Purifikasi dan Karakterisasi Parsial Rekombinan Luciferase-like Monooxygenase Tipe 1 (LLM1)

HOSSANA ABRAHAM A K, M. Saifur Rohman,. M.Si., M. Eng., Ph.D; Ahmad Suparmin., S.P., M.Agr.Sc., Ph.D; Ir. Ngadiman., M.Si., Ph.D

2023 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Luciferase-like monooxygenase (LLM) merupakan salah satu flavoprotein enzim yang termasuk ke dalam golongan luciferase-like family. LLM diketahui memiliki peran sebagai biokatalisator dalam berbagai reaksi oksidasi seperti, epoksidasi, halogenasi, dan oksidasi baeyer villiger. Pada penelitian sebelumnya, ORF llm1 yang berasal dari Priestia megaterium telah berhasil dikloningkan ke dalam vektor pET28a(+) serta diekspresikan dalam Escherichia coli BL21 (DE3). Pada penelitian ini dilakukan pengujian kelarutan, purifikasi, dan karakterisasi parsial rekombinan LLM1. Pada persiapan pre-culture, Escherichia coli BL21 (DE3) yang memiliki pET-llm1 dikultivasi dalam medium Luria Bertani (LB) cair mengandung 50 mikrogram/ml kanamisin pada suhu inkubasi sebesar 37 derajat celcius, agitasi sebesar 150 rpm, selama 18 jam. Sebanyak 2% pre-culture diambil dan diinokulasi kembali pada 50 mL medium LB cair berkanamisin. Kultur diinkubasi selama 1 jam hingga nilai absorbansi (OD600) pertumbuhan kultur mencapai 0.6 - 0.8, kemudian IPTG berbagai perlakuan (0,1 mM; 0,5 mM; dan 1 mM) ditambahkan ke dalam medium. Kultur diinkubasi kembali hingga 3 jam, kemudian sel dipanen dan disonikasi dalam larutan 10 mM Tris-HCl pH 8.8. Hasil berupa LLM1 terlarut dipurifikasi, dialisis, dan dianalisis dengan SDS-PAGE. Karakterisasi biofisik-kemikal parsial dilakukan dengan menggunakan analisis spektrum UV-Vis spektrofotometri. Pengujian stabilitas LLM1 dilakukan dengan mereaksikan protein dengan denaturan guanidine hydrochloride berkonsentrasi 1 M hingga 3 M. Hasil pengujian kelarutan menunjukkan bahwa LLM1 terlarut dalam 10 mM Tris-HCl pH 8.8 pada induksi IPTG 0,1 ; 0,5 ; dan 1 mM berturut - turut 68, 60, dan 69 %. LLM1 dapat dipurifikasi secara parsial menggunakan metode penukar ion. Analisis spektrum absorbansi (OD200-250) menunjukkan bahwa LLM1 memiliki nilai puncak absorbansi 206,5 nm pada kondisi terlipat dan 230 nm pada kondisi terdenaturasi. LLM1 juga terbukti memiliki ketahanan terhadap guanidine hydrochloride dengan konsentrasi maksimal 1,2 M.

Luciferase-like monooxygenase (LLM) is a flavoprotein enzyme that belongs to the luciferase-like family. LLM is known to have a role as a biocatalyst in various oxidation reactions such as epoxidation, halogenation, and baeyer villiger oxidation. In a previous study, ORF llm1 derived from Priestia megaterium has been successfully cloned into pET28a (+) vector and expressed in Escherichia coli BL21 (DE3). In this study, solubility assay, purification, and partial characterization of recombinant LLM1 were carried out. To prepare the pre-culture, Escherichia coli BL21 (DE3) with pET-llm1 was cultivated in liquid Luria Bertani (LB) medium containing 50 mikrogram/ml kanamycin at an incubation temperature of 37 celcius, agitation at 150 rpm, for 18 hours. A total of 2% of the pre-culture was taken and re-inoculated into 50 mL of liquid LB medium with kanamycin. The culture was incubated for 1 hour until the absorbance value (OD600) of culture growth reached 0.6 - 0.8, then IPTG of various concentrations (0.1 mM, 0.5 mM, and 1 mM) was added to the medium. The culture was re-incubated for up to 3 hours, then cells were harvested and sonicated in 10 mM Tris-HCl pH 8.8 solution. The soluble product of LLM1 was purified, dialyzed, and analyzed by SDS-PAGE. Partial biophysical-chemical characterization was performed using spectrophotometric UV-Vis spectra analysis. A stability assay of LLM1 was carried out by interacting the protein with guanidine hydrochloride in concentration of 1 M to 3M. Solubility assay results showed that LLM1 dissolved in 10 mM Tris-HCl pH 8.8 at IPTG induction of 0.1, 0.5, and 1 mM was 68, 60, and 69 %, respectively. LLM1 was able to be partially purified using the ion exchange method. Absorbance spectrum analysis (OD 200-250) showed that LLM1 had a peak absorbance value of 206.5 nm in the folded condition and 230 nm in the denatured condition. LLM1 was also shown to have a tolerance to guanidine hydrochloride with a maximum concentration of 1.2 M.

Kata Kunci : Purifikasi, Karakterisasi, Kelarutan, LLM1