Isolasi Open Reading Frame (ORF) 6-Fosfo-Beta-Glukosidase dari Bacillus sp. T3 dan Karakterisasi Produknya secara In Silico

SIH PIRENANINGTYAS, M. Saifur Rohman, S.P., M.Si., M.Eng., Ph.D.; Ir. Jaka Widada, M.P., Ph.D.

2019 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Selulosa dapat dimanfaatkan di berbagai industri. Namun, proses degradasi selulosa menjadi monosakarida oleh endoglukanase dan eksoglukanase dihambat oleh keberadaan selubiosa. Oleh karena itu, 6-Fosfo-β-glukosidase memegang peranan penting karena dapat memotong ikatan 1,4-β-glikosidik dari selubiosa. Tujuan dari penelitian ini adalah untuk mengisolasi Open reading frame (ORF) 6-Fosfo-β-glukosidase dan melakukan karakterisasi secara in silico dari produk ORF tersebut. ORF 6-Fosfo-β-glukosidase dilakukan sekuensing dan dianalisis dengan menggunakan BLASTx. Kemudian, produk ORF tersebut dikarakterisasi dengan menggunakan ExPASy ProtParam, MEGA7, PSIPRED, Swiss Model, dan PyMOL 2.2. Hasil sekuensing menunjukkan bahwa Bacillus sp. T3 memiliki ukuran nukleotida sebesar 1392 bp. Analisis BLASTx menunjukkan bahwa 6-Fosfo-β-glukosidase memiliki identity sebesar 97% dengan 6-Fosfo-β-glukosidase dari grup Bacillus. Analisis filogenetik juga menyatakan bahwa 6- Fosfo-β-glukosidase Bacillus sp. T3 memiliki kekerabatan yang dekat dengan kelompok termofilik. Analisis fisikokimia dari produk ORF tersebut menunjukkan bahwa 6-Fosfo-β- glukosidase Bacillus sp. T3 memiliki rasio asam amino basa dan asam sebesar 0,774; rasio Arg/Lys sebesar 0,778; dan rasio Ala/Gly sebesar 0,68. Analisis struktural menunjukkan bahwa 6-Fosfo-β-glukosidase Bacillus sp. T3 masuk ke dalam famili GH1, yang memiliki bentuk topologi α/β TIM barrel. Dua residu katalitik dari 6-Fosfo-β-glukosidase Bacillus sp. T3, Glu- 157 dan Glu-365, memiliki orientasi yang sama terhadap residu-residu yang sesuai dari 6-fosfo- β-galaktosidase Geobacillus stearothermophilus (4ZE5). Analisis struktural juga menunjukkan bahwa 6-Fosfo-β-glukosidase dari Bacillus sp. T3 memiliki dua residu penting dalam pengikatan fosfat, yaitu Lys-425 dan Tyr-427. Berdasarkan analisis tersebut, 6-Fosfo-β-glukosidase Bacillus sp. T3 memiliki karakteristik yang mengarah ke karakteristik kelompok bakteri termofil. Namun, perlu dilakukan uji eksperimental untuk mendukung analisis in silico dari 6-Fosfo-β-glukosidase Bacillus sp. T3.

Cellulose can be used in various industries. However, the degradation of cellulose into monosaccharides by endoglucanase and exogucase is inhibited by the presence of cellubiose. Therefore, 6-phospho-β-glucosidase plays an important role because it can cleaves 1,4-β- glycosidic bonds from cellobiose. The aims of this study were to isolate the open reading frame (ORF) of 6-phospho-β-glucosidase and perform in silico characterization the product of the ORF. The ORF 6-phospho-β-glucosidase was sequenced and subjected for BLASTx analysis. The ORF product was then characterized by using ExPASy ProtParam, MEGA7, PSIPRED, Swiss Model, and PyMOL 2.2. Sequencing results showed that the ORF of Bacillus sp. T3 had nucleotides size of 1392 bp. BLASTx analysis clearly showed that the ORF product was 6-phospho-β-glucosidase with 97% identity to the 6-phospho-β-glucosidase of Bacillus group. Phylogenetic analysis also indicated that 6-phospho-β-glucosidase of Bacillus sp. T3 was close to the thermophilic group. Physicochemical analysis of the ORF product showed that 6-phospho-β-glucosidase of Bacillus sp. T3 had base and acid amino acid ratio of 0,774; Arg/Lys ratio of 0,778; and Ala/Gly ratio of 0,68. Structural analysis indicated that 6-phospho-β-glucosidase of Bacillus sp. T3 belongs to the GH1 family, which had protein topology of α/β TIM barrel. The two catalytic residues of 6- phospho-β-glucosidase of Bacillus sp. T3, Glu-157 and Glu-365, were orient similar to the correspond residues of 6-phospho-β-galactosidase of Geobacillus stearothermophilus (4ZE5). Structural analysis also indicated that 6-phospho-β-glucosidase of Bacillus sp. T3 had two important residues for phosphate binding, namely Lys-425 and Tyr-427. Based on this analysis, 6-phospho-β-glucosidase Bacillus sp. T3 has characteristics that lead to the characteristics of thermophilic bacteria. However, an experiment is needed to support the in silico analysis of 6- phospho-β-glucosidase Bacillus sp. T3.

Kata Kunci : ORF, 6-phospho-β-Glucosidase, Bacillus sp. T3, in silico analysis