Isolasi Open Reading Frame Xylanase dan Karakterisasi Produknya secara In Silico dari Bacillus sp. T3

ANNUR MINA RIDHAYANI, M. Saifur Rohman, S.P., M.Si., M.Eng., Ph.D.; Ir. Jaka Widada, M.P., Ph.D.

2019 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Xylanase merupakan enzim glikosida hidrolase yang memiliki kemampuan dalam menghidrolisis ikatan 1,4-beta-xylosidik. Xylanase berperan penting dalam proses degradasi polisakarida terutama hemiselulosa. Perlu dilakukan upaya untuk mengeksplorasi lebih banyak xylanase dari berbagai bakteri untuk mengidentifikasi karakter xylanase yang berpotensi untuk keperluan rekayasa. Pada penelitian ini bertujuan untuk mengisolasi orf xylanase dari Bacillus sp. T3 dan melakukan analisis karakter xylanase yang terekspresikan secara in silico. Analisis in silico meliputi analisis fisikokimia dan analisis struktural. Open reading frame (orf) xylanase dari Bacillus sp. T3 telah berhasil diisolasi dengan menggunakan metode Polymerase Chain Reaction (PCR) menghasilkan pita berukuran +/- 1200 bp. Produk PCR kemudian digunakan untuk sekuensing DNA untuk analisis lebih lanjut. Analisis BLASTx menunjukkan bahwa orf menunjukkan nilai identity sebesar 77 hingga 85% pada endo-1,4-beta-xylanase dari kelompok Bacillus. Produk orf selanjutnya dianalisis dengan menggunakan ExPASy ProtParam, MEGA7, dan ExPASy SWISS MODEL. Xylanase memiliki berat molekul 44,9 kDa dengan nilai pI 6,38. Xylanse dari Bacillus sp. T3 menunjukkan rasio Arg/Lys yang secara signifikan lebih tinggi daripada kelompok bakteri mesofilik, termofilik dan psikrofilik. Ini menunjukkan bahwa xylanse dari Bacillus sp. T3 lebih bersifat stabil daripada xylanase dari bakteri lainnya. Analisis struktural berdasarkan model 3D xylanase dari Bacillus sp. T3 menunjukkan bahwa xylanase memiliki situs pengikatan logam (Ca+) yang terletak pada carbohydrate binding module (CBM). Residu katalitik yang dibentuk oleh Asp-24, Asp-163 dan Glu-226 memiliki orientasi yang mirip dengan Arabinoxylan arabinofuranohydrolase Bacillus subtilis BsAXH-m2,3.

Xylanase is a hydrolase glycoside enzyme which has an ability to hydrolyze 1,4-beta-xylosidik bonds. Xylanase has played an important role for polysaccharide degradation especially hemicellulose. Efforts should be made to explore more xylanases from various bacteria in order to further identify their characters which have potentially useful for engineering purposes. Therefore, the objective of this work were to isolate open reading frame (orf) of xylanase from Bacillus sp. T3 and perform in silico analyses of the xylanase that might be expressed from the orf. In silico analysis that were carried out included physiochemical analysis and structural analysis. The orf of xylanase from Bacillus sp. T3 has been successfully isolated using Polymerase Chain Reaction (PCR) method which resulted band of +/- 1200 bp. The PCR product was then used for DNA sequencing for further analyses. BLASTx analysis indicated that the orf exhibited 77 to 85% identity to the endo-1,4-beta-xylanase of the Bacilus group. The orf product was further analsyses by using MEGA7, ExPASy ProtParam and ExPASy SWISS MODEL. The xylanase has molecular weight of 44.9 kDa with the pI value of 6.38. Xylanse from Bacillus sp T3 exhibited the Arg/Lys ratio which significantly higher than that of mesophilic, thermophilic and psychrophilic group. This indicating that the xylanse from Bacillus sp T3 was intriguingly more stable than that the xylanases from others bacteria. Structural analysis, based on the 3D model of xylanase from Bacillus sp. T3 showed that xylanase has a metal binding site (Ca+) that located on a carbohydrate binding module. The catalytic residues which was formed by Asp-24, Asp-163 and Glu-226 had similar orientation to that of Arabinoxylan arabinofuranohydrolase Bacillus subtilis BsAXH-m2,3.

Kata Kunci : orf, isolasi, analisis in silico, xylanase, Bacillus sp. strain T3.