed by 12% SDS-PAGE.

Kata Kunci : LLM tipe 2, Priestia megaterium, kloning, ekspresi, protein rekombinan