ISOLASI OPEN READING FRAME (ORF) LYTIC POLYSACCHARIDE MONOOXYGENASE DARI Bacillus sp. T3 DAN ANALISIS PRODUKNYA MELALUI IN SILICO

NIKEN KARINA, M. Saifur Rohman, S.P., M.Si., M. Eng., Ph.D

2019 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Lytic Polysaccharide Monooxygenase merupakan enzim pengoksidasi ikatan glikosidik beta-1,4 pada polisakarida yang berperan penting dalam depolimerisasi senyawa kitin dan selulosa. Enzim ini memiliki potensi pemanfaatan untuk menghasilkan biofuel dan senyawa kimia lain yang bersumber dari polisakarida kitin dan selulosa. Penelitian ini bertujuan untuk mengamplifikasi open reading frame (ORF) gen LPMO dari Bacillus sp. T3 dan melakukan analisis in silico produk dari ORF tersebut. Amplifikasi ORF gen LPMO berhasil dilakukan dengan menggunakan metode Polymerase Chain Reaction (PCR). Identifikasi ORF dilakukan dengan menggunakan BLASTx kemudian produk dari ORF tersebut dianalisis menggunakan MEGA7 dan ExPASy ProtParam. Blast NCBI digunakan untuk menganalisis struktur primer, sedangkan PSIPRED digunakan untuk menganalisis struktur sekunder, Swiss Model digunakan untuk prediksi struktur tersier dan PyMOL 2.2 digunakan untuk visualisasi struktur tersier protein LPMO. Berdasarkan hasil amplifikasi putative ORF dan analisis menggunakan Blastx diketahui ORF gen LPMO memiliki ukuran 621 bp. Berdasarkan hasil ExPASy ProtParam diketahui bahwa LPMO dari Bacillus sp. T3 memiliki panjang sekuen 206 asam amino dengan rasio asam amino muatan positif dan negatif sebesar 0,957, rasio Arg/Lys sebesar 0,591 serta rasio Gly/Ala sebesar 0,59. Analisis struktur primer dan sekunder protein membuktikan bahwa protein LPMO dari Bacillus sp. T3 memiliki karakteristik yang berbeda dengan LPMO dari bakteri lainnya. Perbedaan ini terletak pada residu katalitik H125 yang berubah menjadi D125. Adapun struktur tersier protein LPMO dari Bacillus sp. T3 memilki kesamaan dengan struktur tersier protein LPMO dari B.amyloliquefaciens (PDB : 2YOX).

Lytic polysaccharide monooxygenase (LPMO) is an enzyme that depolymerize cellulose or chitin through oxidative mechanism leading to subsequent cleavage of the beta-1,4-glycosidic bond. This enzyme has important role for biofuel and other chemical compunds production from cellulose or chitin polymers. This work was aimed to amplify the open reading frame (ORF) of the LPMO encoding gene from Bacillus sp. T3 and characterize the properties of the product of the ORF by a means of in silico analysis. Amplification of the ORF LPMO encoding gene was successfully carried out using the Polymerase Chain Reaction (PCR) method. The ORF confirmation was done using BLASTx then the ORF products were analyzed using MEGA7 and ExPASy ProtParam. The NCBI blast was also used to analyze the primary structure, while the secondary and tertiary structures of LPMO were analyzed by using PSIPRED and Swiss Model, respectively. To visualize the tree dimensionally structure of LPMO, the PyMOL 2.2 suite was employed. BlastX analysis of the DNA sequencing further confirmed that the PCR product was the open reading frame of LPMO. The size of the ORF was 621 bp in length. Amino acids deduction from the nucleotides sequence of the ORF, which was 206 amino acid residues, was further analyzed by using ProtParam. Based on the amino acids composition, the LPMO of Bacillus sp. T3 had the positive and negative amino acid residue ratio of 0.957, Arg/Lys ratio of 0.591 and Gly/Ala ratio of 0,59. Analysis of primary and secondary structures showed that LPMO from Bacillus sp. T3 had distinct characteristics compared to the LPMO of other bacteria. This difference lies in the catalytic residue of H125, in which the H residue changes to D125. The tertiary structure of LPMO protein from Bacillus sp. T3 was similar to the tertiary structure of LPMO protein from B.amyloliquefaciens (PDB: 2YOX).

Kata Kunci : amplifikasi, analisis in silico, Bacillus sp. T3, LPMO/amplification, in silico analysis, Bacillus sp. T3, LPMO